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1
Ravindran, Priyadarshini, and Andreas W. Püschel. "An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation." Life Science Alliance 6, no. 3 (December 21, 2022): e202201722. http://dx.doi.org/10.26508/lsa.202201722.
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The highly conserved GTPase Cdc42 is an essential regulator of cell polarity and promotes exocytosis through the exocyst complex in budding yeast andDrosophila. In mammals, this function is performed by the closely related GTPase TC10, whereas mammalian Cdc42 does not interact with the exocyst. Axon formation is facilitated by the exocyst complex that tethers vesicles before their fusion to expand the plasma membrane. This function depends on the recruitment of the Exo70 subunit to the plasma membrane. Alternative splicing generates two Cdc42 isoforms that differ in their C-terminal 10 amino acids. Our results identify an isoform-specific function of Cdc42 in neurons. We show that the brain-specific Cdc42b isoform, in contrast to the ubiquitous isoform Cdc42u, can interact with Exo70. Inactivation of Arhgef7 or Cdc42b interferes with the exocytosis of post-Golgi vesicles in the growth cone. Cdc42b regulates exocytosis and axon formation downstream of its activator Arhgef7. Thus, the function of Cdc42 in regulating exocytosis is conserved in mammals but specific to one isoform.
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Jansson, Thomas, Marisol Castillo-Castrejon, Madhulika B. Gupta, Theresa L. Powell, and Fredrick J. Rosario. "Down-regulation of placental Cdc42 and Rac1 links mTORC2 inhibition to decreased trophoblast amino acid transport in human intrauterine growth restriction." Clinical Science 134, no. 1 (January 2020): 53–70. http://dx.doi.org/10.1042/cs20190794.
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Abstract Intrauterine growth restriction (IUGR) increases the risk for perinatal complications and metabolic and cardiovascular disease later in life. The syncytiotrophoblast (ST) is the transporting epithelium of the human placenta, and decreased expression of amino acid transporter isoforms in the ST plasma membranes is believed to contribute to IUGR. Placental mechanistic target of rapamycin Complex 2 (mTORC2) signaling is inhibited in IUGR and regulates the trafficking of key amino acid transporter (AAT) isoforms to the ST plasma membrane; however, the molecular mechanisms are unknown. Cdc42 and Rac1 are Rho-GTPases that regulate actin-binding proteins, thereby modulating the structure and dynamics of the actin cytoskeleton. We hypothesized that inhibition of mTORC2 decreases AAT expression in the plasma membrane and amino acid uptake in primary human trophoblast (PHT) cells mediated by down-regulation of Cdc42 and Rac1. mTORC2, but not mTORC1, inhibition decreased the Cdc42 and Rac1 expression. Silencing of Cdc42 and Rac1 inhibited the activity of the System L and A transporters and markedly decreased the trafficking of LAT1 (System L isoform) and SNAT2 (System A isoform) to the plasma membrane. mTORC2 inhibition by silencing of rictor failed to decrease AAT following activation of Cdc42/Rac1. Placental Cdc42 and Rac1 protein expression was down-regulated in human IUGR and was positively correlated with placental mTORC2 signaling. In conclusion, mTORC2 regulates AAT trafficking in PHT cells by modulating Cdc42 and Rac1. Placental mTORC2 inhibition in human IUGR may contribute to decreased placental amino acid transfer and reduced fetal growth mediated by down-regulation of Cdc42 and Rac1.
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Kolyada, Alexey Y., Kathleen N. Riley, and Ira M. Herman. "Rho GTPase signaling modulates cell shape and contractile phenotype in an isoactin-specific manner." American Journal of Physiology-Cell Physiology 285, no. 5 (November 2003): C1116—C1121. http://dx.doi.org/10.1152/ajpcell.00177.2003.
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Rho family small GTPases (Rho, Rac, and Cdc42) play an important role in cell motility, adhesion, and cell division by signaling reorganization of the actin cytoskeleton. Here, we report an isoactin-specific, Rho GTPase-dependent signaling cascade in cells simultaneously expressing smooth muscle and nonmuscle actin isoforms. We transfected primary cultures of microvascular pericytes, cells related to vascular smooth muscle cells, with various Rho-related and Rho-specific expression plasmids. Overexpression of dominant positive Rho resulted in the formation of nonmuscle actin-containing stress fibers. At the same time, α-vascular smooth muscle actin (αVSMactin) containing stress fibers were disassembled, resulting in a dramatic reduction in cell size. Rho activation also yielded a disassembly of smooth muscle myosin and nonmuscle myosin from stress fibers. Overexpression of wild-type Rho had similar but less dramatic effects. In contrast, dominant negative Rho and C3 exotransferase or dominant positive Rac and Cdc42 expression failed to alter the actin cytoskeleton in an isoform-specific manner. The loss of smooth muscle contractile protein isoforms in pericyte stress fibers, together with a concomitant decrease in cell size, suggests that Rho activation influences “contractile” phenotype in an isoactin-specific manner. This, in turn, should yield significant alteration in microvascular remodeling during developmental and pathologic angiogenesis.
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Fediuk, Jena, Anurag S. Sikarwar, Nora Nolette, and Shyamala Dakshinamurti. "Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling." American Journal of Physiology-Lung Cellular and Molecular Physiology 307, no. 11 (December 1, 2014): L877—L887. http://dx.doi.org/10.1152/ajplung.00036.2014.
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In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms regulating thromboxane-mediated actin polymerization are potential targets for future PPHN pharmacotherapy.
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Wirth, Alexander, Chen Chen-Wacker, Yao-Wen Wu, Nataliya Gorinski, Mikhail A. Filippov, Ghanshyam Pandey, and Evgeni Ponimaskin. "Dual lipidation of the brain-specific Cdc42 isoform regulates its functional properties." Biochemical Journal 456, no. 3 (November 22, 2013): 311–22. http://dx.doi.org/10.1042/bj20130788.
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Here we demonstrated that the brain-specific small GTPase Cdc42-palm can be both palmitoylated and prenylated. We also found that Cdc42-palm is critically involved in the formation of spines in neurons, demonstrating that dual lipidation represents an important regulator of morphogenic signalling.
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Zhou, Rihong, Zhen Guo, Charles Watson, Emily Chen, Rong Kong, Wenxian Wang, and Xuebiao Yao. "Polarized Distribution of IQGAP Proteins in Gastric Parietal Cells and Their Roles in Regulated Epithelial Cell Secretion." Molecular Biology of the Cell 14, no. 3 (March 2003): 1097–108. http://dx.doi.org/10.1091/mbc.e02-07-0425.
