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1
Prasad, Jayendra, and James L. Manley. "Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty." Molecular and Cellular Biology 23, no. 12 (June 15, 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.
2
Dong, Shaoyun, Fenglan Zhang, and Diane M. Beckles. "A Cytosolic Protein Kinase STY46 in Arabidopsis thaliana Is Involved in Plant Growth and Abiotic Stress Response." Plants 9, no. 1 (January 2, 2020): 57. http://dx.doi.org/10.3390/plants9010057.
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Starch provides plants with carbon and energy during stressful periods; however, relatively few regulators of starch metabolism under stress-induced carbon starvation have been discovered. We studied a protein kinase Ser/Thr/Tyr (STY) 46, identified by gene co-expression network analysis as a potential regulator of the starch starvation response in Arabidopsis thaliana. We showed that STY46 was induced by (1) abscisic acid and prolonged darkness, (2) by abiotic stressors, including salinity and osmotic stress, and (3) by conditions associated with carbon starvation. Characterization of STY46 T-DNA knockout mutants indicated that there was functional redundancy among the STY gene family, as these genotypes did not show strong phenotypes. However, Arabidopsis with high levels of STY46 transcripts (OE-25) grew faster at the early seedling stage, had higher photosynthetic rates, and more carbon was stored as protein in the seeds under control conditions. Further, OE-25 source leaf accumulated more sugars under 100 mM NaCl stress, and salinity also accelerated root growth, which is consistent with an adaptive response. Salt-stressed OE-25 partitioned 14C towards sugars and amino acids, and away from starch and protein in source leaves. Together, these findings suggested that STY46 may be part of the salinity stress response pathway that utilizes starch during early plant growth.
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Jiang, Kun, Niketa A. Patel, James E. Watson, Hercules Apostolatos, Eden Kleiman, Olivia Hanson, Masatoshi Hagiwara, and Denise R. Cooper. "Akt2 Regulation of Cdc2-Like Kinases (Clk/Sty), Serine/Arginine-Rich (SR) Protein Phosphorylation, and Insulin-Induced Alternative Splicing of PKCβII Messenger Ribonucleic Acid." Endocrinology 150, no. 5 (October 30, 2008): 2087–97. http://dx.doi.org/10.1210/en.2008-0818.
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Serine/arginine-rich (SR) proteins play essential roles in the constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich (RS) domain by SR protein kinases such as Cdc2-like kinases (Clk/Sty) modulates their subcellular localization and activation. However, it remains unclear how these kinases and their target SR proteins are regulated by extracellular signals. Regulation of protein kinase C βII (PKCβII) pre-mRNA alternative splicing via exon inclusion by Akt2, a central kinase in insulin action, involves phosphorylation of SR proteins. Here we showed that Akt2, in response to insulin, resulted in phosphorylation of Clk/Sty, which then altered SR protein phosphorylation in concert with Akt2. Insulin-stimulated PKCβII pre-mRNA splicing was blocked by Clk/Sty and phosphatidylinositol-3-kinase inhibitors, and diabetic Akt2-null mouse tissues had impaired phospho-Clk/Sty, SR protein phosphorylation, and PKCβII expression. Furthermore, we observed that Akt2 phosphorylated several SR proteins distinct from Clk/Sty in response to insulin. Akt2-catalyzed phosphorylation of Clk/Sty and SR proteins revealed a role for both kinases in splicing regulation indicating dual functions for Akt2 in response to insulin in this pathway.
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Howell, B. W., D. E. Afar, J. Lew, E. M. Douville, P. L. Icely, D. A. Gray, and J. C. Bell. "STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases." Molecular and Cellular Biology 11, no. 1 (January 1991): 568–72. http://dx.doi.org/10.1128/mcb.11.1.568-572.1991.
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We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.
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Howell, B. W., D. E. Afar, J. Lew, E. M. Douville, P. L. Icely, D. A. Gray, and J. C. Bell. "STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases." Molecular and Cellular Biology 11, no. 1 (January 1991): 568–72. http://dx.doi.org/10.1128/mcb.11.1.568.
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We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.
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Prasad, Jayendra, Karen Colwill, Tony Pawson, and James L. Manley. "The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing." Molecular and Cellular Biology 19, no. 10 (October 1, 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.
7
Song, Weimeng, Li Hu, Zhihui Ma, Lei Yang, and Jianming Li. "Importance of Tyrosine Phosphorylation in Hormone-Regulated Plant Growth and Development." International Journal of Molecular Sciences 23, no. 12 (June 13, 2022): 6603. http://dx.doi.org/10.3390/ijms23126603.
