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Sylvain Meloche - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii stimulates serine phosphorylation of the adaptor protein nck physical association with the serine threonine kinases pak1 and casein kinase i
Biochemical Journal, 1999Co-Authors: Laure Voisin, Louise Larose, Sylvain MelocheAbstract:Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
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Angiotensin II stimulates serine phosphorylation of the adaptor protein Nck: physical association with the serine/threonine kinases Pak1 and casein kinase I.
Biochemical Journal, 1999Co-Authors: Laure Voisin, Louise Larose, Sylvain MelocheAbstract:Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
Laure Voisin - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii stimulates serine phosphorylation of the adaptor protein nck physical association with the serine threonine kinases pak1 and casein kinase i
Biochemical Journal, 1999Co-Authors: Laure Voisin, Louise Larose, Sylvain MelocheAbstract:Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
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Angiotensin II stimulates serine phosphorylation of the adaptor protein Nck: physical association with the serine/threonine kinases Pak1 and casein kinase I.
Biochemical Journal, 1999Co-Authors: Laure Voisin, Louise Larose, Sylvain MelocheAbstract:Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
D S Waddell - One of the best experts on this subject based on the ideXlab platform.
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Ligand-dependent ubiquitination of Smad3 is regulated by casein kinase 1 gamma 2, an inhibitor of TGF-β signaling
Oncogene, 2008Co-Authors: D S Waddell, W Wang, Z Wang, N T Liberati, S Yong, X-f WangAbstract:Transforming growth factor-beta (TGF-β) elicits a variety of cellular activities primarily through a signaling cascade mediated by two key transcription factors, Smad2 and Smad3. Numerous regulatory mechanisms exist to control the activity of Smad3, thereby modulating the strength and specificity of TGF-β responses. In search for potential regulators of Smad3 through a yeast two-hybrid screen, we identified casein kinase 1 gamma 2 (CKIγ2) as a novel Smad3-interacting protein. In mammalian cells, CKIγ2 selectively and constitutively binds Smad3 but not Smad1, -2 or -4. Functionally, CKIγ2 inhibits Smad3-mediated TGF-β responses including induction of target genes and cell growth arrest, and this inhibition is dependent on CKIγ2 kinase activity. Mechanistically, CKIγ2 does not affect the basal levels of Smad proteins or activity of the receptors. Rather, CKIγ2 preferentially promotes the ubiquitination and degradation of activated Smad3 through direct phosphorylation of its MH2 domain at Ser418. Importantly, mutation of Ser418 to alanine or aspartic acid causes an increase or decrease of Smad3 activity, respectively, in the presence of TGF-β. CKIγ2 is the first kinase known to mark activated Smad3 for destruction. Given its negative function in TGF-β signaling and its reported overexpression in human cancers, CKIγ2 may act as an oncoprotein during tumorigenesis.
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ligand dependent ubiquitination of smad3 is regulated by casein kinase 1 gamma 2 an inhibitor of tgf beta signaling
Oncogene, 2008Co-Authors: D S Waddell, W Wang, Z Wang, N T Liberati, S Yong, Xiaofan WangAbstract:Transforming growth factor-beta (TGF-β) elicits a variety of cellular activities primarily through a signaling cascade mediated by two key transcription factors, Smad2 and Smad3. Numerous regulatory mechanisms exist to control the activity of Smad3, thereby modulating the strength and specificity of TGF-β responses. In search for potential regulators of Smad3 through a yeast two-hybrid screen, we identified casein kinase 1 gamma 2 (CKIγ2) as a novel Smad3-interacting protein. In mammalian cells, CKIγ2 selectively and constitutively binds Smad3 but not Smad1, -2 or -4. Functionally, CKIγ2 inhibits Smad3-mediated TGF-β responses including induction of target genes and cell growth arrest, and this inhibition is dependent on CKIγ2 kinase activity. Mechanistically, CKIγ2 does not affect the basal levels of Smad proteins or activity of the receptors. Rather, CKIγ2 preferentially promotes the ubiquitination and degradation of activated Smad3 through direct phosphorylation of its MH2 domain at Ser418. Importantly, mutation of Ser418 to alanine or aspartic acid causes an increase or decrease of Smad3 activity, respectively, in the presence of TGF-β. CKIγ2 is the first kinase known to mark activated Smad3 for destruction. Given its negative function in TGF-β signaling and its reported overexpression in human cancers, CKIγ2 may act as an oncoprotein during tumorigenesis.
