Articles de revues : « SR Protein Specific Kinase 2 »

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Wang, Huan-You, Wen Lin, Jacqueline A. Dyck, Joanne M. Yeakley, Zhou Songyang, Lewis C. Cantley et Xiang-Dong Fu. « SRPK2 : A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells ». Journal of Cell Biology 140, n^o 4 (23 février 1998) : 737–50. http://dx.doi.org/10.1083/jcb.140.4.737.

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Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

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McKee, Charia, Dan J. Bare et Xun Ai. « Stress-driven cardiac calcium mishandling via a kinase-to-kinase crosstalk ». Pflügers Archiv - European Journal of Physiology 473, n^o 3 (15 février 2021) : 363–75. http://dx.doi.org/10.1007/s00424-021-02533-2.

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AbstractCalcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca2+-ATPase and increased diastolic SR calcium leak are involved in the development of maladaptive cardiac remodeling under pathological conditions. Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca2+ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca2+ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.

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Karakama, Yuko, Naoya Sakamoto, Yasuhiro Itsui, Mina Nakagawa, Megumi Tasaka-Fujita, Yuki Nishimura-Sakurai, Sei Kakinuma et al. « Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase ». Antimicrobial Agents and Chemotherapy 54, n^o 8 (24 mai 2010) : 3179–86. http://dx.doi.org/10.1128/aac.00113-10.

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ABSTRACT Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.

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Meldrum, D. R., J. C. Cleveland, M. B. Mitchell, B. C. Sheridan, F. Gamboni-Robertson, A. H. Harken et A. Banerjee. « Protein kinase C mediates Ca2(+)-induced cardioadaptation to ischemia-reperfusion injury ». American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 271, n^o 3 (1 septembre 1996) : R718—R726. http://dx.doi.org/10.1152/ajpregu.1996.271.3.r718.

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Although protein kinase C (PKC)-mediated cardioadaptation to ischemia-reperfusion (IR) is accompanied by increased intracellular Ca2+ concentration, it is unknown whether a preischemia sarcoplasmic reticulum (SR) Ca2+ release affects PKC-mediated post-IR functional protection. To study this, crystalloid-perfused (Langendorff) Sprague-Dawley rat hearts were used to assess the effects of a ryanodine (Ry)-induced preischemia Ca2+ load (Ry, 5 nM/2 min, retrograde coronary) 10 min before global IR (20 min). Ry was administered with and without each of two different PKC inhibitors (20 microM chelerythrine and 150 nM bisindolylmaleimide I-HCl). Ry improved myocardial functional recovery (developed pressure, end-diastolic pressure, coronary flow, and creatine kinase activity), which was eliminated after PKC inhibition. Immunohistochemical staining for PKC isoforms demonstrated that Ry induces specific PKC translocation of alpha-, delta-, and zeta-isoforms. We conclude that 1) a preischemia Ca2+ load from the SR results in post-IR myocardial functional protection 2) Ca(2+)-induced functional protection is PKC regulated via the translocation of specific isoforms, and 3) Ca(2+)-induced cardioadaptation to IR injury may have important therapeutic implications prior to planned ischemic events such as cardiac allograft preservation and cardiac bypass surgery.

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Manna, Pulak R., Youngah Jo et Douglas M. Stocco. « Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2 : role of protein kinase A and protein kinase C signaling ». Journal of Endocrinology 193, n^o 1 (avril 2007) : 53–63. http://dx.doi.org/10.1677/joe-06-0201.

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The steroidogenic acute regulatory (StAR) protein plays a central role in the regulation of steroid biosynthesis. While steroidogenesis is influenced by many processes, their modes of actions, in a few cases, remain obscure. In this study, we explored the mechanism of action of one such signaling pathway, the extracellular signal-regulated kinase 1/2 (ERK1/2), in regulating StAR expression and steroidogenesis in conjunction with the protein kinase A (PKA) and protein kinase C (PKC) pathways. Using MA-10 mouse Leydig tumor cells, we demonstrate that the activation of PKC and PKA signaling, by phorbol-12-myristate-13-acetate (PMA) and dibutyryl cAMP (dbcAMP)/human chorionic gonadotropin (hCG) respectively, was able to phosphorylate ERK1/2, an event markedly decreased by an upstream kinase inhibitor, U0126. Treatment with PMA enhanced StAR protein expression (associated with a slight increase in progesterone synthesis) but not its phosphorylation (P-StAR), which, in contrast, coordinately increased in response to dbcAMP/hCG. Inhibition of ERK1/2 activity by U0126 decreased PMA-treated StAR expression but increased dbcAMP/hCG-mediated StAR and P-StAR; however, progesterone levels were attenuated. U0126 was found to affect StAR expression and steroidogenesis both at the transcriptional and translational levels. Further studies demonstrated that the effect of U0126 on PMA- and dbcAMP/hCG-mediated StAR expression and steroid synthesis was tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) and scavenger receptor class B type 1 (SR-B1). In fact, both DAX-1 and SR-B1 appear to play important roles in hormone-regulated steroidogenesis. These findings clearly demonstrate that the ERK1/2 signaling cascade involved in regulating StAR expression and steroid synthesis is mediated by multiple factors and pathways and is stimulus specific in mouse Leydig cells.

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Vasanji, Zainisha, Elliott J. F. Cantor, Danijel Juric, Mellissa Moyen et Thomas Netticadan. « Alterations in cardiac contractile performance and sarcoplasmic reticulum function in sucrose-fed rats is associated with insulin resistance ». American Journal of Physiology-Cell Physiology 291, n^o 4 (octobre 2006) : C772—C780. http://dx.doi.org/10.1152/ajpcell.00086.2005.

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Diabetes mellitus (DM) causes the development of a specific cardiomyopathy that results from the metabolic derangements present in DM and manifests as cardiac contractile dysfunction. Although myocardial dysfunction in Type 1 DM has been associated with defects in the function and regulation of the sarcoplasmic reticulum (SR), very little is known about SR function in Type 2 DM. Accordingly, this study examined whether abnormalities in cardiac contractile performance and SR function occur in the prestage of Type 2 DM (i.e., during insulin resistance). Sucrose feeding was used to induce whole body insulin resistance, whereas cardiac contractile performance was assessed by echocardiography and SR function was measured by SR calcium (Ca2+) uptake. Sucrose-fed rats exhibited hyperinsulinemia, hyperglycemia, and hyperlipidemia relative to control rats. Serial echocardiographic assessments in the sucrose-fed rats revealed early abnormalities in diastolic function followed by late systolic dysfunction and concurrent alterations in myocardial structure. The hearts of the 10-wk sucrose-fed rats showed depressed SR function demonstrated by a significant reduction in SR Ca2+ uptake. The decline in SR Ca2+ uptake was associated with a significant decrease in the cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of phospholamban. The results show that abnormalities in cardiac contractile performance and SR function occur at an insulin-resistant stage before the manifestation of overt Type 2 DM.

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Allemand, Eric, Renata Gattoni, Henri-Marc Bourbon, James Stevenin, Javier F. Cáceres, Johann Soret et Jamal Tazi. « Distinctive Features of Drosophila Alternative Splicing Factor RS Domain : Implication for Specific Phosphorylation, Shuttling, and Splicing Activation ». Molecular and Cellular Biology 21, n^o 4 (15 février 2001) : 1345–59. http://dx.doi.org/10.1128/mcb.21.4.1345-1359.2001.

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ABSTRACT The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5′ alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

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Damiani, E., et A. Margreth. « Subcellular fractionation to junctional sarcoplasmic reticulum and biochemical characterization of 170 kDa Ca2+- and low-density-lipoprotein-binding protein in rabbit skeletal muscle ». Biochemical Journal 277, n^o 3 (1 août 1991) : 825–32. http://dx.doi.org/10.1042/bj2770825.

