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)

This study examines whether the serine/threonine protein kinase, Akt, is involved in the crosstalk between the ErbB2 and estrogen receptor-alpha (ER-alpha) pathways. Treatment of MCF-7 cells with 10(-9) M heregulin-beta1 (HRG-beta1) resulted in a rapid phosphorylation of Akt and a 15-fold increase in Akt activity. Akt phosphorylation was blocked by inhibitors of phosphatidylinositol 3-kinase (PI 3-K), by antiestrogens, the protein tyrosine kinase inhibitor, genistein, and by AG825, a selective ErbB2 inhibitor; but not by AG30, a selective EGFR inhibitor. Akt phosphorylation by HRG-beta1 was abrogated by an arginine to cysteine mutation (R25C) in the pleckstrin homology (PH) domain of Akt, and HRG-beta1 did not induce Akt phosphorylation in the ER-negative variant of MCF-7, MCF-7/ADR. Transient transfection of ER-alpha into these cells restored Akt phosphorylation by HRG-beta1, suggesting the requirement of ER-alpha. HRG-beta1 did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. Stable transfection of the cells with a dominant negative Akt or with the R25C-Akt mutant, as well as PI 3-K inhibitors, blocked the effect of HRG-beta1 on ER-alpha expression and activity and on the growth of MCF-7 cells. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of HRG-beta1. Experiments employing selective ErbB inhibitors demonstrate that the effect of HRG-beta1 on ER-alpha expression and activity is also mediated by ErbB2 and not by EGFR, demonstrating that ErbB2 is the primary mediator of the effects of HRG-beta1 on ER-alpha regulation. Taken together, our data suggest that HRG-beta1, bound to the ErbB2 ErbB3 heterodimer, in the presence of membrane ER-alpha, interacts with and activates PI 3-K/Akt. Akt leads to nuclear ER-alpha phosphorylation, thereby altering its expression and transcriptional activity.
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PMID:Heregulin-beta1 regulates the estrogen receptor-alpha gene expression and activity via the ErbB2/PI 3-K/Akt pathway. 1575 10

Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that has a catalytic domain homologous to that of Akt, but lacks the pleckstrin homology domain present in Akt. Akt reportedly plays a key role in various cellular actions, including glucose transport, glycogen synthesis, DNA synthesis, anti-apoptotic activity, and cell proliferation. In this study, we attempted to reveal the different roles of SGK and Akt by overexpressing active mutants of Akt and SGK. We found that adenovirus-mediated overexpression of myristoylated (myr-) forms of Akt resulted in high glucose transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic transformation in soft agar assays. The active mutant of SGK (D-SGK, substitution of Ser422 with Asp) and myr-SGK were shown to phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in a manner very similar to that observed for myr-Akt. However, despite the comparable degree of GSK3 phosphorylation between myr-Akt and d-SGK or myr-SGK, d-SGK and myr-SGK failed to enhance glucose transport activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt cannot be attributed to ability or inability to translocate to the membrane thorough the pleckstrin homology domain, but rather must be attributable to differences in the relatively narrow substrate specificities of these kinases. In addition, our observations strongly suggest that phosphorylation of GSK3 is either not involved in or not sufficient for GLUT4 translocation, DNA synthesis, or oncogenic transformation. Thus, the identification of substrates selectively phosphorylated by Akt, but by not SGK, may provide clues to clarifying the pathway leading from Akt activation to these cellular activities.
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PMID:Differing roles of Akt and serum- and glucocorticoid-regulated kinase in glucose metabolism, DNA synthesis, and oncogenic activity. 1273 7

A key component of the insulin-signalling pathway, the protein kinase Akt, was identified and cloned as a cDNA from ovaries of the mosquito Aedes aegypti. An ortholog gene was found in the Anopheles gambiae genome database, and like other Akts, both mosquito Akts possess pleckstrin homology domains for membrane binding and a serine/threonine kinase domain. When Ae. aegypti ovaries were treated with bovine insulin in vitro, a putative Akt was threonine-phosphorylated, as expected for Akts. AaegAKT was only expressed in embryos for the first 6 h after oviposition and in ovaries before and during a gonotrophic cycle.
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PMID:Molecular analysis of the serine/threonine kinase Akt and its expression in the mosquito Aedes aegypti. 1275 55