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Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.
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Fotiadou, Poppy P., Chiaki Takahashi, Hasan N. Rajabi, and Mark E. Ewen. "Wild-Type NRas and KRas Perform Distinct Functions during Transformation." Molecular and Cellular Biology 27, no. 19 (July 16, 2007): 6742–55. http://dx.doi.org/10.1128/mcb.00234-07.
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ABSTRACT The ras proto-oncogenes, of which there are four isoforms, are molecular switches that function in signal transduction pathways to control cell differentiation, proliferation, and survival. How the Ras isoforms orchestrate cellular processes that affect behavior is poorly understood. Further, why cells express two or more Ras isoforms is unknown. Here, using a genetically defined system, we show that the presence of both wild-type KRas and NRas isoforms is required for transformation because they perform distinct nonoverlapping functions: wild-type NRas regulates adhesion, and KRas coordinates motility. Remarkably, we find that Ras isoforms achieve functional specificity by engaging different signaling pathways to affect the same cellular processes, thereby coordinating cellular outcome. Although we find that signaling from both isoforms intersects in actin and microtubule cytoskeletons, our results suggest that KRas signals through Akt and Cdc42 while NRas signals through Raf and RhoA. Our analyses suggest a previously unappreciated convergence of different Ras isoforms on the dynamics of the processes involved in transformation.
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CARGINALE, Vincenzo, Rosaria SCUDIERO, Clemente CAPASSO, Antonio CAPASSO, Peter KILLE, Guido di PRISCO, and Elio PARISI. "Cadmium-induced differential accumulation of metallothionein isoforms in the Antarctic icefish, which exhibits no basal metallothionein protein but high endogenous mRNA levels." Biochemical Journal 332, no. 2 (June 1, 1998): 475–81. http://dx.doi.org/10.1042/bj3320475.
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Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.
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Chen, Hung-Hsi, Yu-Chiuan Wang, and Ming-Ji Fann. "Identification and Characterization of the CDK12/Cyclin L1 Complex Involved in Alternative Splicing Regulation." Molecular and Cellular Biology 26, no. 7 (April 1, 2006): 2736–45. http://dx.doi.org/10.1128/mcb.26.7.2736-2745.2006.
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ABSTRACT CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12L). A shorter isoform of CDK12, CDK12S, that differs from CDK12L only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.
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Scalia, Pierluigi, Carmen Merali, Carlos Barrero, Antonio Suma, Vincenzo Carnevale, Salim Merali, and Stephen J. Williams. "Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma." International Journal of Molecular Sciences 24, no. 8 (April 17, 2023): 7380. http://dx.doi.org/10.3390/ijms24087380.
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EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein–protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as “DTX3c”), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.
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Huang, Shu-Ching, Eva S. Liu, Siu-Hong Chan, Indira D. Munagala, Heidi T. Cho, Ramasamy Jagadeeswaran, and Edward J. Benz. "Mitotic Regulation of Protein 4.1R Involves Phosphorylation by cdc2 Kinase." Molecular Biology of the Cell 16, no. 1 (January 2005): 117–27. http://dx.doi.org/10.1091/mbc.e04-05-0426.
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The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.
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Lahti, J. M., J. Xiang, L. S. Heath, D. Campana, and V. J. Kidd. "PITSLRE protein kinase activity is associated with apoptosis." Molecular and Cellular Biology 15, no. 1 (January 1995): 1–11. http://dx.doi.org/10.1128/mcb.15.1.1.
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Minimal ectopic expression of a 58-kDa protein kinase (PITSLRE beta 1), distantly related to members of the cdc2 gene family, induces telophase delay, abnormal chromosome segregation, and decreased growth rates in Chinese hamster ovary cells. Here we show that this decrease in cell growth rate is due to apoptosis. Apoptosis is also induced by ectopic expression of an amino-terminal deletion mutant containing the catalytic and C-terminal domains of PITSLRE beta 1 but not by other mutants lacking histone H1 kinase activity or by other members of the cdc2 gene family. However, unlike the wild-type PITSLRE beta 1 over-expressors, ectopic expression of the N-terminal PITSLRE beta 1 mutant does not result in telophase delay or abnormal chromosome segregation. These results suggested that the function of this protein kinase could be linked to apoptotic signaling. To test this hypothesis, we examined levels of PITSLRE mRNA, steady-state protein, and enzyme activity in human T cells undergoing apoptosis after activation with the anti-Fas monoclonal antibody (MAb). All were substantially elevated shortly after Fas MAb treatment. In addition to new transcription and translation, proteolysis contributed to the increased steady-state levels of a novel 50-kDa PITSLRE protein, as suggested by the diminution of larger PITSLRE isoforms observed in the same cells. Indeed, treatment of the Fas-activated T cells with a serine protease inhibitor prevented apoptotic death and led to the accumulation of larger, less active PITSLRE kinase isoforms but not the enzymatically active 50-kDa PITSLRE isoform. Finally, induction of apoptosis by glucocorticoids in the same cell line, as well as by Fas MAb treatment of another T-cell line, led to a similar induction of 50-kDa PITSLRE protein levels over time. These findings suggest that (i) PITSLRE kinase(s) may lie within apoptotic signaling pathway(s), (ii) serine protease activation may be an early event in Fas-activated apoptosis of human T cells, and (iii) some PITSLRE kinase isoforms may be targets of apoptotic proteases.
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Zhang, Mingliang, Wei Guo, Jun Qian, and Benzhong Wang. "Negative regulation of CDC42 expression and cell cycle progression by miR-29a in breast cancer." Open Medicine 11, no. 1 (January 1, 2016): 78–82. http://dx.doi.org/10.1515/med-2016-0015.
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AbstractObjectiveThe inhibitory role of microRNA-29a (miR-29a) has been assessed in breast cancer cells. Herein, we analyze the underlying mechanisms of its role in cell cycle progression in breast cancer cells.MethodsWe applied real-time polymerase chain reaction (PCR) to detect the expression of miR-29 in breast cancer cell lines. Then one of the cell lines, MDA-MB-453, was transfected with mimics of miR-29a. The cell cycle was analyzed by fluorescence-activated cell sorting after staining the cells with propidium iodide. Real-time PCR, luciferase assay and western blot were used together to verify the regulation of the predicted target, cell division cycle 42 (CDC42) by miR-29a.ResultsMiR-29s were decreased in our selected mammary cell lines, among which miR-29a was the dominant isoform. Overexpression of miR-29a caused cell cycle arrest at the G0/G1 phase. We further found that miR-29a could target the expression of CDC42, which is a small GTPase associated with cell cycle progression.ConclusionWe suggest that miR-29a exerts its tumor suppressor role in breast cancer cells partially by arresting the cell cycle through negative regulation of CDC42.