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Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1–2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.
8
NAYLER, Oliver, Stefan STAMM, and Axel ULLRICH. "Characterization and comparison of four serine- and arginine-rich (SR) protein kinases." Biochemical Journal 326, no. 3 (September 15, 1997): 693–700. http://dx.doi.org/10.1042/bj3260693.
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Phosphorylated serine- and arginine-rich (SR) proteins are components of the spliceosomal complex, and have been implicated in the control of alternative splicing. Kinases that regulate the phosphorylation and possibly the intranuclear distribution of SR proteins may therefore contribute to changes in choice of splice site. We have cloned three mouse cDNAs with high sequence identity to the family of LAMMER kinases (i.e. kinases carrying the conserved signature EHLAMMERILG in the catalytic domain). A comparison of their amino acid sequences revealed two related subfamilies with high evolutionary conservation. We have compared the expression patterns of these proteins in mouse tissues and transformed cell lines with that of a previously cloned family member (mCLK1/STY), and detected various transcripts for each gene. This underlines previous findings of alternative splicing of mclk1/STY. Our results suggest that the proportions of products for each gene are regulated independently. We further demonstrate that all variants encode autophosphorylating proteins that can phosphorylate several biochemically purified SR proteins in vitro, leading to hyperphosphorylation of at least one SR protein in vivo. The observed tissue distributions and substrate specificities suggest that these kinases may all be constituents of a network of regulatory mechanisms that enable SR proteins to control RNA splicing.
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Kornbluth, S., B. Sebastian, T. Hunter, and J. Newport. "Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14." Molecular Biology of the Cell 5, no. 3 (March 1994): 273–82. http://dx.doi.org/10.1091/mbc.5.3.273.
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The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.
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Liu, X. Q., and T. Pawson. "The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains." Molecular and Cellular Biology 11, no. 5 (May 1991): 2511–16. http://dx.doi.org/10.1128/mcb.11.5.2511-2516.1991.
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GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.
11
Liu, X. Q., and T. Pawson. "The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains." Molecular and Cellular Biology 11, no. 5 (May 1991): 2511–16. http://dx.doi.org/10.1128/mcb.11.5.2511.
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GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.
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Alcázar, Isabela, Miriam Marqués, Amit Kumar, Emilio Hirsch, Matthias Wymann, Ana C. Carrera, and Domingo F. Barber. "Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation." Journal of Experimental Medicine 204, no. 12 (November 12, 2007): 2977–87. http://dx.doi.org/10.1084/jem.20070366.
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Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation.
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Marth, J. D., J. A. Cooper, C. S. King, S. F. Ziegler, D. A. Tinker, R. W. Overell, E. G. Krebs, and R. M. Perlmutter. "Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck)." Molecular and Cellular Biology 8, no. 2 (February 1988): 540–50. http://dx.doi.org/10.1128/mcb.8.2.540-550.1988.
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The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.
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Marth, J. D., J. A. Cooper, C. S. King, S. F. Ziegler, D. A. Tinker, R. W. Overell, E. G. Krebs, and R. M. Perlmutter. "Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck)." Molecular and Cellular Biology 8, no. 2 (February 1988): 540–50. http://dx.doi.org/10.1128/mcb.8.2.540.
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The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.
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O'neill, L. "The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence." Biochemical Society Transactions 28, no. 5 (October 1, 2000): 557–63. http://dx.doi.org/10.1042/bst0280557.
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The pro-inflammatory cytokine interleukin-1 (IL-1) signals via the Type-1 IL-1 receptor (IL-1RI), inducing an increase in the expression of many genes with roles in immunity and inflammation. The signalling pathways involve two adapter proteins, MyD88 and Tollip, which via two IL-1 receptor-associated kinases (IRAK and IRAK-2) activate transcription factors such as nuclear factor-κB and protein kinases such as p38 mitogen-activated protein kinase. A role for the low-molecular-mass G-proteins Rac, Ras and Rap in these processes has also been indicated. IL-1RI is the founder of a diverse superfamily of receptors, which all share a cytosolic domain, termed the Toll/IL-1 receptor (TIR) domain. The superfamily can be divided broadly into three subgroups. The first of these is most similar to IL-1RI and includes the receptor for IL-18 and the Th2 cell regulator T1/ST2. The second subgroup is most similar to the Drosophila melanagaster protein Toll and includes Toll-like receptor 2 (TLR2), which is required for host defence against Gram-positive bacteria and fungi, and TLR4, which is required for lipopolysaccharide responsiveness, and thus is involved in host defence against Gram-negative bacteria. There are also a number of TLRs in plants and insects, all involved in host defence. The third subgroup contains nonreceptor proteins which possess a TIR domain and are cytosolic. MyD88 is a member, and it presumably complexes with IL-1RI via a TIR-TIR interaction. The other two members are proteins encoded by the vaccinia virus, A46R and A52R, which block TIR-dependent signalling. This receptor superfamily therefore appears to play a central role in inflammation and host defence against infection, pointing to the TIR domain as a critical molecular player in the innate immune response.