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Skeletal-muscle sarcoplasmic reticulum (SR) comprises two distinct domains, corresponding to the free membrane of longitudinal SR (LSR) and the junctional membrane region of the terminal cisternae (TC), respectively. The junctional membrane contains the ryanodine receptor (RyR)/Ca(2+)-release channel and additional minor protein components that still require biochemical investigation, in relation to excitation-contraction coupling. Recent findings suggested the involvement in this process of a 170 kDa protein [Kim, Caswell, Talvenheimo & Brandt (1990) Biochemistry 29, 9281-9289], also characterized as a phosphoprotein in junctional TC in independent studies [Chu, Submilla, Inesi, Jay & Campbell (1990) Biochemistry 29, 5899-5905]. We show that this protein is a specific substrate of exogenous cyclic AMP-dependent protein kinase, that it is exposed to the outer surface of intact TC vesicles, and that it co-localizes with the RyR to the junctional membrane. Comparative analysis of LSR and TC subfractions for the 160 kDa glycoprotein sarcalumenin, using Western-blot techniques and specific monoclonal antibodies or concanavalin A as a ligand, revealed that the distribution of this protein within the SR corresponds inversely to both that of the RyR and of the 170 kDa protein. The 170 kDa protein, like sarcalumenin, stains blue with the cationic dye Stains-All and binds 45Ca2+ on blots, but it is uniquely distinguished by its ability to bind 125I-labelled low-density lipoprotein. The similarity of these properties, as well as the pI and solubility properties, to those described for the SR protein, recently purified and cloned and named histidine-rich Ca(2+)-binding protein [HCP; Hofmann, Brown, Lee, Pathak, Anderson & Goldstein (1989) J. Biol. Chem. 264, 8260-8270], makes it very likely that our protein and HCP may indeed be identical. The protein described in the present study differs from sarcalumenin because its migration in SDS/PAGE is accelerated in the presence of Ca2+, a previously reported property of other Ca(2+)-binding proteins [leMaire, Lund, Viel, Champeil & Moller (1989) J. Biol. Chem. 265, 1111-1123], arguing for Ca(2+)-induced protein-conformational changes. Kinase-dependent phosphorylation of our protein is another distinguishing feature, which, although not previously reported for HCP, is consistent with the presence of potential serine/threonine phosphorylation sites in the middle portion of the cloned HCP molecule. The finding that HCP, contrary to early views, selectively binds to the cytoplasmic side of the junctional membrane, together with its newly characterized properties, seem to provide new clues as to a possible role in electromechanical coupling and/or Ca2+ release.

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Fujioka, Y., T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi, M. Tsuda, T. Takada et M. Kasuga. « A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. » Molecular and Cellular Biology 16, n^o 12 (décembre 1996) : 6887–99. http://dx.doi.org/10.1128/mcb.16.12.6887.

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Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2) domains play important roles in growth factor and cytokine signal transduction pathways. A protein of approximately 115 to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1 cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein, termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin, and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases, such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases. In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth factor- and cell adhesion-induced cell signaling.

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Kuranaga, Yuki, Nobuhiko Sugito, Haruka Shinohara, Takuya Tsujino, Kohei Taniguchi, Kazumasa Komura, Yuko Ito, Tomoyoshi Soga et Yukihiro Akao. « SRSF3, a Splicer of the PKM Gene, Regulates Cell Growth and Maintenance of Cancer-Specific Energy Metabolism in Colon Cancer Cells ». International Journal of Molecular Sciences 19, n^o 10 (2 octobre 2018) : 3012. http://dx.doi.org/10.3390/ijms19103012.

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Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.

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Velagapudi, Srividya, Mustafa Yalcinkaya, Antonio Piemontese, Roger Meier, Simon Flyvbjerg Nørrelykke, Damir Perisa, Andrzej Rzepiela et al. « VEGF-A Regulates Cellular Localization of SR-BI as Well as Transendothelial Transport of HDL but Not LDL ». Arteriosclerosis, Thrombosis, and Vascular Biology 37, n^o 5 (mai 2017) : 794–803. http://dx.doi.org/10.1161/atvbaha.117.309284.

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Objective— Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. Approach and Results— Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125 I-HDL but not 125 I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. Conclusions— The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.

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POINOT-CHAZEL, Caroline, Marielle PORTIER, Monsif BOUABOULA, Natalio VITA, Florence PECCEU, Danielle GULLY, John G. MONROE, Jean-Pierre MAFFRAND, Gérard LE FUR et Pierre CASELLAS. « Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24 ». Biochemical Journal 320, n^o 1 (15 novembre 1996) : 145–51. http://dx.doi.org/10.1042/bj3200145.

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Neurotensin (NT) is a neuropeptide that is important in a variety of biological processes such as signal transduction and cell growth. NT effects are mediated by a single class of cell-surface receptors, known as neurotensin receptors (NTRs), which exhibit structural features of the G-protein-coupled receptors superfamily. We investigated NTR signalling properties with Chinese hamster ovary (CHO) cells stably transformed with human NTR (hNTR). First, we showed that NTR stimulation by NT induced the activation of the mitogen-activated protein kinases (MAPKs) in time- and dose-dependent manners. Both p42 and p44 MAPK isoforms were retarded in gel-shift assays, which was consistent with their activation by phosphorylation. In addition we showed that NT caused a prolonged activation of MAPK as measured by in-gel kinase assay. Secondly, we demonstrated that NT induced the expression of the growth-related gene Krox-24 at the protein level, as assessed by Western-blot analysis, and at the transcriptional level, as demonstrated in CHO cells transfected with hNTR and a reporter gene for Krox-24. Activation of MAPK and induction of Krox-24 were both prevented by the NTR antagonist SR 48692, confirming the specific action on NTR. Furthermore we observed coupling of NTR to a mitogenic pathway and Krox-24 induction in the human adenocarcinoma cell line HT29, which naturally expresses NTRs. Considering coupling pathways between NTR stimulation and MAPK activation, we observed a partial inhibition by pertussis toxin (PTX) and a complete blockade by the protein kinase C (PKC) inhibitor GF 109203X. Taken together, these results suggest that (1) stimulation of NTR activates the MAPK pathway by mechanisms involving dual coupling to both PTX-sensitive and PTX-insensitive G-proteins as well as PKC activation, and (2) these effects are associated with the induction of Krox-24, which might be a target of MAPK effector.

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Jensen, Thomas E., Adam J. Rose, Ylva Hellsten, Jørgen F. P. Wojtaszewski et Erik A. Richter. « Caffeine-induced Ca2+ release increases AMPK-dependent glucose uptake in rodent soleus muscle ». American Journal of Physiology-Endocrinology and Metabolism 293, n^o 1 (juillet 2007) : E286—E292. http://dx.doi.org/10.1152/ajpendo.00693.2006.

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Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because α-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-β (ACCβ) Ser221 and immunoprecipitated α1-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated α2-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCβ and α1-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 μM), the CaM-competitive inhibitor KN-93 (10 μM), or the SR Ca2+ release blocking agent dantrolene (10 μM) all inhibited ACCβ phosphorylation and α1-AMPK phosphorylation, suggesting that SR Ca2+ release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca2+-activated CaMKK may control α1-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.

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Liu, Xuefeng, Akila Mayeda, Mingfang Tao et Zhi-Ming Zheng. « Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3′ Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway ». Journal of Virology 77, n^o 3 (1 février 2003) : 2105–15. http://dx.doi.org/10.1128/jvi.77.3.2105-2115.2003.

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ABSTRACT Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5′ splice site alternatively splices to a proximal 3′ splice site (at nucleotide 3225) to express L2 or to a distal 3′ splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3′ splice sites and have been identified as regulating alternative 3′ splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3′ splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3′ splice site while increasing use of the distal 3′ splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3′ splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3′ splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.

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Portal, Daniel, Guillermo S. Lobo, Sebastián Kadener, Jayendra Prasad, Joaquı́n M. Espinosa, Claudio A. Pereira, Zhaohua Tang et al. « Trypanosoma cruzi TcSRPK, the first protozoan member of the SRPK family, is biochemically and functionally conserved with metazoan SR protein-specific kinases ». Molecular and Biochemical Parasitology 127, n^o 1 (mars 2003) : 9–21. http://dx.doi.org/10.1016/s0166-6851(02)00299-2.