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Ivaska, Johanna, Hilkka Reunanen, Jukka Westermarck, Leeni Koivisto, Veli-Matti Kähäri, and Jyrki Heino. "Integrin α2β1 Mediates Isoform-Specific Activation of p38 and Upregulation of Collagen Gene Transcription by a Mechanism Involving the α2 Cytoplasmic Tail." Journal of Cell Biology 147, no. 2 (October 18, 1999): 401–16. http://dx.doi.org/10.1083/jcb.147.2.401.
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Two collagen receptors, integrins α1β1 and α2β1, can regulate distinct functions in cells. Ligation of α1β1, unlike α2β1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by α2β1 integrin, we have overexpressed wild-type α2, or chimeric α2 subunit with α1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous α2β1. The chimeric α2/α1 chain formed a functional heterodimer with β1. In contrast to α2/α1 chimera, forced expression of α2 integrin resulted in upregulation of α1 (I) collagen gene transcription in response to three-dimensional collagen, indicating that the cytoplasmic domain of α2 integrin was required for signaling. Furthermore, signals mediated by α2β1 integrin specifically activated the p38α isoform, and selective p38 inhibitors blocked upregulation of collagen gene transcription. Dominant negative mutants of Cdc42, MKK3, and MKK4 prevented α2β1 integrin–mediated activation of p38α. RhoA had also some inhibitory effect, whereas dominant negative Rac was not effective. Our findings show the isoform-specific activation of p38 by α2β1 integrin ligation and identify Cdc42, MKK3, and MKK4 as possible downstream effectors. These observations reveal a novel signaling mechanism of α2β1 integrin that is distinct from ones previously described for other integrins.
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Wang, Lin, William A. Rudert, Anatoly Grishin, Patrice Dombrosky-Ferlan, Kevin Sullivan, Xiaoying Deng, David Whitcomb, and Seth Corey. "Identification and genetic analysis of human and mouse activated Cdc42 interacting protein-4 isoforms." Biochemical and Biophysical Research Communications 293, no. 5 (May 2002): 1426–30. http://dx.doi.org/10.1016/s0006-291x(02)00398-4.
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Jaiswal, Mamta, Eyad Kalawy Fansa, Radovan Dvorsky, and Mohammad Reza Ahmadian. "New insight into the molecular switch mechanism of human Rho family proteins: shifting a paradigm." Biological Chemistry 394, no. 1 (January 1, 2013): 89–95. http://dx.doi.org/10.1515/hsz-2012-0207.
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Abstract Major advances have been made in understanding the structure, function and regulation of the small GTP-binding proteins of the Rho family and their involvement in multiple cellular process and disorders. However, intrinsic nucleotide exchange and hydrolysis reactions, which are known to be fundamental to Rho family proteins, have been partially investigated in the case of RhoA, Rac1 and Cdc42, but for others not at all. Here we present a comprehensive and quantitative analysis of the molecular switch functions of 15 members of the Rho family that enabled us to propose an active GTP-bound state for the rather uncharacterized isoforms RhoD and Rif under equilibrium and quiescent conditions.
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Smith-Donald, Benjamin A., and Bernard Roizman. "The Interaction of Herpes Simplex Virus 1 Regulatory Protein ICP22 with the cdc25C Phosphatase Is Enabled In Vitro by Viral Protein Kinases US3 and UL13." Journal of Virology 82, no. 9 (February 13, 2008): 4533–43. http://dx.doi.org/10.1128/jvi.02022-07.
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ABSTRACT Earlier studies have shown that ICP22 and the UL13 protein kinase but not the US3 kinase are required for optimal expression of a subset of late (γ2) genes exemplified by UL38, UL41, and US11. In primate cells, ICP22 mediates the disappearance of inactive isoforms of cdc2 and degradation of cyclins A and B1. Active cdc2 acquires a new partner, the viral DNA synthesis processivity factor UL42. The cdc2-UL42 complex recruits and phosphorylates topoisomerase IIα for efficient expression of the γ2 genes listed above. In uninfected cells, the cdc25C phosphatase activates cdc2 by removing two inhibitory phosphates. The accompanying report shows that in the absence of cdc25C, the rate of degradation of cyclin B1 is similar to that occurring in infected wild-type mouse embryo fibroblast cells but the levels of cdc2 increase, and the accumulation of a subset of late proteins and virus yields are reduced. This report links ICP22 with cdc25C. We show that in infected cells, ICP22 and US3 protein kinase mediate the phosphorylation of cdc25C at its C-terminal domain. In in vitro assays with purified components, both UL13 and US3 viral kinases phosphorylate cdc25C and ICP22. cdc25C also interacts with cdc2. However, in infected cells, the ability of cdc25C to activate cdc2 by dephosphorylation of the inactive cdc2 protein is reduced. Coupled with the phosphorylation of cdc25C by the US3 kinase, the results raise the possibility that herpes simplex virus 1 diverts cdc25C to perform functions other than those performed in uninfected cells.
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Priante, Ettore, Edoardo Pietropoli, Elisabetta Piva, Gianfranco Santovito, Sophia Schumann, and Paola Irato. "Cadmium–Zinc Interaction in Mus musculus Fibroblasts." International Journal of Molecular Sciences 23, no. 19 (October 9, 2022): 12001. http://dx.doi.org/10.3390/ijms231912001.
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This work aimed to evaluate the effects of zinc (Zn) relating to cadmium (Cd)-induced toxicity and the role played by MTF-1. This transcription factor regulates the expression of genes encoding metallothioneins (MTs), some Zn transporters and the heavy chain of γ-glutamylcysteine synthetase. For this reason, two cell lines of mouse fibroblasts were used: a wild-type strain and a knockout strain to study the effects. Cells were exposed to complete medium containing: (1) 50 μM ZnSO4 (Zn), (2) 1 μM CdCl2 (Cd 1), (3) 2 μM CdCl2 (Cd 2), (4) 50 μM ZnSO4 + 1 μM CdCl2 (ZnCd 1) and (5) 50 μM ZnSO4 + 2 μM CdCl2 (ZnCd 2) for 4, 18 and 24 h. Following exposure, cell viability, the intracellular content of metals, glutathione (GSH) and MT and the gene expression of the two isoforms of MT was evaluated. The results obtained suggest that a lower Cd content in the co-treatments is responsible for the protection offered by Zn due to the probable competition for a common transporter. Furthermore, Zn determines an increase in GSH in co-treatments compared to treatments with Cd alone. Finally, the MTF-1 factor is essential for the expression of MT-1 but not of MT-2 nor probably for the heavy chain of γ-glutamylcysteine synthetase.
