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:2.7.11.2 (PDK1)
2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.
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PMID:The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. 1150 Mar 65

Involvement of voltage-gated (Kv) potassium channels in IGF-1-induced proliferation of HEK293 cells was studied by patch-clamp, RT-PCR and FACS analysis. IGF-1 up-regulated outwardly rectifying whole-cell K+ current starting after 1 h of incubation and reaching a maximum after 4-6 h. The IGF-1-stimulated current was voltage-gated with an activation threshold of -30 mV to -40 mV, a half-maximal activation at +5.3+/-1.8 mV, and time constants for activation and inactivation of 4.5+/-0.4 ms and 43.5+/-5.6 ms ( n=10), respectively. The current was inhibited by TEA, margatoxin, agitoxin-2 and stichodactyla toxin. PCR amplification of different Kv subunits from HEK293 cDNA demonstrated the expression of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv3.1 and Kv3.4 mRNA. Quantitative RT-PCR showed up-regulation of Kv1.1, 1.2 and 1.3 mRNA by IGF-1. The effect of IGF-1 on K+ current was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), wortmannin and LY294002, and mimicked by overexpression of human 3-phosphoinositide-dependent protein kinase-1 (hPDK1) or serum- and glucocorticoid-dependent kinase-1 (hSGK1), both sequential downstream targets of PI3-kinase. IGF-1-induced proliferation of HEK293 cells was inhibited by both K+ channel blockers and inhibitors of PI3-kinase. In conclusion, IGF-1 through PI3-kinase, PDK1 and SGK1 up-regulates Kv channels, an effect required for the proliferative action of the growth factor.
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PMID:IGF-1 up-regulates K+ channels via PI3-kinase, PDK1 and SGK1. 1190 30

The SGK1 protein belongs to the AGC gene family of kinases that are regulated by phosphorylation mediated by PDK1. SGK1 regulation is accomplished by several pathways including growth-factor and stress-mediated signaling. We have expanded the analysis of SGK1 regulation in epithelial cells. We used HA-tagged SGK1 to transiently transfect MDCK cells and study the regulation of SGK1 upon stimulation with HGF, cAMP or upon adhesion of the cells to immobilized fibronectin. In addition, we studied the regulation of SGK1 activity by small GTP-binding proteins of the Rho family. Treatment of MDCK cells with HGF leads to a time-dependent activation of SGK1 that is blocked by wortmanin. This activation requires the conserved phosphorylation site present in the activation loop of the kinase (T256 in SGK1) and the phosphorylation site present in a hydrophobic domain at its C-terminus (S422 in SGK1), which are targets for PDK1/PDK2-mediated regulation of SGK1. We tested whether SGK1 could be activated by cAMP as it contains a putative PKA site. We were unable to demonstrate a significant activation of HA-SGK1 by cAMP stimulation under conditions where we detect cAMP-mediated phosphorylation of the transcription factor CREB. Cotransfection of SGK1 with activated small GTP-binding proteins revealed that Rac1, but not Rho or Rap1, induces activation of SGK1. However, this activation was wortmanin insensitive and dominant-negative Rac1 did not inhibit the HGF-mediated activation of SGK1. Adhesion of MDCK cells to immobilized fibronectin also leads to activation of SGK1. However, it appears that the integrin-mediated activation of HA-SGK1 differs from AKT activation in the fact that AKT phosphorylation was blocked by wortmanin (or LY294002) whereas HA-SGK1 was not. The adhesion-dependent activation, however, requires the intact phosphorylation sites of SGK1. Co-transfection of HA-SGK1 with RacV12 results in increased activity in adherent cells compared with HA-SGK1 alone. Since RacN17 failed to inhibit adhesion dependent-activation of SGK1, it suggests that integrin activation is achieved by a parallel Rac-independent pathway. The activation of SGK1 by HGF and integrin provides a link between HGF-mediated protection of MDCK from de-attachment induced apoptosis (anoikis). We demonstrate that dephosphorylation of the transcription factor FKRHL1 induced by cell de-attachment is prevented by activated SGK1, suggesting that SGK1 regulates cell survival pathways. In summary, we demonstrate that SGK1 activation could be achieved through signaling pathways involved in the regulation of cell survival, cell-cell and cell-matrix interactions. SGK1 activation can be accomplished via HGF, PI-3K-dependent pathways and by integrin-mediated, PI-3K independent pathways. In addition, activation of SGK1 by the small GTP-binding protein Rac1 has been observed.
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PMID:Activation of SGK1 by HGF, Rac1 and integrin-mediated cell adhesion in MDCK cells: PI-3K-dependent and -independent pathways. 1195 29

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.
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PMID:A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. 1237 40