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Meads, Mark B., Paula Oliveira, Allison Distler, Maria Silva, Karen Burger, Bin Fang, Aunshka Collins et al. « Identification of Target Pathways Induced By the Multiple Myeloma Tumor Microenvironment Using Activity-Based Protein Profiling and Ex Vivo Protein Kinase Inhibitor Screening ». Blood 128, n^o 22 (2 décembre 2016) : 3288. http://dx.doi.org/10.1182/blood.v128.22.3288.3288.

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Abstract Multiple myeloma (MM) is a heterogeneous plasma cell neoplasm that remains all but incurable despite recent advances in treatment. Indeed, nearly all patients eventually experience disease progression or relapse due to a reservoir of residual myeloma cells that appear to persist through pro-survival signaling from interactions with the tumor microenvironment (TME), leading to eventual clonal expansion. Thus, identifying targets that are induced in MM by the TME may reveal new and important targets amenable to therapeutic intervention. To develop a non-biased method to screen bone marrow specimens from myeloma patients for activated targets throughout the course of disease, we used a combination of activity-based protein profiling (ABPP) and a high-throughput protein kinase inhibitor (PKI) screen using a platform that recapitulates the TME. Target validation was then performed using ex vivo functional screens of pathways using MM patient specimens. The MM cell lines MM1.S, H929, and OPM2 were grown in mono-culture or co-culture with HS5 bone marrow stroma cells for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe (ActivX, Thermo). Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer). Using this method, 176, 136, and 85 kinases out of a total of 1511, 1409, and 1281 proteins were preferentially enriched by 2-fold change from MM1.S, H929, and OPM2 myeloma cells grown in co-culture conditions with HS5 bone marrow stroma, respectively. Of these, 42 kinases were common to all three and 87 were common to two of three MM cell lines. Kinases were chosen for target validation after pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database to identify signaling networks. To identify functionally relevant signaling networks identified via ABPP experiments, the same MM cell lines were simultaneously screened with 30 protein kinase inhibitors (PKIs) in a novel high throughput viability assay. This label-free method measures the viability of MM cells grown in a collagen matrix with bone marrow stroma cells in 384-well plates to simulate the TME by capturing brightfield images every 30 minutes for 96h using a motorized microscope equipped with an incubation chamber. Digital image analysis software measures live cell numbers by detecting membrane motion and generates viability curves as a function of drug concentration and exposure time (Khin et al. Cancer Research 2014). This functional screen confirming known MM survival networks, validated 12 kinases/PKIs in the context of the TME and highlighted novel targetable pathways. To provide an additional level of screening, the same PKIs were tested in CD138-MACS-selected cells from 15 MM patient specimens in a high-throughput viability assay. Eight PKIs targeting IGFR, PLK1, Abl, mTOR, FAK/Pyk2, ALK, Akt, and Casein Kinase-1δ (CK1δ)/CK1ε also showed significant activity in the 15 primary MM specimens. Our three-tiered pharmaco-proteomic screen identified eight kinases critical to MM survival in the context of the TME. Notably, a highly specific in-house inhibitor of Casein Kinase 1δ/CK1ε, SR-3029, which targets the Wnt/β-catnenin pathway, was identified as the most effective compound assessed as a single agent in our ex vivo viability assay in all patients with an average 36h LD50 of 290nM. This compound is under further investigation in MM (Submitted Abstract: Burger, et al, ASH 2016). Additional studies are underway to functionally interrogate the pathways identified in this screen, including ErbB1/EGFR, EphA1 and AMPK. Future work will optimize this method for evaluation of primary bone marrow specimens with ABPP followed by functional validation to better predict potential clinical response at different disease stages. We anticipate that this iterative "at the moment of care" approach is critical because drug resistant tumor phenotypes fluctuate with therapy, and this strategy can track and define clinically relevant changes in tumor cells in situ after the selection pressures applied by exposure to therapy. Disclosures Shain: Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Signal Genetics: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Okoro, Emmanuel Ugochukwu. « TNFα-Induced LDL Cholesterol Accumulation Involve Elevated LDLR Cell Surface Levels and SR-B1 Downregulation in Human Arterial Endothelial Cells ». International Journal of Molecular Sciences 22, n^o 12 (9 juin 2021) : 6236. http://dx.doi.org/10.3390/ijms22126236.

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Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFα) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFα promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFα could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFα-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFα included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFα-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFα on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFα did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFα promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.

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Naqvi, Ammar S., Brian Ennis, Run Jin, Karina L. Conkrite, Komal S. Rathi, Adam A. Kraya, Poonam Sonawane, Phillip B. Storm, Adam C. Resnick et Jo Lynne Rokita. « Abstract 2725 : Molecular mechanisms of aberrant splicing in pediatric high grade gliomas ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 2725. http://dx.doi.org/10.1158/1538-7445.am2022-2725.

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Abstract Pediatric brain cancer is the number one cause of disease-related death in children. While much of the somatic mutation variation in these tumors have been profiled, there have been few studies exploring their transcriptional splicing programs. Here, we characterize aberrant alternative splicing across pediatric brain tumors. We identified three splicing-based clusters that spanned across seven broad histological tumor classifications. Patients in clusters 1 and 2 had significantly lower overall survival (p < 0.001) than those in cluster 3. High-grade gliomas (HGGs) displayed the greatest splicing heterogeneity, spanning all three clusters. We focused on midline HGGs (n = 84), identifying 19,275 recurrent and significant (20% change from control, P < 0.05, FDR < 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. We also identified several splicing factor genes, including SRSF11, that were differentially expressed (p < 0.05), suggesting a potential tumor-specific novel mechanism of splicing dysregulation. Strikingly, 27% (5,157) of these splicing events resulted in either a gain or loss of a functional site in 3,294 genes: disulfide bonding (n = 391), localization signaling (n = 216), modifications, such as phosphorylation sites (n = 1,670), and other, such as ion-binding sites (n = 4,245). We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of the SR family of master splicing regulators, exhibit significantly increased exon 4 inclusion. This leads to a gain of two known phosphorylation sites in CLK1, increased protein expression, and hyper-phosphorylation of SR proteins, inducing cooperative binding of SR proteins to RNA. To assess the impact of this CLK1 event, we compared splicing in tumors with high exon 4 inclusion (n = 5) to those with low exon 4 inclusion (n = 5) and discovered 862 differential splicing events (DSE). Samples with high CLK1 exon 4 inclusion had significantly lower percent spliced in events, suggesting that increased activity of CLK1 resulting from the gain of the exon 4 promotes overall exon skipping. Additionally, more than 80% of the DSE (n = 695), including those in known brain tumor oncogenes such as CDC27 and E2H2, were recurrently mis-spliced in midline HGG tumors, suggesting that CLK1 splicing dysregulation leads to a global aberrant splicing phenotype in HGGs. Finally, we determined a subset of events significantly associated with prolonged survival, suggesting that stratification of patients based on splicing phenotypes can be utilized as a prognostic indicator. In summary, we demonstrate aberrant splicing in pediatric HGGs as an alternative mechanism driving tumorigenesis and future work will focus on urgently needed therapeutic targeting of these pathways toward our overall goal of improving survival outcomes in children with brain tumors. Citation Format: Ammar S. Naqvi, Brian Ennis, Run Jin, Karina L. Conkrite, Komal S. Rathi, Adam A. Kraya, Poonam Sonawane, Phillip B. Storm, Adam C. Resnick, Jo Lynne Rokita. Molecular mechanisms of aberrant splicing in pediatric high grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2725.

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Hayakawa, Fumihiko, Keiki Sugimoto, Shingo Kurahashi, Takumi Sumida et Tomoki Naoe. « A Novel STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition in a Wide Range of Hematopoietic Malignancies without Growth Suppression of Normal Hematopoietic Cells ». Blood 118, n^o 21 (18 novembre 2011) : 577. http://dx.doi.org/10.1182/blood.v118.21.577.577.