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Kang, Min, Aera Bang, Ok Choi, and Seung Han. "Comparative analysis of two murine CDC25B isoforms." Archives of Biological Sciences 69, no. 1 (2017): 35–44. http://dx.doi.org/10.2298/abs160315062k.
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CDC25B phosphatase plays a pivotal role in the cell cycle process by dephosphorylating and activating the CDC2 kinase of maturation-promoting factor (MPF). In mice, two transcripts of Cdc25B are generated by the alternative splicing of one gene. We compared the properties of these two forms of CDC25B. When the expression pattern of Cdc25B was examined using RT-PCR, both forms were detected in almost all mouse tissues tested. The expression of two forms of the CDC25B protein in various mouse tissues was confirmed using Western blotting with generated isoform specific antibodies. CDC25B1 tends to accumulate more in the cytosol than CDC25B2 does, and they have different binding capacity for 14-3-3 proteins. CDC25B1 was more effective in dephosphorylating in vitro substrate para-nitrophenyl phosphate and showed higher activity in the modified histone H1 kinase assay than CDC25B2. These results suggest that the two forms of CDC25B play different roles in cell cycle regulation.
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Tcherkezian, Joseph, Eric I. Danek, Sarah Jenna, Ibtissem Triki, and Nathalie Lamarche-Vane. "Extracellular Signal-Regulated Kinase 1 Interacts with and Phosphorylates CdGAP at an Important Regulatory Site." Molecular and Cellular Biology 25, no. 15 (August 1, 2005): 6314–29. http://dx.doi.org/10.1128/mcb.25.15.6314-6329.2005.
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ABSTRACT Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.
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Lampe, P. D., W. E. Kurata, B. J. Warn-Cramer, and A. F. Lau. "Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase." Journal of Cell Science 111, no. 6 (March 15, 1998): 833–41. http://dx.doi.org/10.1242/jcs.111.6.833.
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The gap junction protein connexin43 is a phosphoprotein that typically migrates as three bands (nonphosphorylated, P1 and P2) during polyacrylamide gel electrophoresis. The electrophoretic mobility of connexin43 from mitotic cells was distinctly reduced to a form (P3) that migrated slower than P2 from Rat1 cells prepared by shakeoff of nocodazole-treated and untreated cultures. Mitotic FT210 cells, which contain a temperature-sensitive mutation in the p34(cdc2) kinase, showed abundant levels of the P3 connexin43 when maintained at the permissive temperature where p34(cdc2) is active. In contrast, nocodozole-treated FT210 cells grown at the nonpermissive temperature did not contain P3 connexin43. These results indicated that generation of the P3 connexin43 was dependent upon active p34(cdc2)/cyclin B kinase. Although the p34(cdc2)kinase phosphorylated connexin43 in vitro on peptides containing serine 255, the major phosphotryptic peptides in P3 connexin43 from mitotic cells appeared to be the consequence of another protein kinase(s), which may be activated by the p34(cdc2)/cyclin B kinase. The P3 connexin43 exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly stained cytoplasmic structures. These events may be part of the dramatic structural changes observed in mitotic cells undergoing cell rounding and cytokinesis. Results of initial studies using inhibitors of protein degradative and synthetic pathways suggested the likelihood that protein degradation and synthesis participate in the disappearance of the P3 connexin43 and restoration of the pattern of connexin43 isoforms observed in nonmitotic cells.
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Rousseau, Véronique, Olivier Goupille, Nathalie Morin, and Jean-Vianney Barnier. "A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases." Journal of Biological Chemistry 278, no. 6 (December 2, 2002): 3912–20. http://dx.doi.org/10.1074/jbc.m207251200.
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Lorenzi, Matthew V., Paola Castagnino, Qiong Chen, Yasuhiro Hori, and Toru Miki. "Distinct expression patterns and transforming properties of multiple isoforms of Ost, an exchange factor for RhoA and Cdc42." Oncogene 18, no. 33 (August 1999): 4742–55. http://dx.doi.org/10.1038/sj.onc.1202851.
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Weernink, Paschal A. Oude, Konstantinos Meletiadis, Silvia Hommeltenberg, Matthias Hinz, Hisamitsu Ishihara, Martina Schmidt, and Karl H. Jakobs. "Activation of Type I Phosphatidylinositol 4-Phosphate 5-Kinase Isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42." Journal of Biological Chemistry 279, no. 9 (December 17, 2003): 7840–49. http://dx.doi.org/10.1074/jbc.m312737200.
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Chew, Catherine S., Curtis T. Okamoto, Xunsheng Chen, and Ruby Thomas. "Drebrin E2 is differentially expressed and phosphorylated in parietal cells in the gastric mucosa." American Journal of Physiology-Gastrointestinal and Liver Physiology 289, no. 2 (August 2005): G320—G331. http://dx.doi.org/10.1152/ajpgi.00002.2005.
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Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.
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Medina, Julia I., Eliud Hernández, Cornelis Vlaar, and Suranganie Dharmawardhane. "Abstract 347: Comparative activity of MBQ-167 metabolites in metastatic breast cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 347. http://dx.doi.org/10.1158/1538-7445.am2022-347.