Aldosterone increases within 30 min renal Na+reabsorption and K+secretion by a mechanism that is triggered at the level of gene transcription. Thus, gene products that are rapidly up- or down-regulated transmit this effect to the transport machinery within the distal nephron target cells. One such rapidly up-regulated gene product is a structural element of the transport machinery, namely the a subunit of ENaC. Its amount might in certain conditions play a rate limiting role for Na+transport. Cell-surface localization and function of ENaC and of the Na,K-ATPase are also tightly controlled by a complex regulatory network and aldosterone appears to acutely regulate the expression of elements of this network such as the small G-protein K-Ras (in A6 cells) and the kinase SGK1 (also in ENaC-expressing cells of the mammalian distal nephron). The kinase SGK1 is an early aldosterone-induced protein that relays signals from pathways that are transmitted via PDK1/2 and possibly PKA. Active SGK1 has been shown to increase ENaC and Na,K-ATPase cell-surface expression in Xenopus oocytes. This effect at the level of ENaC has been recently shown to be mediated by the ubiquitin ligase Nedd4-2 which is a direct target of SGK1. Once phosphorylated by SGK1, Nedd4-2 is prevented from interacting with ENaC and thus from decreasing ENaC cell-surface expression. This SGK1-Nedd4-2-ENaC pathway is the first direct link between aldosterone-induced transcriptional regulation and the function of the Na+transport machinery to be unravelled. The physiological importance of this pathway for mediating the aldosterone response in different target epithelia remains to be verified in vivo, in particular in view of the axial gradient of ENaC apical translocation observed along the aldosterone-sensitive distal nephron.
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PMID:SGK1: aldosterone-induced relay of Na+ transport regulation in distal kidney nephron cells. 1264 99

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.
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PMID:WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate. 1461 43

The stimulation of cell proliferation by insulin like growth factor IGF-1 has previously been shown to depend on activation of voltage gated K(+) channels. The signaling involved in activation of voltage gated K(+) channel Kv1.3 includes the phosphatidylinositol-3 (PI3) protein kinase, 3-phosphoinositide dependent protein kinase PDK1 and the serum and glucocorticoid inducible kinase SGK1. However, nothing is known about mechanisms mediating the stimulation of Kv1.3 by SGK1. Most recently, SGK1 has been shown to phosphorylate and thus inactivate the ubiquitin ligase Nedd4-2. The present study has been performed to explore whether the regulation of Kv1.3 involves Nedd4-2. To this end Kv1.3 has been expressed in Xenopus oocytes with or without coexpression of Nedd4-2 and/or constitutively active (S422D)SGK1. In oocytes expressing Kv1.3 but not in water injected oocytes, depolarization from a holding potential of -80 mV to +20 mV triggers rapidly inactivating currents typical for Kv1.3. Coexpression of Nedd4-2 decreases, coexpression of (S422D)SGK1 enhances the currents significantly. The effects of either Nedd4-2 or of SGK1 are abrogated by destruction of the respective catalytic subunits ((C938S)Nedd4-2 or (K127N)SGK1). Further experiments revealed that wild type SGK1 and SGK3 and to a lesser extent SGK2 are similarly effective in stimulating Kv1.3 in both, presence and absence of Nedd4-2. It is concluded that Kv1.3 is downregulated by Nedd4-2 and stimulates by SGK1, SGK2, and SGK3. The data thus disclose a novel mechanism of Kv1.3 channel regulation.
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PMID:Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1. 1504 1

The DAF-2 insulin receptor-like signaling pathway controls metabolism, development, longevity, and stress response in C. elegans. Here we show that SGK-1, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase SGK, acts in parallel to the AKT kinases to mediate DAF-2 signaling. Loss of sgk-1 results in defective egg-laying, extended generation time, increased stress resistance, and an extension of life span. SGK-1 forms a protein complex with the AKT kinases, and is activated by and strictly depends on PDK-1. All three kinases of this complex are able to directly phosphorylate DAF-16/FKHRL1, yet have different functions in DAF-2 signaling. Whereas AKT-1 and AKT-2 are more important for regulating dauer formation, SGK-1 is the crucial factor for the control of development, stress response, and longevity. Our data also suggest the existence of a second pathway from DAF-2 to DAF-16 that does not depend on AKT-1, AKT-2, and SGK-1.
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PMID:C. elegans SGK-1 is the critical component in the Akt/PKB kinase complex to control stress response and life span. 1506 96

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by PKBa (protein kinase Ba), but not by SGK1 (serum- and glucocorticoid-induced kinase 1), and identified it as the cytoskeletal protein FLNc (filamin C). PKBa phosphorylated FLNc at Ser2213 in vitro, which lies in an insert not present in the FLNa and FLNb isoforms. Ser2213 became phosphorylated when C2C12 myoblasts were stimulated with insulin or epidermal growth factor, and phosphorylation was prevented by low concentrations of wortmannin, at which it is a relatively specific inhibitor of phosphoinositide 3-kinase. PD 184352 [an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade] and/or rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] had no effect. Insulin also induced the phosphorylation of FLNc at Ser2213 in cardiac muscle in vivo, but not in cardiac muscle that does not express PDK1 (3-phosphoinositide-dependent kinase 1), the upstream activator of PKB. These results identify the muscle-specific isoform FLNc as a new physiological substrate for PKB.
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PMID:Identification of filamin C as a new physiological substrate of PKBalpha using KESTREL. 1546 88

A substrate for PKBalpha (protein kinase Balpha) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKBalpha, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser52 in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser58). CRHSP24 became phosphorylated at Ser52 when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similar phosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK1 (3-phosphoinositide-dependent kinase 1) mutant (PDK1[L155E]) that activates PKBalpha normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser52 in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKBalpha, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser30, Ser32 and Ser41 in vitro, and Ser41 was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser52 by PKBalpha in response to IGF-1, at Ser52 by PKBalpha and RSK in response to EGF, and at Ser41 in the absence of IGF-1/EGF by a DYRK isoform or another proline-directed protein kinase(s).
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PMID:Identification of calcium-regulated heat-stable protein of 24 kDa (CRHSP24) as a physiological substrate for PKB and RSK using KESTREL. 1591 Feb 84


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