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Abstract Abstract 577 Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like JAK family kinases and Src family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials. Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases and Src family kinases. In HEL92.1.7 cells with constitutively active mutation of JAK2, OPB-31121 treatment inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). Src-dependent constitutive phosphorylation of STAT3 was also inhibited by OPB-31121 without inhibition of Src in H1650 cells that had active mutantation of EGF receptor. In addition, STAT3 immunoprecipitated from OPB-31121-treated cells was neither phosphorylated by JAK2 nor Lyn, a Src family kinase, in vitro without decrease in auto phosphorylation of upstream kinases, OPB-31121 demonstrated strong growth inhibitory effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including myeloma, AML with JAK2 mutation and CML. It is revealed that STAT3 is constitutively activated by oncogenic autocrine of IL-6 pathway or tyrosine kinase signal from oncoprotein in these cell lines. We also demonstrated growth inhibition or reduction of cell lines including HEL92.1.7 (AML with JAK2 mutation, T/C: 16%), KU812 (CML, T/C: 2%), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation, T/C: 5.9%) in NOD/SCID mice. For further analyses, we used human leukemia model mice where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth reduction of leukemia cells of BCR-ABL-positive ALL (T/C: 4%, Figure B), CML-BC with T315I mutation in BCR-ABL (T/C: 15.3%), and AML (T/C: 15.9%). Notably, OPB-31121-induced growth reduction was extremely selective for leukemia cells. Normal hematopoietic cells of mice were hardly affected by OPB-31121, whereas, cytarabine showed non-specific growth suppression of both leukemia cells and normal hematopoietic cells (Figure C). The safety of OPB-31121 on normal hematopoietic cells was also confirmed by colony formation assay, where OPB-31121 hardly affected colony formation of human cord blood cells at 100 nM. For further analyses, we transplanted human cord blood cells into NOG mice and investigated the growth inhibitory effect of OPB-31121 on normal hematopoietic cells in vivo. No significant growth suppression of human normal hematopoietic cells was observed in OPB-31121 treated mice (T/C: 99.9%, Figure D). Taken together, we conclude that OPB-31121 holds promise as a non-myelosuppressive therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase I/II trial in Japan. Disclosures: Hayakawa: Otsuka Pharmaceutical Co. Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co. Ltd.: Employment. Sumida:otsuka Pharmaceutical Co. Ltd.: Employment. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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Mylonis, Ilias, et Thomas Giannakouros. « Protein kinase CK2 phosphorylates and activates the SR protein-specific kinase 1 ». Biochemical and Biophysical Research Communications 301, n^o 3 (février 2003) : 650–56. http://dx.doi.org/10.1016/s0006-291x(02)03055-3.

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Kuroyanagi, Noriyo, Hiroshi Onogi, Takashi Wakabayashi et Masatoshi Hagiwara. « Novel SR-Protein-Specific Kinase, SRPK2, Disassembles Nuclear Speckles ». Biochemical and Biophysical Research Communications 242, n^o 2 (janvier 1998) : 357–64. http://dx.doi.org/10.1006/bbrc.1997.7913.

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Ding, Jian-Hua, Xiang-Yang Zhong, Jonathan C. Hagopian, Marissa M. Cruz, Gourisankar Ghosh, James Feramisco, Joseph A. Adams et Xiang-Dong Fu. « Regulated Cellular Partitioning of SR Protein-specific Kinases in Mammalian Cells ». Molecular Biology of the Cell 17, n^o 2 (février 2006) : 876–85. http://dx.doi.org/10.1091/mbc.e05-10-0963.

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Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.

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NORI, Alessandra, Pei-Ju LIN, Arianna CASSETTI, Antonello VILLA, K. Ulrich BAYER et Pompeo VOLPE. « Targeting of alpha-kinase-anchoring protein (alphaKAP) to sarcoplasmic reticulum and nuclei of skeletal muscle ». Biochemical Journal 370, n^o 3 (15 mars 2003) : 873–80. http://dx.doi.org/10.1042/bj20021624.

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The sarcoplasmic reticulum (SR) plays a key role in excitation/contraction coupling of skeletal muscle. The SR is composed of two continuous yet heterogeneous membrane compartments, the free or longitudinal SR and cisternal SR. Cisternal SR is made up of free SR membrane, enriched in Ca2+ pumps, and junctional SR (jSR) membrane, enriched in ryanodine-sensitive Ca2+-release channels, and contains calsequestrin within its lumen. Protein phosphorylation mediated by the Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) has significant, distinct regulatory roles in both Ca2+ uptake and Ca2+ release. Kinase-anchoring proteins (KAPs) constitute a novel mechanism for achieving cell compartmentalization of effectors in phosphorylation pathways. Here, targeting of αKAP, a CaM kinase II-anchoring protein encoded within the α-CaM kinase II gene, was studied in transgenic skeletal muscle fibres of the adult rat soleus. The transgenes were epitope-tagged versions of αKAP and of a deletion mutant, allowing their specific immunodetection against the wild-type background. Our results show that αKAP is largely localized at the free SR and thus near the Ca2+ pump, a protein that can be modulated by CaM kinase II phosphorylation. Only minor co-localization was observed with the jSR ryanodine-sensitive Ca2+-release channel, which is a potential CaM kinase II target. In non-muscle cells, recombinant αKAP is targeted to endoplasmic reticulum (ER). Both ER and SR targeting requires the N-terminal hydrophobic region of αKAP. An unexpected additional specific localization that does not require the N-terminus was found in the nucleus, providing a first clue of how CaM kinase II can fulfil its nuclear functions in skeletal muscle.

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Prasad, Jayendra, Karen Colwill, Tony Pawson et James L. Manley. « The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity : Both Hyper- and Hypophosphorylation Inhibit Splicing ». Molecular and Cellular Biology 19, n^o 10 (1 octobre 1999) : 6991–7000. http://dx.doi.org/10.1128/mcb.19.10.6991.

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ABSTRACT The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

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Tang, Zhaohua, Mitsuhiro Yanagida et Ren-Jang Lin. « Fission Yeast Mitotic Regulator Dsk1 Is an SR Protein-specific Kinase ». Journal of Biological Chemistry 273, n^o 10 (6 mars 1998) : 5963–69. http://dx.doi.org/10.1074/jbc.273.10.5963.

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Tang, Zhaohua, Maria Luca, Laura Taggart-Murphy, Jessica Portillio, Cathey Chang, Ayse Guven, Ren-Jang Lin, Johanne Murray et Antony Carr. « Interacting factors and cellular localization of SR protein-specific kinase Dsk1 ». Experimental Cell Research 318, n^o 16 (octobre 2012) : 2071–84. http://dx.doi.org/10.1016/j.yexcr.2012.05.020.

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Prasad, Jayendra, et James L. Manley. « Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty ». Molecular and Cellular Biology 23, n^o 12 (15 juin 2003) : 4139–49. http://dx.doi.org/10.1128/mcb.23.12.4139-4149.2003.

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ABSTRACT SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

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Zhu, Haifeng, et Hesham M. Amin. « NPM-ALK Oncogene Upregulates the Expression of Inducible Nitric Oxide Synthase in T-Cell Lymphoma Through STAT3/MicroRNA-26a-Dependent Mechanism ». Blood 118, n^o 21 (18 novembre 2011) : 1383. http://dx.doi.org/10.1182/blood.v118.21.1383.1383.