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Abstract Metastasis continues to be the most difficult phase for cancer treatment due to few targeted therapy options. Therefore, we focus on targeting the Rho GTPases Rac1 and Cdc42, key drivers of the primary steps of metastasis: cell migration and invasion. We developed MBQ-167 as a small molecule dual inhibitor for Rac1 and Cdc42 activation with IC50s of 103 and 78 nM, respectively. In metastatic human breast cancer cells, MBQ-167 inhibits the Rac/Cdc42 downstream effector p21-activated kinase (PAK)/LIM kinase/Cofilin signaling, lamellipodia extension, and thus, cell polarity, and migration. In addition, in metastatic breast cancer cells, MBQ-167 inhibits cell proliferation and cell cycle progression to ultimately induce apoptosis via anoikis. MBQ-167 inhibits tumor growth and metastasis in experimental and spontaneous HER-positive and Triple negative breast cancer (TNBC) mouse models. MBQ-167 has 35% bioavailability and is not toxic in rodent or canine models up to 1000 mg/kg. Therefore, we are further developing MBQ-167 as a lead anti metastatic cancer compound. The objective in this study is to elucidate potential activity of MBQ-167 metabolites. Prior work has identified MBQ-167 metabolites via LC MS/MS from a mammalian liver microsome (rats and human) assay. Four of the MBQ-167 metabolites with the highest MS peak area, were analyzed in metastatic breast cancer cell lines, for cell viability via MTT assays and apoptosis via caspase 3/7 assays. We report that the metabolites M6, M7, M8, and M9 did not inhibit cell viability or induce apoptosis at 250 or 500 nM in HER2-type MDA-MB-435, and TNBC MDA-MB-231 and MDA-MB-468 breast cancer cell lines, compared to MBQ-167. We next analyzed whether these metabolites could inhibit the activation of Rac and Cdc42 by analyzing the phosphorylation status of its downstream effector p-21 activated kinase isoforms 1/2/3 (PAK1/2/3). When MBQ-167 and metabolites at 250nM were incubated for 24h in HER2-type MDA-MB-435 metastatic cancer cells, and the lysates western blotted with antibodies to total PAK1/2/3 or phospho-Pak1/2/3, we found a decrease in the phosphorylation levels of PAK in response to MBQ-167 and metabolite M6. However, since the peak area of M6 in the microsome assay is only 6% compared to MBQ-167, we conclude that MBQ-167 is the major active compound with anti-metastatic cancer properties. Citation Format: Julia I. Medina, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane. Comparative activity of MBQ-167 metabolites in metastatic breast cancer [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 347.
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Shi, Geng-Xian, Won Seok Yang, Ling Jin, Michelle L. Matter, and Joe W. Ramos. "RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases." Proceedings of the National Academy of Sciences 115, no. 2 (December 26, 2017): E190—E199. http://dx.doi.org/10.1073/pnas.1708584115.
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Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 or Cdc42. We further show that epidermal growth factor or FBS stimulation induces association of endogenous RSK2 with LARG and LARG with RhoA. In response to these stimuli, RSK2 phosphorylates LARG at Ser1288 and thereby activates RhoA. Phosphorylation of RSK2 at threonine 577 is essential for activation of LARG-RhoA. Moreover, RSK2-mediated motility signaling depends on RhoA and -B, but not RhoC. These results establish a unique RSK2-dependent LARG-RhoA signaling module as a central organizer of directed cell migration and invasion.
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Greciano, Patricia G., Jose V. Moyano, Mary M. Buschmann, Jun Tang, Yue Lu, Jean Rudnicki, Aki Manninen, and Karl S. Matlin. "Laminin 511 partners with laminin 332 to mediate directional migration of Madin–Darby canine kidney epithelial cells." Molecular Biology of the Cell 23, no. 1 (January 2012): 121–36. http://dx.doi.org/10.1091/mbc.e11-08-0718.
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Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front–rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin–Darby canine kidney cells by suppressing expression of their α subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell–cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin α3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin α5 and laminin α3 restores directional migration and cell–cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins.
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Itakura, Asako, Joseph E. Aslan, Branden T. Kusanto, Kevin G. Phillips, Robert H. Insall, Jonathan Chernoff, and Owen J. T. McCarty. "p21-Activated Kinases Regulate Directional Migration and Cytoskeletal Organization in Human Neutrophils." Blood 120, no. 21 (November 16, 2012): 834. http://dx.doi.org/10.1182/blood.v120.21.834.834.
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Abstract Abstract 834 Neutrophil chemotaxis is controlled by coordinated processes of directional sensing, polarization and motility. This study was designed to characterize the role of p21-activated kinases (PAKs) during the chemotaxis of human primary neutrophils. PAKs are known as effectors of the Rho GTPases Rac and Cdc42. It has been shown that PAK1 and PAK2 are strongly activated downstream of the f-Met-Leu-Phe (fMLP) receptor via Rac (Huang et al., MCB 1998). PAK1 is known to localize in lamellipodia at the leading edge of human neutrophils (Dharmawardhane et al., JLB 1999) and mediate persistent directional migration via Cdc42 in a neutrophil-like cell line (Li et al., Cell 2003). However, little is known about the specific role of PAK isoforms in spatial/temporal regulation of cytoskeletal dynamics in human neutrophils. Our data show that human neutrophils express PAK1, 2 and 4. Under an fMLP gradient, human neutrophils developed morphological polarity with a distinct leading edge and rear, and migrated up the fMLP gradient at the speed of 7.5 ± 0.56 μm/min. Inhibition of Rac or PI3K impaired directionality but did not significantly affect migration speed of chemotaxing neutrophils (6.3 ± 0.56 μm/min or 6.2 ± 0.85 μm/min, respectively). In contrast, neutrophils treated with the PAK inhibitor, PF3758309 (PF), displayed random migration, less polarization and reduced motility (3.1 ± 0.21 μm/min). These results suggest that PAK regulates neutrophil chemotaxis independently of the Rac-PI3K axis. The presence of PF did not abrogate intracellular Ca2+mobilization in fMLP-driven chemotactic condition. Instead, the decreased migratory ability by PAK inhibition was associated with multiple Ca2+ spikes. Immunofluorescence imaging shows that PAK2 but not PAK1, was phosphorylated and translocated from cytosol to actin-rich leading edge in the proximity to GTP-bound Rac within 3 min of fMLP stimulation. Notably, PF treatment resulted in partial neutrophil spreading and actin/myosin II translocation in the absence of extracellular stimuli, suggesting that basal level of PAK phosphorylation may be required for cytoskeletal integrity of resting neutrophils. Neutrophils pretreated with PF displayed less activation and translocation of PAK2 and Rac. In summary, our data demonstrate for the first time the distinct roles of PAK isoforms in human neutrophil morphological polarity and directional migration and suggest that PAK2 is activated downstream of fMLP receptor through Rho-family small GTPases. Disclosures: No relevant conflicts of interest to declare.
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Rullo, Jacob, Henry Becker, Sharon J. Hyduk, Janice C. Wong, Genevieve Digby, Pamma D. Arora, Adrianet Puig Cano, John Hartwig, Christopher A. McCulloch, and Myron I. Cybulsky. "Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion." Journal of Cell Biology 197, no. 1 (April 2, 2012): 115–29. http://dx.doi.org/10.1083/jcb.201107140.
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Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces.
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Brachmann, Saskia M., Claudine M. Yballe, Metello Innocenti, Jonathan A. Deane, David A. Fruman, Sheila M. Thomas, and Lewis C. Cantley. "Role of Phosphoinositide 3-Kinase Regulatory Isoforms in Development and Actin Rearrangement." Molecular and Cellular Biology 25, no. 7 (April 1, 2005): 2593–606. http://dx.doi.org/10.1128/mcb.25.7.2593-2606.2005.
