Serum and glucocorticoid-inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF-1 or pervanadate induced a 3-to 12-fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3-kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other secondmessenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6-fold activation of SGK activity, whereas kinase-dead PDK1 caused no activation. GST-pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a ϳ10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.The signaling pathway centered on protein kinase B (PKB, 1 also called Akt) has emerged as a critical mediator of diverse cellular processes including metabolism, gene expression, migration, angiogenesis, proliferation, and cell survival (1, 2). PKB is tightly controlled and the consequences of its deregulation have been implicated in the development of cancers and diabetes (1, 2). The activity of PKB is markedly stimulated in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Upon stimulation, PKB is recruited to the plasma membrane through the binding of its N-terminal pleckstrin homology (PH) domain to phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), a lipid product of PI3K. PKB␣ is then activated by phosphorylation on two residues: Thr-308 in the activation loop and Ser-473 in the hydrophobic motif of the C-terminal tail (3). There is convincing evidence that Thr-308 is phosphorylated by 3-phosphoinositol-dependent kinase 1 (PDK1) (4, 5). In embryonic stem cells in which the PDK1 gene has been genetically disrupted, PKB is resistant to growth factor stimulation as consequence of loss of Thr-308 phosphorylation (5), but phosphorylation on Ser-473 still occurs. Like PKB, PDK1 also contains a PH domain that binds to PIP 3 (4, 6). Phosphorylation of Thr-308 in vivo is dependent on PI3K activity, but it is unclear if this requirement is necessary for the unfolding of PKB to allow access of PDK1 to Thr-308 site or direct activation of PDK1 through its PH domain (6, 7). Other results indicate that PI3K is important for PKB on Ser-473 because analysis of knock-in embryonic stem cells expressing PDK1 with a mutation in its PH domain revealed that PKB is not activated by insulin-like growth factor-1 (IGF-1), whereas ribosomal S6 kinase (RSK) is activated normally, indicating the importance of colocalization of PKB with PDK1 at the plasma membrane (8).Identification of the kinase responsible for phosphorylating Ser-473 has been a major challenge for a number of years but remains elusive. Several kinases have been r...

Protein kinase B (PKB) has emerged as the focal point for many signal transduction pathways, regulating multiple cellular processes such as glucose metabolism, transcription, apoptosis, cell proliferation, angiogenesis, and cell motility. In addition to acting as a kinase toward many substrates involved in these processes, PKB forms complexes with other proteins that are not substrates, but rather act as modulators of PKB activity and function. In this review, we discuss the implications of these data in understanding the multitude of functions predicted for PKB in cells.

We have established that the serum-and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5 deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation of sgk gene promoter activity. Gel shift analysis identified four major DNAprotein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction of sgk promoter activity or protein production. Using an in vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosinephosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5-to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased ϳ6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.3-Phosphoinositide-dependent protein kinase-1 (PDK1) 1 appears to play a central regulatory role in many cell-signaling pathways (1-12). Several substrates of PDK1 have so far been identified, including protein kinase B (PKB), p70 ribosomal protein S6 kinase (p70S6K), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), serum and glucocorticoidinducible kinase (SGK), p90 ribosomal protein S6 kinase (RSK), and p21-activated kinase-1 (1, 2, 9, 13-31). However, most of these putative PDK1 substrates were identified using in vitro phosphorylation experiments, and not all the substrate proteins appear to be physiological targets for PDK1 in vivo. Experiments with embryonic stem (ES) cells ablated for both alleles of PDK1 revealed that PKB, p70S6K, and RSK are no longer phosphorylated or activated by agonists that are potent stimuli in normal ES cells, providing in vivo evidence that these three enzymes are dependent on PDK1 for activation (32). The regulation of the other potential substrates of PDK1 is apparently unaffected in the knock-out cell line (32). PKB, p70S6K, RSK, and SGK, members of the AGC family of protein kinases, are apparently maintained in the cytoplasm in an inactive state. Upon stimulation of receptor tyrosine kinases, phosphoinositide 3-kinase (PI3K) becomes activated, generating the phospholipid second messengers, PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 . These lipids then mediate the phosphorylation and activation of PDK1 targets through diverse mechanisms (...