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Abstract Abstract 1383 The oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma tumors. NPM-ALK promotes cellular survival and transformation, and initiates lymphomagenesis. To induce its effects, NPM-ALK interacts with a comprehensive network of oncogenes and tumor suppressor genes. Previous studies have identified upstream and downstream members of this network that modulate the effects of NPM-ALK. However, the exact mechanisms by which NPM-ALK induces its effects are not completely identified. Nitric oxide (NO) is a gaseous molecule and a highly active free radical. It plays critical, yet versatile, roles in physiological cellular functions including survival, adhesion, migration, and angiogenesis. It also has important contributions to tumor progression and metastasis. NO is generated from L-arginine via 3 distinct isoforms of the enzyme NO synthase including the inducible form (iNOS). The expression and role of iNOS in NPM-ALK-expressing T-cell lymphoma is not known. We found that iNOS mRNA and protein are expressed in the NPM-ALK-expressing T-cell lymphoma cell lines Karpas 299, DEL, and SR-786. In agreement with a functional role of iNOS in these cells, the selective iNOS inhibitor 1400W and the NO scavenger CPITO decreased the proliferation of the NPM-ALK-expressing T-cell lymphoma cell lines (p < 0.05). In addition, the NO donor SNAP recovered the decrease in the proliferation of these cell lines after treatment with the ALK inhibitors TAE684 and PF-2341066 (p < 0.05). Interestingly, iNOS protein was totally absent in normal human T lymphocytes in spite of a notable increase in iNOS mRNA. These observations suggested that the downregulation of iNOS protein in T lymphocytes vs. its high levels in the NPM-ALK-expressing T-cell lymphoma cell lines might occur posttranscriptionally. Because microRNA are major posttranscriptional regulators of protein expression, we set to analyze possible aberrancies in microRNA. First, we performed an array study comparing the expression of microRNA in T lymphocytes vs. NPM-ALK-expressing T cell lymphoma cell lines. Three web-based algorithms [TargetScan (http:// genes.mit.edu/targetscan/), miRanda (http:// www.microrna.org/microrna/home.do), and PicTar (http:// pictar.mdc-berlin.de/] identified microRNA-26a (miR-26a) to potentially bind with iNOS 3'-UTR. Importantly, statistical analysis of the array data showed that the expression of miR-26a is much more pronounced in T lymphocytes in comparison with the lymphoma cell lines (p < 0.00001), and real-time qPCR further confirmed these results. To examine the functional interactions between miR-26a and iNOS 3'-UTR, we performed a luciferase assay in 293T cells after transfection with iNOS 3'-UTR reporter gene. Wild type miR-26a, and not mutated miR-26a, induced a marked decrease in the luciferase activity of iNOS 3'-UTR reporter gene (p < 0.05). We then questioned whether NPM-ALK underlies the aberrancies in the expression of miR-26a and iNOS. Specific targeting of NPM-ALK by siRNA increased miR-26a and simultaneously decreased iNOS protein levels in the lymphoma cell lines. Next, we reasoned to examine how NPM-ALK induces its effects on this system. A transcription factor screening showed that miR-26a gene promoter, which is located within the promoter of a host gene, CTDSPL, contains 2 consensus sequences where it can potentially bind with STAT3, a major downstream target of NPM-ALK. Chromatin immunoprecipitation studies illustrated the binding between STAT3 and CTDSPL promoter. To analyze the functional interactions between STAT3 and miR-26a, a luciferase assay performed in NPM-ALK-expressing T-cell lymphoma cell lines showed that the downregulation of STAT3 by siRNA increased CTDSPL promoter activity. In addition, downregulation of STAT3 protein or the decrease in STAT3 phosphorylation by STAT3 or NPM-ALK siRNA, respectively, increased miR-26a levels and decreased iNOS protein expression in NPM-ALK-expressing T-cell lymphoma cell lines. Our study proposes novel mechanisms by which NPM-ALK contributes to the survival of T-cell lymphoma. We are currently analyzing the expression of miR-26a and iNOS in primary human lymphoma tumors, and examining the effects of transfecting NPM-ALK-expressing T-cell lymphoma cell lines with miR-26a on cell proliferation, adhesion, and migration. Disclosures: No relevant conflicts of interest to declare.

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Tang, Zhaohua, Tiffany Kuo, Jenny Shen et Ren-Jang Lin. « Biochemical and Genetic Conservation of Fission Yeast Dsk1 and Human SR Protein-Specific Kinase 1 ». Molecular and Cellular Biology 20, n^o 3 (1 février 2000) : 816–24. http://dx.doi.org/10.1128/mcb.20.3.816-824.2000.

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ABSTRACT Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore,dsk1 + gene determines the phenotype ofprp2 + overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the double-deletion mutant. In conjunction with the resemblance of dsk1 + andSRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms.

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Yamamura, Ken, Charles Steenbergen et Elizabeth Murphy. « Protein kinase C and preconditioning : role of the sarcoplasmic reticulum ». American Journal of Physiology-Heart and Circulatory Physiology 289, n^o 6 (décembre 2005) : H2484—H2490. http://dx.doi.org/10.1152/ajpheart.00590.2005.

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Activation of protein kinase C (PKC) is cardioprotective, but the mechanism(s) by which PKC mediates protection is not fully understood. Inasmuch as PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca2+ and because altered SR Ca2+ handling during ischemia is involved in cardioprotection, we examined the role of PKC-mediated alterations of SR Ca2+ in cardioprotection. Using isolated adult rat ventricular myocytes, we found that addition of 1,2-dioctanoyl- sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death associated with simulated ischemia and reperfusion, also reduced SR Ca2+. Cell death was 57.9 ± 2.9% and 47.3 ± 1.8% in untreated and DOG-treated myocytes, respectively ( P < 0.05). Using fura 2 fluorescence to monitor Ca2+ transients and caffeine-releasable SR Ca2+, we examined the effect of DOG on SR Ca2+. Caffeine-releasable SR Ca2+ was significantly reduced (by ∼65%) after 10 min of DOG treatment compared with untreated myocytes ( P < 0.05). From our examination of the mechanism by which PKC alters SR Ca2+, we present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser16. This effect is mediated by PKC-ε, because a PKC-ε-selective inhibitory peptide blocked the DOG-mediated decrease in phosphorylation of PLB and abolished the DOG-induced reduction in caffeine-releasable SR Ca2+. Using immunoprecipitation, we further demonstrated that DOG increased the association between protein phosphatase 1 and PLB. These data suggest that activated PKC-ε reduces SR Ca2+ content through PLB dephosphorylation and that reduced SR Ca2+ may be important in cardioprotection.

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Osada, Mitsuru, Thomas Netticadan, Kenichi Kawabata, Kohji Tamura et Naranjan S. Dhalla. « Ischemic preconditioning prevents I/R-induced alterations in SR calcium-calmodulin protein kinase II ». American Journal of Physiology-Heart and Circulatory Physiology 278, n^o 6 (1 juin 2000) : H1791—H1798. http://dx.doi.org/10.1152/ajpheart.2000.278.6.h1791.

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Although Ca2+/calmodulin-dependent protein kinase II (CaMK II) is known to modulate the function of cardiac sarcoplasmic reticulum (SR) under physiological conditions, the status of SR CaMK II in ischemic preconditioning (IP) of the heart is not known. IP was induced by subjecting the isolated perfused rat hearts to three cycles of brief ischemia-reperfusion (I/R; 5 min ischemia and 5 min reperfusion), whereas the control hearts were perfused for 30 min with oxygenated medium. Sustained I/R in control and IP groups was induced by 30 min of global ischemia followed by 30 min of reperfusion. The left ventricular developed pressure, rate of the left ventricular pressure, as well as SR Ca2+-uptake activity and SR Ca2+-pump ATPase activity were depressed in the control I/R hearts; these changes were prevented upon subjecting the hearts to IP. The beneficial effects of IP on the I/R-induced changes in contractile activity and SR Ca2+ pump were lost upon treating the hearts with KN-93, a specific CaMK II inhibitor. IP also prevented the I/R-induced depression in Ca2+/calmodulin-dependent SR Ca2+-uptake activity and the I/R-induced decrease in the SR CaMK II activity; these effects of IP were blocked by KN-93. The results indicate that IP may prevent the I/R-induced alterations in SR Ca2+ handling abilities by preserving the SR CaMK II activity, and it is suggested that CaMK II may play a role in mediating the beneficial effects of IP on heart function.

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TANG, Zhaohua, Norbert F. KÄUFER et Ren-Jang LIN. « Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation ». Biochemical Journal 368, n^o 2 (1 décembre 2002) : 527–34. http://dx.doi.org/10.1042/bj20021133.

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The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1—Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.