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ABSTRACT Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85α gene (p85α−/− p55α−/− p50α−/−) or in mice lacking the p85β gene (p85β−/−) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor α null (PDGFRα−/−) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRα signaling at this developmental stage. p85α−/− p55α+/+ p50α+/+ p85β−/− mice had similar but less severe defects, indicating that p85α and p85β have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85α and p85β gene products (p85α−/− p55α−/− p50α−/− p85β−/−) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85α or p85β rescues the membrane ruffling defect. Surprisingly, reintroduction of p50α also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50α, are not required for this response.
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Lee, Joo Youn, Ji-Sook Yun, Woo-Keun Kim, Hang-Suk Chun, Hyeonseok Jin, Sungchan Cho, and Jeong Ho Chang. "Structural Basis for the Selective Inhibition of Cdc2-Like Kinases by CX-4945." BioMed Research International 2019 (August 18, 2019): 1–10. http://dx.doi.org/10.1155/2019/6125068.
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Cdc2-like kinases (CLKs) play a crucial role in the alternative splicing of eukaryotic pre-mRNAs through the phosphorylation of serine/arginine-rich proteins (SR proteins). Dysregulation of this processes is linked with various diseases including cancers, neurodegenerative diseases, and many genetic diseases. Thus, CLKs have been regarded to have a potential as a therapeutic target and significant efforts have been exerted to discover an effective inhibitor. In particular, the small molecule CX-4945, originally identified as an inhibitor of casein kinase 2 (CK2), was further revealed to have a strong CLK-inhibitory activity. Four isoforms of CLKs (CLK1, CLK2, CLK3, and CLK4) can be inhibited by CX-4945, with the highest inhibitory effect on CLK2. This study aimed to elucidate the structural basis of the selective inhibitory effect of CX-4945 on different isoforms of CLKs. We determined the crystal structures of CLK1, CLK2, and CLK3 in complex with CX-4945 at resolutions of 2.4 Å, 2.8 Å, and 2.6 Å, respectively. Comparative analysis revealed that CX-4945 was bound in the same active site pocket of the CLKs with similar interacting networks. Intriguingly, the active sites of CLK/CX-4945 complex structures had different sizes and electrostatic surface charge distributions. The active site of CLK1 was somewhat narrow and contained a negatively charged patch. CLK3 had a protruded Lys248 residue in the entrance of the active site pocket. In addition, Ala319, equivalent to Val324 (CLK1) and Val326 (CLK2), contributed to the weak hydrophobic interactions with the benzonaphthyridine ring of CX-4945. In contrast, the charge distribution pattern of CLK2 was the weakest, favoring its interactions with benzonaphthyridine ring. Thus, the relatively strong binding affinities of CX-4945 with CLK2 are consistent with its strong inhibitory effect defined in the previous study. These results may provide insights into structure-based drug discovery processes.
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Koh, Cheng-Gee, Ed Manser, Zhou-Shen Zhao, Chee-Peng Ng, and Louis Lim. "β1PIX, the PAK-interacting exchange factor, requires localization via a coiled-coil region to promote microvillus-like structures and membrane ruffles." Journal of Cell Science 114, no. 23 (December 1, 2001): 4239–51. http://dx.doi.org/10.1242/jcs.114.23.4239.
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PIX is a Rho-family guanine nucleotide exchange factor that binds PAK. We previously described two isoforms of PIX that differ in their N termini. Here, we report the identification of a new splice variant of βPIX, designated β2PIX, that is the dominant species in brain and that lacks the region of ∼120 residues with predicted coiled-coil structure at the C terminus of β1PIX. Instead, β2PIX contains a serine-rich C terminus. To determine whether these splice variants differ in their cellular function, we studied the effect of expressing these proteins in HeLa cells. We found that the coiled-coil region plays a key role in the localization of β1PIX to the cell periphery and is also responsible for PIX dimerization. Overexpression of β1, but not β2PIX, drives formation of membrane ruffles and microvillus-like structures (via activation of Rac1 and Cdc42, respectively), indicating that its function requires localized activation of these GTPases. Thus, β1PIX, like other RhoGEFs, exerts specific morphological functions that are dependent on its intracellular location and are mediated by its C-terminal dimerization domain.
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CALOCA, Maria Jose, HongBin WANG, and Marcelo G. KAZANIETZ. "Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor." Biochemical Journal 375, no. 2 (October 15, 2003): 313–21. http://dx.doi.org/10.1042/bj20030727.
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The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.
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Gabrielli, B. G., C. P. De Souza, I. D. Tonks, J. M. Clark, N. K. Hayward, and K. A. Ellem. "Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells." Journal of Cell Science 109, no. 5 (May 1, 1996): 1081–93. http://dx.doi.org/10.1242/jcs.109.5.1081.
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The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.
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Sarowar, Tasnuva, and Andreas M. Grabrucker. "Actin-Dependent Alterations of Dendritic Spine Morphology in Shankopathies." Neural Plasticity 2016 (2016): 1–15. http://dx.doi.org/10.1155/2016/8051861.
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Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains—the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, andβPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiplein vitroandin vivomodels. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.
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Llano, Olaya, Sergey Smirnov, Shetal Soni, Andrey Golubtsov, Isabelle Guillemin, Pirta Hotulainen, Igor Medina, Hans Gerd Nothwang, Claudio Rivera, and Anastasia Ludwig. "KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX." Journal of Cell Biology 209, no. 5 (June 8, 2015): 671–86. http://dx.doi.org/10.1083/jcb.201411008.
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Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.
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Driggers, Paul H., James H. Segars, and Domenica M. Rubino. "The Proto-oncoprotein Brx Activates Estrogen Receptor β by a p38 Mitogen-activated Protein Kinase Pathway." Journal of Biological Chemistry 276, no. 50 (September 28, 2001): 46792–97. http://dx.doi.org/10.1074/jbc.m106927200.
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The estrogen receptors (ERs) are ligand-inducible transcription factors that play key roles in the control of growth and differentiation in reproductive tissues. We showed that the novel Dbl family proto-oncoprotein Brx enhances ligand-dependent activity of ERα via a Cdc42-dependent pathway. Brx also significantly enhances ligand-dependent activity of ERβ. This enhancement is not affected by inhibition of p44/42 mitogen-activated protein kinase (MAPK) activation by PD98059. However, addition of the p38 MAPK inhibitor SB202190 abrogates the enhancement of ERβ activity by Brx, showing that p38 MAPK activity is required for the enhancement of ERβ function by Brx. In COS-7 cells, transfection of Brx leads to activation of endogenous p38 MAPK activity. Co-expression of the β2 isoform of human p38 MAPK and a constitutively active form of the p38 MAPK kinase MKK6 (MKK6-EE) synergistically augments ligand-dependent activity of ERβ. Our findings suggest that p38 MAPKs may be important regulators of ERβ activity.
