Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1-225, including the Ca2+/calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the serotonin-induced ERK1/2 phosphorylation was Ca2+-dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras-dependent ERK1/2 activation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.
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PMID:Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells. 1585 14

Inhibition of general transcription and translation occurs during mitosis to preserve the high energy requirements needed for the dynamic structural changes that are occurring at this time of the cell cycle. Although the mitotic kinase Cdc2 appears to directly phosphorylate and inhibit key proteins directly involved in transcription and translation, the role of Cdc2 in regulating up-stream growth factor receptor-mediated signal transduction pathways is limited. In the present study, we examined mechanisms involved in uncoupling receptor-mediated activation of the extracellular signal-regulated (ERK) signaling pathway in mitotic cells. Treatment with epidermal growth factor (EGF) failed to activate the ERK pathway in mitotic cells, although partial activation of ERK could be achieved in mitotic cells treated with phorbol 12-myristate 13-acetate (PMA). The discrepancy between EGF and PMA-mediated ERK activation suggested that multiple events in the ERK pathway were regulated during mitosis. We show that Cdc2 inhibits EGF-mediated ERK activation through direct interaction and phosphorylation of several ERK pathway proteins, including the guanine nucleotide exchange factor, Sos-1, and Raf-1 kinase. Inhibition of Cdc2 activity with roscovitine in mitotic cells restored ERK activation by EGF and PMA. Similarly, mitotic inhibition of ERK activity in cells expressing active mutants of H-Ras and Raf-1 kinase could also be reversed following Cdc2 inhibition. In contrast, ERK activation in cells expressing active MEK1 was not inhibited during mitosis or affected by roscovitine. These data suggest that Cdc2 inhibits growth factor receptor-mediated ERK activation during mitosis by primarily targeting signaling proteins that are upstream of MEK1.
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PMID:Cdc2-mediated inhibition of epidermal growth factor activation of the extracellular signal-regulated kinase pathway during mitosis. 1588 52

This study demonstrates that cyclic AMP (cAMP) production is induced by lipopolysaccharide (LPS) stimulation and activates two different pathways in murine BV2 microglial cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. When cells were treated with various cAMP level modulators, nitric oxide (NO) production increased as the result of posttreatment with Type IV phosphodiesterase (PDE4) inhibitor, rolipram or dibutyryl-cAMP (dbcAMP), at 2 hr after LPS stimulation. Intracellular cAMP increased due to LPS stimulation and the cAMP modulators phosphorylate transcription factor CREB, which is enhanced in turn by posttreatment with dbcAMP. In contrast, the Epac-specific cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) activates Rap1 in the BV2 cells, but does not induce PKA activation, as judged by CREB phosphorylation. NO production was enhanced by posttreatment with dbcAMP but not by treatment with 8CPT-2Me-cAMP. This suggests that LPS-stimulated NO production is mainly PKA-dependent and also that Epac1-mediated Rap1 activation is not required for the induction of NO production.
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PMID:Epac1-mediated Rap1 activation is not required for the production of nitric oxide in BV2, murine microglial cells. 1593 67

Cyclic AMP regulates Ca(2+)-dependent exocytosis through a classical protein kinase A (PKA)-dependent and an alternative cAMP-guanine nucleotide exchange factor (GEF)/Epac-dependent pathway in many secretory cells. Although increased cAMP is believed to double secretory output in isolated pituitary cells, the direct target(s) for cAMP action and a detailed and high-time resolved analysis of the effect of intracellular cAMP levels on the secretory activity in melanotrophs are still lacking. We investigated the effect of 200 microM cAMP on the kinetics of secretory vesicle depletion in mouse melanotrophs from fresh pituitary tissue slices. The whole-cell patch-clamp technique was used to depolarize melanotrophs and increase the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Exogenous cAMP elicited an about twofold increase in cumulative membrane capacitance change and approximately 34% increase of high-voltage activated Ca(2+) channel amplitude. cAMP-dependent mechanisms did not affect [Ca(2+)](i), since the application of forskolin failed to change [Ca(2+)](i) in melanotrophs, a phenomenon readily observed in anterior lobe. Depolarization-induced secretion resulted in two distinct kinetic components: a linear and a threshold component, both stimulated by cAMP. The linear component (ATP-independent) probably represented the exocytosis of the release-ready vesicles, whereas the threshold component was assigned to the exocytosis of secretory vesicles that required ATP-dependent reaction(s) and > 800 nM [Ca(2+)](i). The linear component was modulated by 8-pCPT-2Me-cAMP (Epac agonist), while either H-89 (PKA inhibitor) or Rp-cAMPS (the competitive antagonist of cAMP binding to PKA) completely prevented the action of cAMP on the threshold component. In line with this, 6-Phe-cAMP, (PKA agonist), increased the threshold component. From our study, we suggest that the stimulation of cAMP production by application of oestrogen, as found in pregnant mice, increases the efficacy of the hormonal output through both PKA and cAMP-GEFII/Epac2-dependent mechanisms.
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PMID:cAMP increases Ca2+-dependent exocytosis through both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices. 1599 84

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.
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PMID:A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein. 1604 15