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Eisa, Ahmed, Bettina Bölter, and Serena Schwenkert. "The ACT domain in chloroplast precursor–phosphorylating STY kinases binds metabolites and allosterically regulates kinase activity." Journal of Biological Chemistry 294, no. 46 (October 8, 2019): 17278–88. http://dx.doi.org/10.1074/jbc.ra119.010298.
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Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase–chorismate mutase–tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo. Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.
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HIMPEL, Sunke, Pascal PANZER, Klaus EIRMBTER, Hanna CZAJKOWSKA, Muhammed SAYED, Len C. PACKMAN, Tom BLUNDELL, et al. "Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A." Biochemical Journal 359, no. 3 (October 25, 2001): 497–505. http://dx.doi.org/10.1042/bj3590497.
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Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.
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Stern, D. F., P. Zheng, D. R. Beidler, and C. Zerillo. "Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine." Molecular and Cellular Biology 11, no. 2 (February 1991): 987–1001. http://dx.doi.org/10.1128/mcb.11.2.987-1001.1991.
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A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.
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Stern, D. F., P. Zheng, D. R. Beidler, and C. Zerillo. "Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine." Molecular and Cellular Biology 11, no. 2 (February 1991): 987–1001. http://dx.doi.org/10.1128/mcb.11.2.987.
Full textAbstract:
A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.
20
Courtneidge, Sara A., Stefano Fumagalli, Manfred Koegl, Giulio Superti-Furga, and Geraldine M. Twamley-Stein. "The Src family of protein tyrosine kinases: regulation and functions." Development 119, Supplement (December 1, 1993): 57–64. http://dx.doi.org/10.1242/dev.119.supplement.57.
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Most of the nine members of the Src family of tyrosine kinases arc restricted in their expression, often to cells of the haematopoietic lineage, while some, particularly Src, Fyn and Yes, are more unbiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts with platelet-derived growth factor (PDGF) causes Src, Fyn and Yes to become activated, and to associate transiently with the PDGF receptor. To address the role of Src, Fyn and Yes in the response to PDGF, we have used a dominant negative approach, in which cells were engineered to express catalytically inactive forms of Src kinases. These cells were unable to enter S phase in response to PDGF, and we therefore conclude that Src family tyrosine kinases are required in order for the PDGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphorylation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that phosphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses activity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in S. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in the SH2 domain were as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain : phosphorylated tail interaction repressing Src activity. However, we have also found that Src molecules containing mutations in the SH3 domain are not regulated by Csk, suggesting that the SH3 domain also functions in the intramolecular regulation of Src activity.
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Majumdar, A. P. "Role of tyrosine kinases in gastrin induction of ornithine decarboxylase in colonic mucosa." American Journal of Physiology-Gastrointestinal and Liver Physiology 259, no. 4 (October 1, 1990): G626—G630. http://dx.doi.org/10.1152/ajpgi.1990.259.4.g626.
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An organ culture system was utilized to evaluate the role of tyrosine kinases (Tyr-k) and tyrosine-specific phosphorylation of proteins in gastrin regulation of ornithine decarboxylase (ODC) activity in colonic mucosa. Exposure of colonic mucosal explants to gastrin (50-100 ng G-17 I/ml) resulted in a profound stimulation of both Tyr-k and ODC activities compared with the corresponding basal levels. Whereas the maximal stimulation (ranging between 70 and 150%) of Tyr-k occurred within 10-15 min of exposure to gastrin, ODC activity was significantly stimulated (180%) 2 h after exposure to the hormone, and at 4 h it was found to be 750% above the corresponding basal level. Difluoromethylornithine (DFMO; 2 mM), an irreversible inhibitor of ODC, completely abolished the gastrin-mediated stimulation of ODC but not Tyr-k activity. On the other hand, genistein (100 micrograms/ml), a specific inhibitor of Tyr-k, caused a total suppression of the gastrin-induced stimulation of both Tyr-k and ODC. Gastrin also stimulated tyrosyl phosphorylation of a colonic mucosal membrane protein with molecular mass of 57 kDa, and genistein greatly attenuated this effect. We conclude that gastrin stimulates colonic mucosal ODC in vitro, and Tyr-k may be required for the regulation of this process.