Louise Larose - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii stimulates serine phosphorylation of the adaptor protein nck physical association with the serine threonine kinases pak1 and casein kinase i
Biochemical Journal, 1999Co-Authors: Laure Voisin, Louise Larose, Sylvain MelocheAbstract:Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
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Angiotensin II stimulates serine phosphorylation of the adaptor protein Nck: physical association with the serine/threonine kinases Pak1 and casein kinase I.
Biochemical Journal, 1999Co-Authors: Laure Voisin, Louise Larose, Sylvain MelocheAbstract:Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cells. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21-activated kinase 1 (Pak1), which was found to form a stable complex with Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
David M Virshup - One of the best experts on this subject based on the ideXlab platform.
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nuclear entry of the circadian regulator mper1 is controlled by mammalian casein kinase i ɛ
Molecular and Cellular Biology, 2000Co-Authors: Erica Vielhaber, Erik J Eide, Ann Rivers, David M VirshupAbstract:The circadian rhythm is an intrinsic 24-h cycle that, in species from Neurospora to humans, is generated by an intracellular oscillating negative feedback loop that controls the periodic transcription of both regulatory and output genes. The molecular mechanism generating the circadian rhythm has been the object of intense study (reviewed in references 12, 42, and 57). Genetic investigations in the fruit fly Drosophila melanogaster, augmented by studies of circadian rhythm mutants in mammals, have led to a rapidly evolving understanding of the workings of the metazoan central clock. In Drosophila, a heterodimeric transcription factor composed of CLOCK and CYCLE binds to E-box-containing promoters and drives expression of the negative regulators PERIOD (dPER) and TIMELESS (dTIM). dPER and dTIM accumulate in the cytoplasm until they heterodimerize. Heterodimerization serves to mask their cytoplasmic localization domains, allowing the complex to enter the nucleus (46). Nuclear dPER-dTIM heterodimers repress the activity of the CLOCK/CYCLE transcription factor, thus causing a decrease in dPER and dTIM expression (10). Although the mammalian circadian clock is composed of proteins homologous to those found in Drosophila, the mechanisms for regulating circadian rhythm in mammals appear to be more complex and in many aspects quite different from those in Drosophila. The increased complexity in the mammalian system is due in part to the expansion of the per gene family. Three mammalian period genes have been cloned; all are rhythmically expressed in the anatomic location of the central clock, the suprachiasmatic nucleus (SCN), as well as in diverse peripheral tissues (including heart, liver, and muscle) and cultured fibroblasts, with peak levels of transcripts detected during the circadian day in the mouse (1, 2, 47, 55, 62). The three mper genes differ in their transcriptional regulation. Several reports suggest that mper1 is expressed 4 to 8 h before mper2 and mper3 (1, 25, 53). Regulated nuclear entry of the PER proteins is a common element in many but not all (50) of the observed circadian regulators (12, 42, 57). In the mouse, periodic nuclear accumulation of mPER1 protein has been demonstrated in the mouse SCN, peaking 4 to 6 h after mper1 mRNA expression (19). How and if mPER nuclear entry is regulated is less clear. In the murine system, heterodimerization of mPER proteins with mTIM has been controversial, being found by some but not all observers (48, 54, 61). However, each of the mPER proteins can homodimerize with itself and heterodimerize with other mPER proteins. Forced expression of mPER proteins alone can partially repress CLOCK/BMAL1-activated transcription (BMAL1 is the mammalian homolog of CYCLE) in the absence of coexpressed mTIM (25, 48). Recently, Kume et al. (31) reported that coexpression of cryptochrome proteins mCRY1 and mCRY2 facilitated the nuclear entry