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)

The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3' phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.
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PMID:Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B. 1216 17

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.
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PMID:Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. 1217 97

We investigated whether the effect of troglitazone on glucose disposal is associated with altered insulin signaling. Nondiabetic first-degree relatives of type 2 diabetic patients (age 30 +/- 2 years, BMI 30 +/- 1 kg/m(2); n = 20) were randomized in a double-blind manner to 3 months of troglitazone (200 mg/day) or placebo treatment. Before and after treatment, 3-h euglycemic-hyperinsulinemic glucose clamps (40 mU. m(-2). min(-1)) were performed, and muscle biopsies were obtained immediately before and after the clamps. In the biopsies, insulin receptor kinase (IRK) activity, insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity, Ser(473) and Thr(308) phosphorylation of protein kinase B (PKB), and protein expression of IRS-1, IRS-2, phosphoinositol-dependent kinase-1 (PDK-1), PKB, and GLUT-4 were determined. After troglitazone treatment, insulin-stimulated glucose disposal was increased compared with pretreatment and placebo (279 +/- 37 vs. 211 +/- 26 and 200 +/- 25 mg. m(-2). min(-1); both P < 0.05). IRK and PI3K activities were not altered by troglitazone, but PKB Ser(473) phosphorylation was enhanced compared with pretreatment and placebo at the clamp insulin level (138 +/- 36 vs. 77 +/- 16 and 55 +/- 13 internal standard units; both P < 0.05) and with pretreatment at the basal level (31 +/- 9 vs. 14 +/- 4 internal standard units; P < 0.05). PKB Thr(308) phosphorylation also tended to be higher, but this was not statistically significant. Troglitazone did not alter insulin receptor number or IRS-1, IRS-2, PKB, PDK-1, or GLUT-4 protein expression. We conclude that increased PKB phosphorylation may contribute to the insulin-sensitizing effects of thiazolidinediones in human skeletal muscle.
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PMID:Troglitazone treatment increases protein kinase B phosphorylation in skeletal muscle of normoglycemic subjects at risk for the development of type 2 diabetes. 1219 60

Signaling events involving angiotensin IV (ANG IV)-mediated pulmonary artery endothelial cell (PAEC) proliferation were examined. ANG IV significantly increased upstream phosphatidylinositide (PI) 3-kinase (PI3K), PI-dependent kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), and protein kinase B-alpha/Akt (PKB-alpha) activities, as well as downstream p70 ribosomal S6 kinase (p70S6K) activities and/or phosphorylation of these proteins. ANG IV also significantly increased 5-bromo-2'-deoxy-uridine incorporation into newly synthesized DNA in a concentration- and time-dependent manner. Pretreatment of cells with wortmannin and LY-294002, inhibitors of PI3K, or rapamycin, an inhibitor of the mammalian target of rapamycin kinase and p70S6K, diminished the ANG IV-mediated activation of PDK-1 and PKB-alpha as well as phosphorylation of p70S6K. Although an inhibitor of mitogen-activated protein kinase kinase, PD-98059, but not rapamycin, blocked ANG IV-induced phosphorylation of ERK1/2, both PD-98059 and rapamycin independently caused partial reduction in ANG IV-mediated cell proliferation. However, simultaneous treatment with PD-98059 and rapamycin resulted in total inhibition of ANG IV-induced cell proliferation. These results demonstrate that ANG IV-induced DNA synthesis is regulated in a coordinated fashion involving multiple signaling modules in PAEC.
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PMID:Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation. 1222 47

Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.
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PMID:Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells. 1259 28

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.
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PMID:Regulation of the phosphatidylinositol 3-kinase, Akt/protein kinase B, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland. 1266 83

Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.
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PMID:Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways. 1276 74

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.
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PMID:Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition. 1289 59

We determined basal and insulin-stimulated responses on signaling intermediates in soleus skeletal muscle from male Wistar and diabetic Goto-Kakizaki (GK) rats. Rats were infused with glucose (5 or 20 mm) for 3 h, followed by a continuous infusion of saline or insulin (3 U/kg.h) for 20 min. Under euglycemic and hyperglycemic conditions, basal and insulin-stimulated action on phosphatidylinositol (PI) 3-kinase, protein kinase B/Akt, and ERK were reduced in GK rats, whereas insulin-stimulated protein kinase C (PKC)zeta activity was not altered. Interestingly, basal PKCzeta activity was increased under hyperglycemic conditions in GK and Wistar rats. This finding of increased PKCzeta activity was confirmed in vitro in isolated soleus muscle exposed to high extracellular glucose, and occurred concomitant with an increase in PI-dependent kinase 1 (PDK-1) activity. The glucose effects were not specific to PKCzeta, because an increase in phosphorylation of PKCalpha/beta and PKCdelta, but not PKCtheta, in isolated soleus muscle exposed to 25 mm glucose was observed. In conclusion, insulin signaling defects in diabetic GK rats are not corrected by an acute normalization of glycemia. Interestingly, acute hyperglycemia leads to a parallel increase in PDK-1, PKCalpha/beta, PKCdelta, and PKCzeta phosphorylation/activity via a PI 3-kinase-protein kinase B/Akt-independent mechanism. The long-term consequence of elevated PDK-1 and PKC phosphorylation/activity should be considered in the context of diabetes mellitus, as hyperglycemia is a clinical feature of this disease.
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PMID:Effect of hyperglycemia on signal transduction in skeletal muscle from diabetic Goto-Kakizaki rats. 1296 81

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


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