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Jiang, Mao, Ande Xu et Njanoor Narayanan. « Thyroid hormone downregulates the expression and function of sarcoplasmic reticulum-associated CaM kinase II in the rabbit heart ». American Journal of Physiology-Heart and Circulatory Physiology 291, n^o 3 (septembre 2006) : H1384—H1394. http://dx.doi.org/10.1152/ajpheart.00875.2005.

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Phosphorylation of sarcoplasmic reticulum (SR) Ca2+-cycling proteins by a membrane-associated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a well-documented physiological mechanism for regulation of transmembrane Ca2+ fluxes and the cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of l-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca2+ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca2+ release channel (RyR-CRC), Ca2+-ATPase, and phospholamban (PLN) was significantly lower (30–70%) in JSR and LSR vesicles from hyperthyroid than from euthyroid rabbit heart. Western immunoblotting analysis revealed significantly higher (∼40%) levels of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) in JSR, but not in LSR, from hyperthyroid than from euthyroid rabbit heart. Maximal velocity of Ca2+ uptake was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid compared with euthyroid rabbit hearts. Apparent affinity of the Ca2+-ATPase for Ca2+ did not differ between the two groups. Protein levels of PLN and CaM kinase II were significantly lower (30–40%) in JSR, LSR, and ventricular tissue homogenates from hyperthyroid rabbit heart. These findings demonstrate selective downregulation of expression and function of CaM kinase II in hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment.

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Papoutsopoulou, S. « SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1 ». Nucleic Acids Research 27, n^o 14 (15 juillet 1999) : 2972–80. http://dx.doi.org/10.1093/nar/27.14.2972.

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Yu, Zhuo, Zhi-Hua Wang et Huang-Tian Yang. « Calcium/calmodulin-dependent protein kinase II mediates cardioprotection of intermittent hypoxia against ischemic-reperfusion-induced cardiac dysfunction ». American Journal of Physiology-Heart and Circulatory Physiology 297, n^o 2 (août 2009) : H735—H742. http://dx.doi.org/10.1152/ajpheart.01164.2008.

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Intermittent high-altitude (IHA) hypoxia-induced cardioprotection against ischemia-reperfusion (I/R) injury is associated with the preservation of sarcoplasmic reticulum (SR) function. Although Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and phosphatase are known to modulate the function of cardiac SR under physiological conditions, the status of SR CaMKII and phosphatase during I/R in the hearts from IHA hypoxic rats is unknown. In the present study, we determined SR and cytosolic CaMKII activity during preischemia and I/R (30 min/30 min) in perfused hearts from normoxic and IHA hypoxic rats. The left ventricular contractile recovery, SR CaMKII activity as well as phosphorylation of phospholamban at Thr17, and Ca2+/CaM-dependent SR Ca2+-uptake activity were depressed in the I/R hearts from normoxic rats, whereas these changes were prevented in the hearts from IHA hypoxic rats. Such beneficial effects of IHA hypoxia were lost by treating the hearts with a specific CaMKII inhibitor, KN-93. I/R also depressed cytosolic CaMKII and SR phosphatase activity, but these alterations remained unchanged in IHA hypoxic group. Furthermore, we found that the autophosphorylation at Thr287, which confers Ca2+/CaM-independent activity, was not altered by I/R in both groups. These findings indicate that preservation of SR CaMKII activity plays an important role in the IHA hypoxia-induced cardioprotection against I/R injury via maintaining SR Ca2+-uptake activity.

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Wang, Guanghui, Peng Sun, Zhongjuan Sun, Jindong Zhu, Dan Yu, Zhe Tang, Zonghua Wang, Chenfang Wang et Huawei Zheng. « Sgh1, an SR-like Protein, Is Involved in Fungal Development, Plant Infection, and Pre-mRNA Processing in Fusarium graminearum ». Journal of Fungi 8, n^o 10 (8 octobre 2022) : 1056. http://dx.doi.org/10.3390/jof8101056.

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Serine/arginine (SR) proteins are essential pre-mRNA splicing factors in eukaryotic organisms. Our previous studies have shownthat the unique SR-specific protein kinase Srk1 is important for RNA splicing and gene transcription in Fusarium graminearum, and interacts with two SR proteins, FgSrp1 and FgSrp2. In this study, we have identified an SR-like protein called Sgh1 in F. graminearum, which is orthologous to budding yeast paralogous Gbp2 and Hrb1. Our data have shownthat the Sgh1 is involved in vegetative growth, conidiation, sexual reproduction, DON synthesis, and plant infection. Moreover, the Sgh1 is mainly localized to the nucleus. RNA-seq analysis has shownthat the expression of over 1100 genes and the splicing efficiency in over 300 introns were affected in theΔsgh1 mutant. Although the RS domain and all three of the RRM domains are important for the Sgh1 functions, only the RS domain is responsible for its nuclear localization. Finally, we verified that the Sgh1 interacts with the unique SR-specific kinase Srk1 in F. graminearum by the yeast-two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Taken together, our results have revealed that the Sgh1 regulates the fungal development, plant infection, and the pre-mRNA processing, and the RS domain regulates the function of the Sgh1 by modulating its nucleocytoplasmic shuttling.

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Shi, Wenyu, Suraj Konnath George, Choladda V. Curry, Serhan Alkan et Hesham M. Amin. « Trka, a Novel Binding Partner of NPM-ALK Oncogenic Tyrosine Kinase, Facilitates the Survival of T-Cell Anaplastic Large-Cell Lymphoma ». Blood 124, n^o 21 (6 décembre 2014) : 3108. http://dx.doi.org/10.1182/blood.v124.21.3108.3108.

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Abstract Anaplastic lymphoma kinase-expressing (ALK+) T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer that frequently occurs in children and adolescents. Approximately 85% of the ALK+ ALCL patients harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. The expression of ALK is primarily identified in neural tissues at early stages of human development, which suggests that ALK contributes to neural tissue development probably through interactions with neurotrophic factors. Nerve growth factor (NGF) is the first characterized neurotrophic factor. In addition to its canonical roles in neural tissue development, NGF has gained attention as a promoter of survival of cancer cells. Indeed, deregulation of NGF signaling has been attributed to cell proliferation, invasion, and metastasis. It is believed that NGF’s non-neural functions are essentially mediated through two cognate receptors: TrkA and p75NTR. The expression and role of NGF/TrkA signaling in NPM-ALK+ T-cell lymphoma have not been previously studied. Because of the biological similarities between ALK and NGF/TrkA signaling, we hypothesized that blockade of NGF/TrkA signaling may suppress NPM-ALK+ T-cell lymphoma cell survival, which can have important therapeutic implications. Western blotting and immunohistochemical staining showed that TrkA and NGF proteins are highly expressed in 5 NPM-ALK+ T-cell lymphoma cell lines compared with normal human T lymphocytes. In addition, immunohistochemical staining demonstrated the expression of TrkA in 85% (11/13) of ALK+ T-cell lymphoma patient tumors. Immunoprecipitation showed physical association between TrkA and NPM-ALK. Specific downregulation of NPM-ALK using ALK siRNA was associated with a marked decrease in phosphorylated TrkA, suggesting functional interactions between the two proteins. Thereafter, we used a selective TrkA Inhibitor (catalogue number: 648450; Calbiochem, Billerica, MA) and performed in vitro assays to elucidate the contribution of TrkA signaling on the lymphoma cells. The TrkA Inhibitor decreased efficiently the phosphorylation and kinase activity of TrkA in NPM-ALK+ T-cell lymphoma cells. These effects were associated with decreased NPM-ALK+ T-cell lymphoma cellular proliferation. The TrkA Inhibitor also induced apoptosis and abrogated anchorage-independent colony formation of these cells. Moreover, the TrkA Inhibitor caused downregulation of pNPM-ALK, pIGF-IR, pSTAT3, and pAKT. Caspase-3, BCL-2 and BCL-XL were also reduced, supporting apoptosis occurrence. To rule out nonspecific effects that might have been caused by the TrkA Inhibitor, TrkA siRNA was used in some experiments. TrkA siRNA, but not scrambled siRNA, decreased the viability and proliferation of NPM-ALK+ T-cell lymphoma cells. In addition, TrkA siRNA caused biochemical effects similar to those observed with the TrkA Inhibitor. An ELISA assay demonstrated the presence of NGF in cell culture supernatant of serum-deprived NPM-ALK+ lymphoma cells. Importantly, NGF enhanced the viability and proliferation of NPM-ALK+ T-cell lymphoma cells, and TrkA blocking antibody abrogated these effects. These results suggest the presence of an autocrine NGF/TrkA loop that supports the survival of NPM-ALK+ T-cell lymphoma. We also examined the effects of TrkA Inhibitor in SCID-beige mice with NPM-ALK+ T-cell lymphoma xenografts implanted subcutaneously using SR-786 cells. The tumors were established after 8 days, and mice were randomly treated with vehicle or the TrkA Inhibitor for 21 days (10 mg/kg b.i.d; s.c.). Tumor volumes were remarkably smaller in the TrkA Inhibitor-treated mice compared with vehicle-treated mice. Our data provide novel evidence that TrkA contributes to the survival of NPM-ALK+ T-cell lymphoma, and implicate that targeting TrkA may represent an effective approach to suppress this lymphoma. Disclosures No relevant conflicts of interest to declare.