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Reed, Steven I. "Cooperation between Different Cdc4/Fbw7 Isoforms May Be Associated with 2-Step Inactivation of SCFCdc4 Targets." Cell Cycle 5, no. 17 (August 2, 2006): 1923–24. http://dx.doi.org/10.4161/cc.5.17.3198.
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Kiso, Marina, Sunao Tanaka, Shigehira Saji, Masakazu Toi, and Fumiaki Sato. "Long isoform of VEGF stimulates cell migration of breast cancer by filopodia formation via NRP1/ARHGAP17/Cdc42 regulatory network." International Journal of Cancer 143, no. 11 (October 9, 2018): 2905–18. http://dx.doi.org/10.1002/ijc.31645.
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Advani, Sunil J., Ryan Hagglund, Ralph R. Weichselbaum, and Bernard Roizman. "Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize." Journal of Virology 75, no. 17 (September 1, 2001): 7904–12. http://dx.doi.org/10.1128/jvi.75.17.7904-7912.2001.
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ABSTRACT The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We examined by two-dimensional separations the posttranslational forms of ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL) fibroblasts infected with wild-type virus, mutant R325, lacking the sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid 199 of ICP0 was replaced by alanine. We report the following (i) Both ICP0 and ICP4 were sequentially posttranslationally modified at least until 12 h after infection. In HEL fibroblasts, the processing of ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A′ form noted at 2 h to B′ and C′ forms noted at 4 h to the additional D′ and E′ forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii) Although the overall patterns were similar, the mobility of proteins made in HEp-2 cells differed from those made in HEL fibroblasts. (iii) The processing of ICP0 forms E and F was blocked in HEL fibroblasts infected with R325 or with wild-type virus and treated with roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2, cdk2, and cdk5. R325-infected HEp-2 cells lacked the D′ form of ICP4, and roscovitine blocked the appearance of the most highly charged E′ form of ICP4. (iv) A characteristic of ICP0 is that it is translocated into the cytoplasm of HEL fibroblasts between 5 and 9 h after infection. Addition of MG132 to the cultures late in infection resulted in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure of HEL fibroblasts to MG132 late in infection resulted in the disappearance of the highly charged ICP0 G isoform. The G form of ICP0 was also absent in cells infected with R7914 mutant. In cells infected with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last, cdc2 was active in infected cells, and this activity was inhibited by roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and may represent a contaminating kinase activity. We conclude from these results that the ICP0 G isoform is the cytoplasmic form, that it may be phosphorylated by cdc2, consistent with evidence published earlier (S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc. Natl. Acad. Sci. USA 96:10996–11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by R325 and roscovitine. The latter result suggests that ICP4 may also be a substrate of cdc2 late in infection. Last, additional modifications are superimposed by cell-type-specific enzymes.
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Boyer, Laurent, Laurent Turchi, Benoit Desnues, Anne Doye, Gilles Ponzio, Jean-Louis Mege, Motozo Yamashita, et al. "CNF1-induced Ubiquitylation and Proteasome Destruction of Activated RhoA Is Impaired in Smurf1−/−Cells." Molecular Biology of the Cell 17, no. 6 (June 2006): 2489–97. http://dx.doi.org/10.1091/mbc.e05-09-0876.
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Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-β trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.
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Furukawa, Masayuki, Jörg Ebmeyer, Kwang Pak, Darrell A. Austin, Åsa Melhus, Nicholas J. G. Webster, and Allen F. Ryan. "Jun N-Terminal Protein Kinase Enhances Middle Ear Mucosal Proliferation during Bacterial Otitis Media." Infection and Immunity 75, no. 5 (February 26, 2007): 2562–71. http://dx.doi.org/10.1128/iai.01656-06.
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ABSTRACT Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation.
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Makhoul, Christian, Prajakta Gosavi, Regina Duffield, Bronwen Delbridge, Nicholas A. Williamson, and Paul A. Gleeson. "Intersectin-1 interacts with the golgin GCC88 to couple the actin network and Golgi architecture." Molecular Biology of the Cell 30, no. 3 (February 2019): 370–86. http://dx.doi.org/10.1091/mbc.e18-05-0313.
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The maintenance of the Golgi ribbon relies on a dynamic balance between the actin and microtubule networks; however, the pathways controlling actin networks remain poorly defined. Previously, we showed that the trans-Golgi network (TGN) membrane tether/golgin, GCC88, modulates the Golgi ribbon architecture. Here, we show that dispersal of the Golgi ribbon by GCC88 is dependent on actin and the involvement of nonmuscle myosin IIA. We have identified the long isoform of intersectin-1 (ITSN-1), a guanine nucleotide exchange factor for Cdc42, as a novel Golgi component and an interaction partner of GCC88 responsible for mediating the actin-dependent dispersal of the Golgi ribbon. We show that perturbation of Golgi morphology by changes in membrane flux, mediated by silencing the retromer subunit Vps26, or in a model of neurodegeneration, induced by Tau overexpression, are also dependent on the ITSN-1-GCC88 interaction. Overall, our study reveals a role for a TGN golgin and ITSN-1 in linking to the actin cytoskeleton and regulating the balance between a compact Golgi ribbon and a dispersed Golgi, a pathway with relevance to pathophysiological conditions.
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Carréno, Sébastien, Emmanuelle Caron, Céline Cougoule, Laurent J. Emorine, and Isabelle Maridonneau-Parini. "p59Hck Isoform Induces F-actin Reorganization to Form Protrusions of the Plasma Membrane in a Cdc42- and Rac-dependent Manner." Journal of Biological Chemistry 277, no. 23 (March 19, 2002): 21007–16. http://dx.doi.org/10.1074/jbc.m201212200.
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Azab, Feda, Shireen Vali, Nicholas Potter, Joseph Abraham, Barbara Muz, Pilar De La Puente, Taher Abbasi, Ravi Vij, and Abdel Kareem Azab. "PI3K-Alpha Plays A Major Role In Multiple Myeloma and Its Inhibition With BYL917 Decreases Proliferation, Synergizes With Other Therapies and Overcomes Stroma-Induced Resistance." Blood 122, no. 21 (November 15, 2013): 3215. http://dx.doi.org/10.1182/blood.v122.21.3215.3215.