RGS proteins comprise a large family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. RGS6 is a member of the R7 subfamily of RGS proteins possessing DEP (disheveled/Egl-10/pleckstrin) homology and GGL (G protein gamma-subunit-like) domains in addition to the semiconserved RGS domain. Our previous study documented unusual complexity in splicing of the human RGS6 gene, and we demonstrated localization of various RGS6 splice forms at sites other than the plasma membrane, including the cytoplasm and nucleus, where G proteins are not localized (Chatterjee, T. K., Liu, Z., and Fisher, R. A. (2003) J. Biol. Chem. 278, 30261-30271). Here we provide new evidence that mild heat stress, proteasome-mediated proteotoxic stress, and HSF1 expression induces dramatic relocalization of RGS6 proteins from such sites to nucleoli. This response was observed in COS-7 cells expressing various splice forms of RGS6, was not elicited by other forms of cellular stress and was observed in cells treated with various protein kinase inhibitors or co-expressing a dominant-negative kinase inactive SAPK. The RGS domain of RGS6 was identified as a primary structural module providing support for its stress-induced nucleolar trafficking and various other RGS proteins or their isolated RGS domains similarly undergo nucleolar migration in response to heat or proteotoxic stress or during co-expression of HSF1. The atypical RGS domains of axin and AKAP10 also underwent stress-induced nucleolar trafficking while structural domains outside of the RGS domain of some RGS proteins can override nucleolar trafficking in response to stress. Inhibition of rDNA transcription also promoted nucleolar migration of RGS6, a response previously observed in a subset of nucleolar proteins. The DEP domain of RGS6, but not its RGS domain, conferred structural support for its transcription-linked nucleolar migration. RGS6 exhibited trafficking from subnuclear dots to nucleoli in response to heat-, proteotoxic- or transcription-linked stress. These results provide new evidence that mammalian RGS proteins undergo unique subcellular trafficking in response to specific forms of cellular stress and implicate the RGS family of proteins in cellular stress signaling pathways.
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PMID:Mild heat and proteotoxic stress promote unique subcellular trafficking and nucleolar accumulation of RGS6 and other RGS proteins. Role of the RGS domain in stress-induced trafficking of RGS proteins. 1276 Dec 20

The phosphorylation of heptahelical receptors by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor kinases (GRKs) is a universal regulatory mechanism that leads to desensitization of G protein signaling and to the activation of alternative signaling pathways. We determined the crystallographic structure of bovine GRK2 in complex with G protein beta1gamma2 subunits. Our results show how the three domains of GRK2-the RGS (regulator of G protein signaling) homology, protein kinase, and pleckstrin homology domains-integrate their respective activities and recruit the enzyme to the cell membrane in an orientation that not only facilitates receptor phosphorylation, but also allows for the simultaneous inhibition of signaling by Galpha and Gbetagamma subunits.
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PMID:Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbetagamma. 1276 89

Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1alpha (K46RCK1alpha) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells.
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PMID:Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling. 1457 54

3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a Ser/Thr kinase with an essential role in insulin and growth-factor signalling. PDK1 activity towards protein kinase B (PKB) is partially regulated by its pleckstrin homology (PH) domain, which preferentially binds to 3-phosphoinositides. However, the precise molecular mechanism of this regulation is not well understood. Here, the cloning, purification and crystallization of a 150-amino-acid C-terminal region of PDK1 containing the PH domain is reported. A crystal of the PDK1 PH domain grown in the presence of inositol 1,3,4,5-tetrakisphosphate and derivatized with AuCN diffracted to 1.5 A at a synchrotron source. Diffraction data collected near the Au edge resulted in an anomalous Patterson map with a 30sigma peak.
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PMID:Purification, crystallization and preliminary X-ray diffraction of a proteolytic fragment of PDK1 containing the pleckstrin homology domain. 1474 9

Protein kinase D (PKD) participates in activation of the transcription factor NF-kappaB (nuclear factor kappaB) in cells exposed to oxidative stress, leading to increased cellular survival. We previously demonstrated that phosphorylation of PKD at Tyr463 in the PH (pleckstrin homology) domain is mediated by the Src-Abl pathway and that it is necessary for PKD activation and subsequent NF-kappaB induction. Here we show that activation of PKD in response to oxidative stress requires two sequential signaling events, i.e., phosphorylation of Tyr463 by Abl, which in turn promotes a second step, phosphorylation of the PKD activation loop (Ser738/Ser742). We show that this is mediated by PKCdelta (protein kinase Cdelta), a kinase that is activated by Src in response to oxidative stress. We also show that other PKCs, including PKCepsilon and PKCzeta, do not participate in PKD activation or NF-kappaB induction. We propose a model in which two coordinated signaling events are required for PKD activation. Tyrosine phosphorylation in the PH domain at Tyr463, mediated by the Src-Abl pathway, which in turn facilitates the phosphorylation of Ser738/Ser742 in the activation loop, mediated by the Src-PKCdelta pathway. Once active, the signal is relayed to the activation of NF-kappaB in oxidative stress responses.
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PMID:Protein kinase Cdelta selectively regulates protein kinase D-dependent activation of NF-kappaB in oxidative stress signaling. 1502 53

Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.
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PMID:Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment. 1506 72

We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-tris-phosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.
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PMID:The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. 1511 68


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