of mPER proteins and fully repressed transcription from CLOCK/BMAL1-driven promoters. Thus, PER nuclear entry seems to be periodic and regulated in mammals as well as in Drosophila. Phosphorylation of the components of the circadian clock has been postulated to regulate the duration of the cycle. Treatment of the dinoflagellate Gonyaulax polyedra with either serine/threonine phosphatase or kinase inhibitors alters its circadian rhythm (6, 7). The frequency gene product, a negative regulator of the Neurospora circadian clock, is rhythmically phosphorylated (12), and its phosphorylation regulates both its stability and period duration (34). dCLOCK, dPER, and dTIM are phosphoproteins, and the level of dPER and dTIM phosphorylation increases steadily from the time of their synthesis until their degradation at dawn (13, 32). The first genetic evidence that a specific protein kinase regulates the Drosophila circadian rhythm was the discovery of the novel gene double-time (dbt), encoding a protein serine/threonine kinase (27, 41). dbt is coexpressed with per and tim in the fly brain lateral neurons that regulate circadian rhythm. Different missense alleles of dbt cause marked lengthening or shortening of the circadian period, while homozygosity for the null allele causes pupal lethality (27). Examination of flies with mutations in the dbt gene led Kloss and coworkers (27) to conclude that the DBT kinase phosphorylated and regulated dPER. Drosophila larvae homozygous for the dbt-null allele manifest several distinct phenotypes. First, they accumulate high levels of dPER but not dTIM, suggesting a role for phosphorylation in the degradation of dPER. Second, the dPER that accumulates is hypophosphorylated, indicating a major role for DBT in the phosphorylation of dPER. In a final indication that DBT directly regulates dPER, DBT binds to an amino-terminal fragment of dPER (27). Drosophila DBT is most similar in sequence (86% identical) in its kinase domain to the kinase domains of mammalian casein kinase I ɛ and δ (CKIɛ and CKIδ). The CKI gene family encodes a number of widely expressed kinases that localize to membranes, cytoplasm, and nucleus; and various members of the CKI family have been identified in plants, fungi, and mammals (14, 18, 44, 45). CKIɛ and CKIδ belong to a branch of the family that includes the yeast kinases HRR25 and Hhp1 and Hhp2, implicated in the response to DNA damage (11, 14, 21). Mammalian CKIδ and CKIɛ have closely related 123-amino-acid carboxy-terminal domains that can autoregulate kinase activity in a phosphorylation-dependent manner (5, 16, 17, 44). However, the carboxy-terminal domains of DBT and CKIɛ are unrelated. Accumulating evidence suggests CKI family members can regulate the intracellular localization of specific substrates. For example, mammalian CKIα (71% identical to DBT over the kinase domain) binds to, phosphorylates, and inhibits the nuclear import of the transcription factor NF-AT4 (60). In Drosophila, a CKIα homolog shuttles from the cytoplasm into the larval nuclei in response to gamma irradiation (49). Finally, one of the few identified substrates of HRR25 is the yeast transcriptional regulator SWI6, a protein whose cytoplasmic retention is dependent on phosphorylation (20, 52). Given the differences between the Drosophila and mammalian PER and TIM proteins and the higher level of complexity in the regulation and interactions of the mPER proteins, the interaction between CKI and the mammalian mPER1 protein was investigated. We find that specific and closely related isoforms of CKI bind to and phosphorylate mPER1 both in vitro and in vivo. Unexpectedly, overexpressed mPER1 was found to accumulate in the nuclei of transfected HEK 293 cells. Two distinct mechanisms appear to be capable of regulating mPER1 nuclear entry. First, coexpressed mPER2 prevents mPER1 nuclear accumulation. Second, CKIɛ or CKI∂ coexpression blocks mPER1 nuclear accumulation in a kinase-dependent manner, by masking its nuclear localization signal (NLS). These results suggest that a critical function of both mPER2 and CKI in circadian rhythm is to control the nuclear entry of mPER1.