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Ma, Ke, Yiming Xu, Chenchen Wang, Nan Li, Kexue Li, Yan Zhang, Xiaoyu Li et al. « A cross talk between class a scavenger receptor and receptor for advanced glycation end-products contributes to diabetic retinopathy ». American Journal of Physiology-Endocrinology and Metabolism 307, n^o 12 (15 décembre 2014) : E1153—E1165. http://dx.doi.org/10.1152/ajpendo.00378.2014.

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In response to hyperglycemia in patients with diabetes, many signaling pathways contribute to the pathogenesis of diabetic complications, including diabetic retinopathy (DR). Excessive production of inflammatory mediators plays an important role in this process. Amadori-glycated albumin, one of the major forms of advanced glycated end-products, has been implicated in DR by inducing inflammatory responses in microglia/macrophages. Our goal was to delineate the potential cross talk between class A scavenger receptor (SR-A) and the receptor for advanced glycated end-product (RAGE) in the context of DR. We show here that SR-A ablation caused an exacerbated form of DR in streptozotocin-injected C57BL/6J mice as evidenced by fundus imaging and electroretinography. Immunohistochemical staining and RT-PCR assay indicated that there was augmented activation of proinflammatory macrophages with upregulated synthesis of proinflammatory mediators in the retina in Sr-a−/− mice. Overexpression of SR-A suppressed RAGE-induced mitogen-activated protein kinase (MAPK) signaling, whereas RAGE activation in macrophages favored a proinflammatory (M1) phenotype in the absence of SR-A. Mechanistic analysis on bone marrow-derived macrophages and HEK293 cell line revealed that SR-A interacted with and inhibited the phosphorylation of mitogen-activated protein kinase kinase 7, the major kinase in the RAGE-MAPK-NF-κB signaling, thereby leading to diminished secretion of proinflammatory cytokines. Our findings suggest that the antagonism between SR-A and RAGE contributes to the pathogenesis of DR by nurturing a disease-prone macrophage phenotype. Therefore, specific agonist that boosts SR-A signaling could potentially provide benefits in the prevention and/or intervention of DR.

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Hunter, R. Bridge, Heather Mitchell-Felton, David A. Essig et Susan C. Kandarian. « Expression of endoplasmic reticulum stress proteins during skeletal muscle disuse atrophy ». American Journal of Physiology-Cell Physiology 281, n^o 4 (1 octobre 2001) : C1285—C1290. http://dx.doi.org/10.1152/ajpcell.2001.281.4.c1285.

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Disuse atrophy of skeletal muscle leads to an upregulation of genes encoding sarcoplasmic reticulum (SR) calcium-handling proteins. Because many of the proteins that are induced with endoplasmic reticulum (ER) stress are ER calcium-handling proteins, we sought to determine whether soleus muscle atrophy was associated with a prototypical ER stress response. Seven days of rat hindlimb unloading did not alter expression of ubiquitous ER stress proteins such as Grp78, calreticulin, and CHOP/GADD-153, nor other proteins that have been shown to be activated by ER stressors such as vinculin, the type I d- myo-inositol 1,4,5-trisphosphate receptor, or protein kinase R, a eukaryotic initiation factor 2α kinase. On the other hand, expression of heme oxygenase-1 (HO-1), an antioxidant ER stress protein, was significantly increased 2.2-fold. In addition, unloading led to an increase in calsequestrin, the muscle-specific SR calcium-binding protein, at both the mRNA (68%) and protein (24%) levels. Although disuse atrophy is associated with a significant remodeling of muscle-specific proteins controlling SR calcium flux, it is not characterized by a prototypical ER stress response. However, the upregulation of HO-1 may indicate ER adaptation to oxidative stress during muscle unloading.

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Yoon, Taewon, Young-In Kim, Elizabeth Fitzpatrick et Ae-Kyung Yi. « Protein kinase D1 is essential for Saccharopolyspora rectivirgula-induced pulmonary inflammation and alveolitis. » Journal of Immunology 198, n^o 1_Supplement (1 mai 2017) : 148.18. http://dx.doi.org/10.4049/jimmunol.198.supp.148.18.

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Abstract Saccharopolyspora rectivirgula (SR) causes farmers’ lung disease, a form of hypersensitivity pneumonitis (HP). The initial proinflammatory responses to SR is dependent on Toll-like receptors (TLR) 2 and 9, and their signaling adaptor MyD88. We found previously that protein kinase D1 (PKD1), a serine/threonine kinase, can be activated through a MyD88-dependent TLR signaling pathway and plays a pivotal role in TLR/MyD88-dependent production of proinflammatory mediators. SR activates PKD1 via a MyD88-dependent manner in murine cells and SR-induced alveolitis is substantially suppressed by pretreatment with a PKC/PKD inhibitor Gö6976. However, it is currently unknown whether PKD1 plays a regulatory role in SR-mediated proinflammatory responses and alveolitis in vivo. In the present study, to further understand contribution of PKD1 in the SR-induced pulmonary inflammation, tamoxifen-inducible PKD1-deficient mice were generated. Wild-type mice and PKD1-deficient mice were challenged intranasally with SR. We found that the levels of proinflammatory mediators (including TNFα, IL-6, IL-12, and IFNγ), alveolitis, and neutrophil influx in the bronchoalveolar lavage fluid and interstitial lung tissue after single intranasal expose to SR were significantly lower in PKD1-deficient mice compared to those in wild-type mice. Our results demonstrate that PKD1 is essential for SR-mediated proinflammatory responses and neutrophil influx in the lung. Our findings also suggest that PKD1 might be one of the critical factors that contributes to the development of HP, and could be a therapeutic target for certain forms of HP, such as farmers’ lung disease.

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Tang, Zhaohua, Amy Tsurumi, Sarah Alaei, Christopher Wilson, Cathleen Chiu, Jessica Oya et Benson Ngo. « Dsk1p kinase phosphorylates SR proteins and regulates their cellular localization in fission yeast ». Biochemical Journal 405, n^o 1 (13 juin 2007) : 21–30. http://dx.doi.org/10.1042/bj20061523.

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Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1+: Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.

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Su, Huai-De, Bruce E. Kemp, R. Scott Turner et J. F. Kuo. « Synthetic myelin basic protein peptide analogs are specific inhibitors of phospholipid/calcium-dependent protein kinase (protein kinase C) ». Biochemical and Biophysical Research Communications 134, n^o 1 (janvier 1986) : 78–84. http://dx.doi.org/10.1016/0006-291x(86)90529-2.

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Oh, You-Chang, Wei Li et Jang-Gi Choi. « Saussureae Radix Attenuates Neuroinflammation in LPS-Stimulated Mouse BV2 Microglia via HO-1/Nrf-2 Induction and Inflammatory Pathway Inhibition ». Mediators of Inflammation 2021 (18 mars 2021) : 1–15. http://dx.doi.org/10.1155/2021/6687089.