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Moens, Ugo, and Sergiy Kostenko. "Structure and function of MK5/PRAK: the loner among the mitogen-activated protein kinase-activated protein kinases." Biological Chemistry 394, no. 9 (September 1, 2013): 1115–32. http://dx.doi.org/10.1515/hsz-2013-0149.
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Abstract Mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways that control pivotal cellular processes including proliferation, differentiation, survival, apoptosis, gene regulation, and motility. MAPK pathways consist of a relay of consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinases, and MAPKs. Conventional MAPKs are characterized by a conserved Thr-X-Tyr motif in the activation loop of the kinase domain, while atypical MAPKs lack this motif and do not seem to be organized into the classical three-tiered kinase cascade. One functional group of conventional and atypical MAPK substrates consists of protein kinases known as MAPK-activated protein kinases. Eleven mammalian MAPK-activated protein kinases have been identified, and they are divided into five subgroups: the ribosomal-S6-kinases RSK1-4, the MAPK-interacting kinases MNK1 and 2, the mitogen- and stress-activated kinases MSK1 and 2, the MAPK-activated protein kinases MK2 and 3, and the MAPK-activated protein kinase MK5 (also referred to as PRAK). MK5/PRAK is the only MAPK-activated protein kinase that is a substrate for both conventional and atypical MAPK, while all other MAPKAPKs are exclusively phosphorylated by conventional MAPKs. This review focuses on the structure, activation, substrates, functions, and possible implications of MK5/PRAK in malignant and nonmalignant diseases.
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Sakai, Hiroyasu, Yu Watanabe, Mai Honda, Rika Tsuiki, Yusuke Ueda, Yuki Nagai, Minoru Narita, Miwa Misawa, and Yoshihiko Chiba. "Involvement of the Tyr Kinase/JNK Pathway in Carbachol-induced Bronchial Smooth Muscle Contraction in the Rat." Anesthesiology 118, no. 5 (May 1, 2013): 1076–85. http://dx.doi.org/10.1097/aln.0b013e318286d0ae.
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Abstract Background: Tyrosine (Tyr) kinases and mitogen-activated protein kinases have been thought to participate in the contractile response in various smooth muscles. The aim of the current study was to investigate the involvement of the Tyr kinase pathway in the contraction of bronchial smooth muscle. Methods: Ring preparations of bronchi isolated from rats were suspended in an organ bath. Isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine the phosphorylation of c-Jun N-terminal kinasess (JNKs) in bronchial smooth muscle. Results: To examine the role of mitogen-activated protein kinase(s) in bronchial smooth muscle contraction, the effects of MPAK inhibitors were investigated in this study. The contraction induced by carbachol (CCh) was significantly inhibited by pretreatment with selective Tyr kinase inhibitors (genistein and ST638, n = 6, respectively), and a JNK inhibitor (SP600125, n = 6). The contractions induced by high K+ depolarization (n = 4), orthovanadate (a potent Tyr phosphatase inhibitor) and sodium fluoride (a G protein activator; NaF) were also significantly inhibited by selective Tyr kinase inhibitors and a JNK inhibitor (n = 4, respectively). However, the contraction induced by calyculin-A was not affected by SP600125. On the other hand, JNKs were phosphorylated by CCh (2.2 ± 0,4 [mean±SEM] fold increase). The JNK phosphorylation induced by CCh was significantly inhibited by SP600125 (n = 4). Conclusion: These findings suggest that the Tyr kinase/JNK pathway may play a role in bronchial smooth muscle contraction. Strategies to inhibit JNK activation may represent a novel therapeutic approach for diseases involving airway obstruction, such as asthma and chronic obstructive pulmonary disease.
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Cruz, T. F., G. Mills, K. P. H. Pritzker, and R. A. Kandel. "Inverse correlation between tyrosine phosphorylation and collagenase production in chondrocytes." Biochemical Journal 269, no. 3 (August 1, 1990): 717–21. http://dx.doi.org/10.1042/bj2690717.