The kinase non-catalytic c-lobe domain (KIND) evolved from the catalytic protein kinase fold into a potential protein interaction module for signalling proteins. Spir family actin organizers and the non-receptor phosphatase type 13 (PTP type 13) encode a KIND domain in the very N-terminal parts of the proteins. Here we report the characterization and cloning of a third member of the KIND protein family, which we have named very-KIND (VKIND) because of its two KIND domains. Like the other members of the protein family, VKIND has a KIND domain at the N-terminus. A second KIND domain is located in the central part of the protein. The C-terminal half encodes a guanine nucleotide exchange factor motif for Ras-like GTPases (RasGEF) and a RasGEF N-terminal module (RasGEFN). There is only one VKIND gene in the mammalian genomes and up to now we have found the gene only in vertebrates. During mouse embryogenesis the VKIND gene was specifically expressed in the developing nervous system. In adult mice Northern hybridizations revealed high expression only in brain. Low expression could be detected in ovary. In situ hybridizations showed a specific expression of VKIND in neuronal cells of the granular and Purkinje cell layers of the cerebellum.
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PMID:Very-KIND is a novel nervous system specific guanine nucleotide exchange factor for Ras GTPases. 1609 29

Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.
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PMID:Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration. 1617 81

Stimulus-secretion coupling is an essential process in secretory cells in which regulated exocytosis occurs, including neuronal, neuroendocrine, endocrine, and exocrine cells. While an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) is the principal signal, other intracellular signals also are important in regulated exocytosis. In particular, the cAMP signaling system is well known to regulate and modulate exocytosis in a variety of secretory cells. Until recently, it was generally thought that the effects of cAMP in regulated exocytosis are mediated by activation of cAMP-dependent protein kinase (PKA), a major cAMP target, followed by phosphorylation of the relevant proteins. Although the involvement of PKA-independent mechanisms has been suggested in cAMP-regulated exocytosis by pharmacological approaches, the molecular mechanisms are unknown. Newly discovered cAMP-GEF/Epac, which belongs to the cAMP-binding protein family, exhibits guanine nucleotide exchange factor activities and exerts diverse effects on cellular functions including hormone/transmitter secretion, cell adhesion, and intracellular Ca(2+) mobilization. cAMP-GEF/Epac mediates the PKA-independent effects on cAMP-regulated exocytosis. Thus cAMP regulates and modulates exocytosis by coordinating both PKA-dependent and PKA-independent mechanisms. Localization of cAMP within intracellular compartments (cAMP compartmentation or compartmentalization) may be a key mechanism underlying the distinct effects of cAMP in different domains of the cell.
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PMID:PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis. 1618 14

The A-kinase anchor protein 13 (AKAP13, alias BRX and lbc) tethers cAMP-dependent protein kinase to its subcellular environment and catalyses Rho GTPases activity as a guanine nucleotide exchange factor. The crucial role of members of the Rho family of GTPases in carcinogenesis is well established and targeting Rho proteins with antineoplastic compounds has become a major effort in the fight against cancer. Thus, genetic alterations within the candidate cancer susceptibility gene AKAP13 would be expected to provoke a constitutive Rho signalling, thereby facilitating the development of cancer. Here, we analysed the potential impact of four polymorphic non-conservative amino acid exchanges (Arg494Trp, Lys526Gln, Asn1086Asp and Gly2461Ser) in AKAP13 on familial breast cancer. We performed a case-control study using genomic DNA of BRCA1/2 mutation-negative German female index patients from 601 unrelated families, among a subset of 356 high-risk families, and 1053 German female unrelated controls. The newfound Lys526Gln polymorphism revealed a significant association with familial breast cancer (OR = 1.58, 95% CI = 1.07-2.35) and an even stronger association with high-risk familial breast cancer (OR = 1.85, 95% CI = 1.19-2.88). Haplotype analyses were in line with genotype results displaying a similar significance as analyses of individual polymorphisms. Due to the pivotal role of AKAP13 in the Rho GTPases signalling network, this variant might affect the susceptibility to other cancers as well.
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PMID:Association of genetic variants in the Rho guanine nucleotide exchange factor AKAP13 with familial breast cancer. 1623 58

Rac activation is a key step in chemotaxis of hematopoietic cells, which is both positively and negatively regulated by receptors coupled to heterotrimeric G proteins. P-Rex1, a Rac-specific guanine nucleotide exchange factor, is dually activated by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) and the Gbetagamma subunits of heterotrimeric G proteins. This study explored the regulation of P-Rex1 by phosphorylation with the cAMP-dependent protein kinase (protein kinase A) in vitro and by G(i)- and G(s)-coupled receptors in HEK293T cells. P-Rex1 isolated from Sf9 and HEK293T cells migrates as two distinct bands that are partially phosphorylated. Phosphorylation of P-Rex1 with protein kinase A (PKA) inhibits the PIP(3)- and Gbetagamma-stimulated P-Rex1 guanine nucleotide exchange activity on Rac. The guanine nucleotide exchange factor activity of three different forms of P-Rex1 (native Sf9, de-phosphorylated, and phosphorylated) was examined in the presence of PIP(3) and varying concentrations of Gbeta(1)gamma(2). Gbeta(1)gamma(2) was 47-fold less potent in activating the phosphorylated form of P-Rex1 compared with the de-phosphorylated form. HEK293T cells expressing P-Rex1 were labeled with (32)P and stimulated with lysophosphatidic acid (LPA) to release Gbetagamma or isoproterenol to activate PKA. Treatment with isoproterenol or S(p)-cAMPS, a potent activator of PKA, increased the incorporation of (32)P into P-Rex1. LPA increased the amount of GTP-bound Rac in the cells and isoproterenol reduced basal levels of GTP-bound Rac and blunted the effect of LPA. Treatment of the cells with S(p)-cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for G(s)-linked receptors to regulate the function of P-Rex1 and inhibit its function in cells.
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PMID:Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gbetagamma-mediated regulation of its activity. 1630 20


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