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Abstract Introduction Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is still incurable. The PI3K pathway is activated and correlated with drug resistance in MM. Pan-inhibition of the PI3K pathway resulted in serious side effects, therefore, we focused on specific inhibition of the PI3K-alpha isoform. We investigated the role of PI3K- alpha in the progression and drug resistance of MM, by inhibiting the PI3K- alpha with a novel specific inhibitor BYL719. This is the first study to describe the preclinical effect of BYL719 on MM in particular and in hematologic malignancies in general. Methods and Results We analyzed the gene expression of PI3K isoforms in MM patients based on published datasets from the Gene Expression Omnibus by Zhan et al and found that PI3K-alpha and beta isoforms were highly expressed in MM. However, the fold-change of expression of the alpha isoform in MM patients was higher than the beta isoform, compared to healthy subjects. BYL719 inhibited the survival of MM cells isolated from three MM patients by MTT, at an IC50 around 1uM, but had no effect on normal PBMCs. Furthermore, BYL719 inhibited survival of all MM cell lines in a different manner. In silico-predicted activity of PI3K-alpha in the cell lines was exponentially correlated with the killing induced by the PI3K inhibitor BYL719 in vitro. BYL719 significantly decreased the activation of the PI3K signaling related proteins (pAKT, pS6R, and pGSK) by western blotting. Moreover, BYL719 inhibited cell cycle of MM cells detected by PI staining showed induction of G1-phase cell cycle arrest. BYL719 inhibited MM proliferation of MM1s cells in a dose dependent manner, through decreasing the levels of pCyclin-E1 and pRb, and increasing of P27 levels. The results were in agreement with the in silico studies that predicted the inhibition of other cell-cycle proteins including CDK4-Cyclin D complex, Myc-Max complex and CDK1-Cyclin B complex by BYL719 in a dose dependent manner. Moreover, BYL719 (0-2.5uM) increased the fraction of apoptotic MM cells in a dose depended manner, as detected by Annexin/PI staining. BYL719 induced apoptosis signaling by inducing the cleavage of Caspase-3, Caspase-9 and PARP by western blotting, in a dose dependent manner. We tested the effect of the combination of BYL719 with other drugs (Bortezomib and Carfilzomib) on survival of MM cells by MTT. We found that the combination of the two drugs decreased the surviving fraction of MM cells more than each of the drugs alone. Mechanistically, Bortezomib increased pAKT and pS6R as a resistance mechanism, and BYL719 abolished the Bortezomib-induced increase of pAKT and pS6R. Moreover, BYL719 enhanced the activation of pJNK induced by Carfilzomib and the combination of each drug increased cleavage of PARP, caspase-3 and caspase-9 more than each of the drugs alone. We tested the effect of BYL719 on the interaction of MM cells with BM stromal cells (BMSCs), and it was found that BYL719 decreased the adhesion of MM cells to BMSCs in a dose-dependent manner. Mechanistically, BYL719 decreased the activation of adhesion signaling such as pFAK, pSRC and pCofilin in a dose dependent manner. The in silico studies predicted the inhibition of the small GTPases Rho, Rac and Cdc42. To test the effect of BYL719 on drug resistance induced by the BM stroma, MM cells were co-Cultured with BMSCs, treated with BYL719 in combination with Bortezomib or Carfilzomib. It was found that co-culture with BMSCs increased the surviving fraction of MM cells after treatment with Bortezomib and Carfilzomib, as a drug resistance mechanism. The combination of the two drugs with BYL719 overcame the resistance induced by the stroma and reduced the surviving fraction to the values observed for treatment without presence of stroma. Conclusion This is the first study to describe preclinical effect of BYL719 on MM in particular and hematologic malignancies in general. The PI3K-alpha isoform plays a major role in the progression and drug resistance in MM cells, and it's inhibition with BYL719 reduces proliferation, inhibits cell cycle and induces apoptosis in MM cells. Moreover, it showed that BYL719 synergizes with Bortezomib and Carfilzomib, and overcomes drug resistance induced by BM stroma. Our findings provide a preclinical basis of future clinical trial of BYL719 in MM as a single agent or in combination with other drugs. Disclosures: Vali: Cellworks Group Inc., San Jose, CA, USA: Employment. Abbasi:Cellworks Group Inc: Employment.
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Romanelli, Angela, Kathleen A. Martin, Alex Toker, and John Blenis. "p70 S6 Kinase Is Regulated by Protein Kinase Cζ and Participates in a Phosphoinositide 3-Kinase-Regulated Signalling Complex." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 2921–28. http://dx.doi.org/10.1128/mcb.19.4.2921.
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ABSTRACT p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCζ as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCζ mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCζ mutant (myristoylated PKCζ [myr-PKCζ]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCζ. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCζ in vivo in a growth factor-independent manner, while PDK-1 and PKCζ can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.
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Zeng, Q., D. Lagunoff, R. Masaracchia, Z. Goeckeler, G. Cote, and R. Wysolmerski. "Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II." Journal of Cell Science 113, no. 3 (February 1, 2000): 471–82. http://dx.doi.org/10.1242/jcs.113.3.471.
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The p21-activated kinase (PAK) family includes several enzyme isoforms regulated by the GTPases Rac1 and Cdc42. PAK1, found in brain, muscle and spleen, has been implicated in triggering cytoskeletal rearrangements such as the dissolution of stress fibers and reorganization of focal complexes. The role of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monophosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2's morphological and biochemical effect on intact endothelial cells utilizing microinjection of constitutively active PAK2. Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial cell margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2. Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specific myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preventing the changes in morphology and the actin cytoskeleton. The additional finding that endogenous PAK2 associates with myosin II is consistent with the proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 monophosphorylation of the regulatory light chain.
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Peng, Hong-Juan, Karen M. Henkels, Madhu Mahankali, Mary C. Dinauer, and Julian Gomez-Cambronero. "Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2." Journal of Biological Chemistry 286, no. 18 (March 4, 2011): 16308–20. http://dx.doi.org/10.1074/jbc.m110.206672.
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Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.
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Bruneau, Alix, Jean-Louis Delaunay, Anne-Marie Durand-Schneider, Virginie Vauthier, Amel Ben Saad, Lynda Aoudjehane, Haquima El Mourabit, et al. "MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression." Cells 11, no. 4 (February 10, 2022): 617. http://dx.doi.org/10.3390/cells11040617.
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ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.