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Collagenase production by chondrocytes appears to play a major role in the development of osteoarthritis. Although the mechanisms regulating collagenase production by chondrocytes are not known, incubation of bovine chondrocytes in serum markedly decreases collagenase production. Since serum has been demonstrated to increase levels of phosphotyrosine (P-Tyr) in several cell types, we determined the effect of altering intracellular levels of P-Tyr on collagenase production. Both orthovanadate, a potent inhibitor of tyrosine phosphatases, and serum caused a marked increase in tyrosine phosphorylation. The increase in P-Tyr was associated with a decrease in the production of collagenase, suggesting that two processes may be linked. Orthovanadate caused an increase in P-Tyr in the absence of serum, suggesting that P-Tyr levels in resting chondrocytes are regulated through activity of both tyrosine kinases and phosphatases. Orthovanadate and serum induced a synergistic increase in P-Tyr levels, suggesting that serum functions through increasing kinase activity rather than decreasing phosphatase activity. In the absence of serum, concentrations of orthovanadate which maximally inhibited collagenase production primarily increased phosphorylation of a 36 kDa protein, suggesting that the phosphorylation of this protein may play a major role in regulating collagenase production. Orthovanadate had limited effects on chondrocyte proteoglycan synthesis, morphology or viability in the presence or absence of serum, suggesting that the decrease in collagenase production was not due to non-specific inhibition of protein synthesis or cellular toxicity. Inhibition of tyrosine phosphatases by orthovanadate or activation of tyrosine kinases by addition of serum correlated with the inhibition of collagenase production.
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Eto, Masumi, Shuichi Katsuki, Yoshinori Tanaka, and Kosuke Takeya. "Kinase activity-tagged western blotting assay." BioTechniques 68, no. 4 (April 2020): 211–13. http://dx.doi.org/10.2144/btn-2019-0136.
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Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a nonradioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation–renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP.
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Seok, Seung-Hyeon. "Structural Insights into Protein Regulation by Phosphorylation and Substrate Recognition of Protein Kinases/Phosphatases." Life 11, no. 9 (September 13, 2021): 957. http://dx.doi.org/10.3390/life11090957.
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Protein phosphorylation is one of the most widely observed and important post-translational modification (PTM) processes. Protein phosphorylation is regulated by protein kinases, each of which covalently attaches a phosphate group to an amino acid side chain on a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue of a protein, and by protein phosphatases, each of which, conversely, removes a phosphate group from a phosphoprotein. These reversible enzyme activities provide a regulatory mechanism by activating or deactivating many diverse functions of proteins in various cellular processes. In this review, their structures and substrate recognition are described and summarized, focusing on Ser/Thr protein kinases and protein Ser/Thr phosphatases, and the regulation of protein structures by phosphorylation. The studies reviewed here and the resulting information could contribute to further structural, biochemical, and combined studies on the mechanisms of protein phosphorylation and to drug discovery approaches targeting protein kinases or protein phosphatases.
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Biarc, Jordane, Robert J. Chalkley, A. L. Burlingame, and Ralph A. Bradshaw. "Dissecting the Roles of Tyrosines 490 and 785 of TrkA Protein in the Induction of Downstream Protein Phosphorylation Using Chimeric Receptors." Journal of Biological Chemistry 288, no. 23 (April 15, 2013): 16606–18. http://dx.doi.org/10.1074/jbc.m113.475285.
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Receptor tyrosine kinases generally act by forming phosphotyrosine-docking sites on their own endodomains that propagate signals through cascades of post-translational modifications driven by the binding of adaptor/effector proteins. The pathways that are stimulated in any given receptor tyrosine kinase are a function of the initial docking sites that are activated and the availability of downstream participants. In the case of the Trk receptors, which are activated by nerve growth factor, there are only two established phosphotyrosine-docking sites (Tyr-490 and Tyr-785 on TrkA) that are known to be directly involved in signal transduction. Taking advantage of this limited repertoire of docking sites and the availability of PC12 cell lines stably transfected with chimeric receptors composed of the extracellular domain of the PDGF receptor and the transmembrane and intracellular domains of TrkA, the downstream TrkA-induced phosphoproteome was assessed for the “native” receptor and mutants lacking Tyr-490 or both Tyr-490 and Tyr-785. Basal phosphorylation levels were compared with those formed after 20 min of stimulation with PDGF. Several thousand phosphopeptides were identified after TiO2 enrichment, and many were up- or down-regulated by receptor activation. The modified proteins in the native sample contained many of the well established participants in TrkA signaling. The results from the mutant receptors allowed grouping of these downstream targets by their dependence on the two characterized docking site(s). A clear subset that was not dependent on either Tyr-490 or Tyr-785 emerged, providing direct evidence that there are other sites on TrkA that are involved in downstream signaling.