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The activation of microglial cells and their subsequent neuroinflammatory reactions are related to various degenerative brain diseases. Therefore, the regulation of microglial cell activation is an important point for the research of therapeutic agents for treating or preventing neurodegenerative disorders. Saussureae Radix (SR) is the root of Saussurea lappa Clarke, and it has been used for a long time as an herbal medicine in East Asia to treat indigestion and inflammation of the digestive system. In previous studies, however, the effect of SR ethanolic extract on microglial cell-mediated neuroinflammation was not fully explained. In this study, we explored the antineuroinflammatory activities and molecular mechanisms of SR in microglial cells stimulated with LPS (lipopolysaccharide). Our results illustrated that SR does not cause cytotoxicity and significantly weakens the production of nitric oxide (NO) and inflammatory cytokines. SR treatment also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 2, induced heme oxygenase- (HO-) 1, and activated the nuclear factor erythroid 2-related factor 2 (Nrf-2) pathway. In addition, SR significantly repressed the transcriptional activities of the nuclear factor- (NF-) κB and activator protein- (AP-) 1. Furthermore, SR effectively inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT). Isolation and high-performance liquid chromatography (HPLC) analysis indicated two major sesquiterpenoids (costunolide and dehydrocostuslactone). These compounds significantly inhibited the production of neuroinflammatory mediators and induced HO-1 expression. These findings show that SR could be a potential candidate for the treatment of inflammation-related degenerative brain diseases.

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Gupta, Ramesh C., Sudhish Mishra, Sharad Rastogi, Makoto Imai, Omar Habib et Hani N. Sabbah. « Cardiac SR-coupled PP1 activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts ». American Journal of Physiology-Heart and Circulatory Physiology 285, n^o 6 (décembre 2003) : H2373—H2381. http://dx.doi.org/10.1152/ajpheart.00442.2003.

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Type 1 protein phosphatase (PP1) is a negative regulator of cardiac function. However, studies on the status and regulation of sarcoplasmic reticulum (SR)-associated PP1 activity in failing hearts are limited. We studied PP1 activity and protein and mRNA expression of the catalytic subunit of PP1 (PP1C) and protein levels of PP1-specific inhibitors [inhibitor 1 (Inh-1) and inhibitor 2 (Inh-2)] in the left ventricular (LV) myocardium of 6 dogs with heart failure (HF; LV ejection fraction, 23 ± 2%) and 6 normal dogs. In failing LV tissue, PP1 activity values (expressed as pmol 32P · min–1 · mg of noncollagen protein–1) in the homogenate, crude membranes, cytosol, and purified SR were increased by 52, 54, 55, and 72%, respectively. Trypsin treatment released PP1 but not type 2A protein phosphatase from the SR. In the supernatant of trypsin-treated SR, PP1 activity was ∼24% higher in failing hearts than in normal control hearts. A similar increase in protein expression of PP1C was observed in the nontrypsinized SR. Heat-denatured phosphorylated SR inhibited PP1 activity by 30%, which suggests the presence of Inh-1 or -2 or both in the SR. With the use of a specific antibody, both Inh-1 and -2 proteins were found in the SR; the former was decreased by 56% in the failing SR, whereas the latter did not change. These results suggest that protein phosphatase activity bound to the SR is increased and is predominantly type 1. Increased SR-associated PP1 activity in failing hearts appears to be due partly to increased expression of PP1C and partly to reduced levels of Inh-1 but not Inh-2 protein. Thus inhibition of PP1 activity in the SR appears to be a potential therapeutic target for improving LV function in failing hearts, because it may lead to increased SR Ca2+ uptake, which is impaired in failing hearts.

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Li, Jianzhuo, Xidong Liu, Huiying Chu, Xueqi Fu, Tianbao Li, Lianghai Hu, Shu Xing, Guohui Li, Jingkai Gu et Zhizhuang Joe Zhao. « Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases ». PROTEOMICS 15, n^o 1 (10 décembre 2014) : 68–76. http://dx.doi.org/10.1002/pmic.201400146.

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Bell, Ian, Ashley Martin et Sally Roberts. « The E1^E4 Protein of Human Papillomavirus Interacts with the Serine-Arginine-Specific Protein Kinase SRPK1 ». Journal of Virology 81, n^o 11 (14 mars 2007) : 5437–48. http://dx.doi.org/10.1128/jvi.02609-06.

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ABSTRACT Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1^E4 protein during the productive phase of infection. However, the precise mechanisms of how E1^E4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1^E4. We map critical residues within an arginine-rich domain of HPV1 E1^E4, and in a region known to facilitate E1^E4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1^E4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1^E4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1^E4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1^E4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1^E4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.

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Cryns, Vincent L., Youngsup Byun, Ajay Rana, Harry Mellor, Kevin D. Lustig, Louis Ghanem, Peter J. Parker, Marc W. Kirschner et Junying Yuan. « Specific Proteolysis of the Kinase Protein Kinase C-related Kinase 2 by Caspase-3 during Apoptosis ». Journal of Biological Chemistry 272, n^o 47 (21 novembre 1997) : 29449–53. http://dx.doi.org/10.1074/jbc.272.47.29449.

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Faisy, Christophe, Emmanuel Naline, Jean-Luc Diehl, Xavier Emonds-Alt, Thierry Chinet et Charles Advenier. « In vitro sensitization of human bronchus by β2-adrenergic agonists ». American Journal of Physiology-Lung Cellular and Molecular Physiology 283, n^o 5 (1 novembre 2002) : L1033—L1042. http://dx.doi.org/10.1152/ajplung.00063.2002.

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Incubation of human distal bronchi from 48 patients for 15 h with 10−7 M fenoterol induced sensitization characterized by an increase in maximal contraction to endothelin-1 (ET-1) and acetylcholine (ACh). Incubation of human bronchi with 10−6, 3 × 10−6, and 10−5 M forskolin (an adenyl cyclase activator) reproduced sensitization to ET-1 and ACh. The sensitizing effect of fenoterol was inhibited by coincubation with gliotoxine (a nuclear factor-κB inhibitor), dexamethasone, indomethacin (a cyclooxygenase inhibitor), GR-32191 (a TP prostanoid receptor antagonist), MK-476 (a cysteinyl leukotriene type 1 receptor antagonist), SR-140333 + SR-48968 + SR-142801 (neurokinin types 1, 2, and 3 tachykinin receptor antagonists) with or without HOE-140 (a bradykinin B2receptor antagonist), SB-203580 (an inhibitor of the 38-kDa mitogen-activated protein kinase, p38MAPK), or calphostin C (a protein kinase C blocker). Our results suggest that chronic exposure to fenoterol induces proinflammatory effects mediated by nuclear factor-κB and pathways involving leukotrienes, prostanoids, bradykinin, tachykinins, protein kinase C, and p38MAPK, leading to the regulation of smooth muscle contraction to ET-1 and ACh.

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Yeakley, Joanne M., Hélène Tronchère, James Olesen, Jacqueline A. Dyck, Huan-You Wang et Xiang-Dong Fu. « Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors ». Journal of Cell Biology 145, n^o 3 (3 mai 1999) : 447–55. http://dx.doi.org/10.1083/jcb.145.3.447.

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The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain–mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain–mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein–specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Δ yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain–containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

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Carlson, Cathrine R., Jan Magnus Aronsen, Anna Bergan-Dahl, Marie Christine Moutty, Marianne Lunde, Per Kristian Lunde, Hilde Jarstadmarken et al. « AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR ». Circulation Research 130, n^o 1 (7 janvier 2022) : 27–44. http://dx.doi.org/10.1161/circresaha.120.317976.

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Background: The sarcoplasmic reticulum (SR) Ca 2+ -ATPase 2 (SERCA2) mediates Ca 2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca 2+ release from SR and triggers contraction. Ca 2+ /CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca 2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca 2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca 2+ reuptake by SERCA2 and Ca 2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca 2+ -frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

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