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Pazdrak, K., D. Schreiber, P. Forsythe, L. Justement, and R. Alam. "The intracellular signal transduction mechanism of interleukin 5 in eosinophils: the involvement of lyn tyrosine kinase and the Ras-Raf-1-MEK-microtubule-associated protein kinase pathway." Journal of Experimental Medicine 181, no. 5 (May 1, 1995): 1827–34. http://dx.doi.org/10.1084/jem.181.5.1827.
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Interleukin 5 (IL-5) regulates the growth and function of eosinophils. The objective of this study was to investigate the intracellular signal transduction mechanism of IL-5 in eosinophils. Purified eosinophils were stimulated with IL-5, and the involvement of various kinases was investigated by immunoblotting, immune complex kinase assay, and in situ denatured/renatured kinase assay. We found that IL-5 induced tyrosine phosphorylation and activation of a number of kinases. Two species of lyn kinases (53 and 56 kD) were present in eosinophils. Both forms were Tyr-phosphorylated and activated rapidly within 1 min. Further, lyn kinase was physically associated with the IL-5 beta receptor in eosinophils. Ras was studied by immunoprecipitation followed by thin-layer chromatography. Ras bound higher quantities of [alpha-32P]guanosine 5'triphosphate upon stimulation with IL-5. Raf-1 kinase showed increased Tyr phosphorylation on immunoblotting and increased activity in the immune complex kinase assay. Two species of MEK (MAP or Erk kinase) (41 and 45 kD) were identified in eosinophils, which underwent autophosphorylation upon stimulation. Microtubule-associated protein (MAP) kinase (p44) was Tyr-phosphorylated on immunoblotting and had increased activity in the immune-complex kinase assay. MAP kinases were also studied after metabolic radiolabeling of the cells with [32P]orthophosphates. IL-5 stimulated phosphorylation of MAP kinases in situ. Thus, we have delineated major components of an important signaling pathway in eosinophils. We believe that one of the signals generated by IL-5 receptor activation is propagated through the lyn-Ras-Raf-1-MEK-MAP kinase pathway.
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Durocher, Y., A. Chapdelaine, and S. Chevalier. "Identification of cytosolic protein tyrosine kinases of human prostate by renaturation after SDS/PAGE." Biochemical Journal 284, no. 3 (June 15, 1992): 653–58. http://dx.doi.org/10.1042/bj2840653.
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The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.
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Han, Zhiqiang Stanley, Hervé Enslen, Xiaodi Hu, Xiangjun Meng, I.-Huan Wu, Tamera Barrett, Roger J. Davis, and Y. Tony Ip. "A Conserved p38 Mitogen-Activated Protein Kinase Pathway Regulates Drosophila Immunity Gene Expression." Molecular and Cellular Biology 18, no. 6 (June 1, 1998): 3527–39. http://dx.doi.org/10.1128/mcb.18.6.3527.
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ABSTRACT Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-κB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novelDrosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of bothDrosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophilaindeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide.
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Fabian, J. R., I. O. Daar, and D. K. Morrison. "Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase." Molecular and Cellular Biology 13, no. 11 (November 1993): 7170–79. http://dx.doi.org/10.1128/mcb.13.11.7170-7179.1993.
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The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.
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Fabian, J. R., I. O. Daar, and D. K. Morrison. "Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase." Molecular and Cellular Biology 13, no. 11 (November 1993): 7170–79. http://dx.doi.org/10.1128/mcb.13.11.7170.
Full textAbstract:
The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.
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Schaller, M. D., J. D. Hildebrand, J. D. Shannon, J. W. Fox, R. R. Vines, and J. T. Parsons. "Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src." Molecular and Cellular Biology 14, no. 3 (March 1994): 1680–88. http://dx.doi.org/10.1128/mcb.14.3.1680-1688.1994.
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The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
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Schaller, M. D., J. D. Hildebrand, J. D. Shannon, J. W. Fox, R. R. Vines, and J. T. Parsons. "Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src." Molecular and Cellular Biology 14, no. 3 (March 1994): 1680–88. http://dx.doi.org/10.1128/mcb.14.3.1680.
Full textAbstract:
The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
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Schlaepfer, D. D., and T. Hunter. "Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases." Molecular and Cellular Biology 16, no. 10 (October 1996): 5623–33. http://dx.doi.org/10.1128/mcb.16.10.5623.
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Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.
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Zhan, Xiao-Li, Yulong Hong, Tianqing Zhu, Aaron P. Mitchell, Robert J. Deschenes, and Kun-Liang Guan. "Essential Functions of Protein Tyrosine Phosphatases Ptp2 and Ptp3 and Rim11 Tyrosine Phosphorylation inSaccharomyces cerevisiaeMeiosis and Sporulation." Molecular Biology of the Cell 11, no. 2 (February 2000): 663–76. http://dx.doi.org/10.1091/mbc.11.2.663.
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Tyrosine phosphorylation plays a central role in eukaryotic signal transduction. In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation. Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases. Here we report that deletion of PTP2 and PTP3results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation. Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes. We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Δptp3Δ homozygous deletion cells under sporulation conditions. The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine. Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation. We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3. Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates. Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation
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Brott, B. K., S. Decker, M. C. O'Brien, and R. Jove. "Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein." Molecular and Cellular Biology 11, no. 10 (October 1991): 5059–67. http://dx.doi.org/10.1128/mcb.11.10.5059-5067.1991.
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GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.
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Brott, B. K., S. Decker, M. C. O'Brien, and R. Jove. "Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein." Molecular and Cellular Biology 11, no. 10 (October 1991): 5059–67. http://dx.doi.org/10.1128/mcb.11.10.5059.
Full textAbstract:
GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.
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Cooper, J. A., and C. S. King. "Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src." Molecular and Cellular Biology 6, no. 12 (December 1986): 4467–77. http://dx.doi.org/10.1128/mcb.6.12.4467-4477.1986.
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Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.
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Cooper, J. A., and C. S. King. "Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src." Molecular and Cellular Biology 6, no. 12 (December 1986): 4467–77. http://dx.doi.org/10.1128/mcb.6.12.4467.
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Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.
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Piedra, Jose, Susana Miravet, Julio Castaño, Héctor G. Pálmer, Nora Heisterkamp, Antonio García de Herreros, and Mireia Duñach. "p120 Catenin-Associated Fer and Fyn Tyrosine Kinases Regulate β-Catenin Tyr-142 Phosphorylation and β-Catenin-α-Catenin Interaction." Molecular and Cellular Biology 23, no. 7 (April 1, 2003): 2287–97. http://dx.doi.org/10.1128/mcb.23.7.2287-2297.2003.
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ABSTRACT β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and α-catenin. We show here that interaction of β-catenin with α-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little α-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-α-catenin interaction.
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Fujii, M., D. Shalloway, and I. M. Verma. "Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos." Molecular and Cellular Biology 9, no. 6 (June 1989): 2493–99. http://dx.doi.org/10.1128/mcb.9.6.2493-2499.1989.
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A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.
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Fujii, M., D. Shalloway, and I. M. Verma. "Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos." Molecular and Cellular Biology 9, no. 6 (June 1989): 2493–99. http://dx.doi.org/10.1128/mcb.9.6.2493.
Full textAbstract:
A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.
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Yaciuk, P., J. K. Choi, and D. Shalloway. "Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A." Molecular and Cellular Biology 9, no. 6 (June 1989): 2453–63. http://dx.doi.org/10.1128/mcb.9.6.2453-2463.1989.
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The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.
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Yaciuk, P., J. K. Choi, and D. Shalloway. "Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A." Molecular and Cellular Biology 9, no. 6 (June 1989): 2453–63. http://dx.doi.org/10.1128/mcb.9.6.2453.
Full textAbstract:
The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.
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RAKSHAMBIKAI, R., N. SRINIVASAN, and RUPALI A. GADKARI. "REPERTOIRE OF PROTEIN KINASES ENCODED IN THE GENOME OF ZEBRAFISH SHOWS REMARKABLY LARGE POPULATION OF PIM KINASES." Journal of Bioinformatics and Computational Biology 12, no. 01 (January 28, 2014): 1350014. http://dx.doi.org/10.1142/s0219720013500145.
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In recent times, zebrafish has garnered lot of popularity as model organism to study human cancers. Despite high evolutionary divergence from humans, zebrafish develops almost all types of human tumors when induced. However, mechanistic details of tumor formation have remained largely unknown. Present study is aimed at analysis of repertoire of kinases in zebrafish proteome to provide insights into various cellular components. Annotation using highly sensitive remote homology detection methods revealed "substantial expansion" of Ser/Thr/Tyr kinase family in zebrafish compared to humans, constituting over 3% of proteome. Subsequent classification of kinases into subfamilies revealed presence of large number of CAMK group of kinases, with massive representation of PIM kinases, important for cell cycle regulation and growth. Extensive sequence comparison between human and zebrafish PIM kinases revealed high conservation of functionally important residues with a few organism specific variations. There are about 300 PIM kinases in zebrafish kinome, while human genome codes for only about 500 kinases altogether. PIM kinases have been implicated in various human cancers and are currently being targeted to explore their therapeutic potentials. Hence, in depth analysis of PIM kinases in zebrafish has opened up new avenues of research to verify the model organism status of zebrafish.
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Colwill, Karen, Lana L. Feng, Joanne M. Yeakley, Gerald D. Gish, Javier F. Cáceres, Tony Pawson, and Xiang-Dong Fu. "SRPK1 and Clk/Sty Protein Kinases Show Distinct Substrate Specificities for Serine/Arginine-rich Splicing Factors." Journal of Biological Chemistry 271, no. 40 (October 4, 1996): 24569–75. http://dx.doi.org/10.1074/jbc.271.40.24569.
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WOLLBERG, Patrik, Johan LENNARTSSON, Eva GOTTFRIDSSON, Akihiko YOSHIMURA, and Lars RÖNNSTRAND. "The adapter protein APS associates with the multifunctional docking sites Tyr-568 and Tyr-936 in c-Kit." Biochemical Journal 370, no. 3 (March 15, 2003): 1033–38. http://dx.doi.org/10.1042/bj20020716.
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The adapter protein APS has previously been shown to be involved in recruiting the ubiquitin E3 ligase c-Cbl to the insulin receptor, the platelet-derived growth factor β-receptor and the erythropoietin receptor, leading to increased degradation of the receptors and inhibition of mitogenesis. Here we demonstrate, by use of immobilized synthetic phosphopeptides corresponding to various autophosphorylated tyrosine residues in the receptor for stem-cell factor (c-Kit), that APS preferentially associates with phosphorylated Tyr-568 and Tyr-936. Tyr-568 has previously been identified as the binding site of the Src family of tyrosine kinases, the Csk-homologous kinase CHK, and the protein tyrosine phosphatase SHP-2. We have recently demonstrated that Tyr-936 is an autophosphorylation site involved in binding the adapter proteins Grb2 and Grb7. We could further demonstrate that the critical determinant for binding of APS is the presence of either a leucine or an isoleucine residue in the position +3 to the phosphorylated tyrosine. This allowed us to design mutants that selectively failed to associate with APS, while still associating with Src family members, SHP-2 and Grb2, respectively.
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CLARI, Giulio, and Vittorio MORET. "Comparative characterization of membrane-associated and cytosolic Tyr-protein kinases in human erythrocytes." European Journal of Biochemistry 179, no. 3 (February 1989): 581–88. http://dx.doi.org/10.1111/j.1432-1033.1989.tb14586.x.
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Bordin, Luciana, Anna Maria Brunati, Arianna Donella-Deana, Bruno Baggio, Antonio Toninello, and Giulio Clari. "Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes." Blood 100, no. 1 (July 1, 2002): 276–82. http://dx.doi.org/10.1182/blood.v100.1.276.
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Abstract Tyr phosphorylation of the multifunctional transmembrane protein band 3 has been implicated in several erythrocyte functions and disorders. We previously demonstrated that pervanadate treatment of human erythrocytes induces band-3 Tyr phosphorylation, which is catalyzed by the sequential action of tyrosine kinase Syk and tyrosine kinase(s) belonging to the Src family. In this study, we show that Tyr phosphorylation of band 3, elicited by pervanadate, N-ethylmaleimide, or diamide, greatly increases band-3 interaction with the tyrosine phosphatase SHP-2 in parallel with the translocation of SHP-2 to erythrocyte membranes. These events seem to be mediated by Src-like catalyzed phosphorylation of band 3 because both SHP-2 translocation to cellular membranes and its interaction with Tyr-phosphorylated protein are greatly counteracted by PP2, a specific inhibitor of Src kinases. Binding-competition experiments demonstrate that SHP-2 recruitment to band 3 occurs via its SH2 domain(s). In particular, our data support the view that SHP-2 docks specifically with P-Y359 of band 3. Experiments performed with intact erythrocytes in the presence of the SHP-2 inhibitor calpeptin suggest that, once recruited to Tyr-phosphorylated band 3, the tyrosine phosphatase dephosphorylates the protein. P-Y8, 21, and 904 are the residues affected by SHP-2, as judged by 32P-peptide mapping of band 3 digested with trypsin. These results indicate that in treated erythrocytes, recruitment of cytosolic SHP-2 to band 3 is a prerequisite for the subsequent dephosphorylation of the transmembrane protein.