Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.     

Nuclear mitogen-activated protein kinase activation by protein kinase czeta during reoxygenation after ischemic hypoxia

We examined the upstream kinases for mitogen-activated protein kinase (MAPK) activation during ischemic hypoxia and reoxygenation using H9c2 cells derived from rat cardiomyocytes. Protein kinase C (PKC)zeta, an atypical PKC isoform mainly expressed in rat heart, has been shown to act as an upstream kinase of MAPK during ischemic hypoxia and reoxygenation by analyses with PKC inhibitors, antisense DNA, a dominant negative kinase defective mutant, and constitutively active mutants of PKCzeta. Immunocytochemical observations show PKCzeta staining in the nucleus during ischemic hypoxia and reoxygenation when phosphorylated MAPK is also detected in the nucleus. This nuclear localization of PKCzeta is inhibited by treatment with wortmannin, a phosphoinositide 3-kinase inhibitor that also inhibits MAPK activation in a dose-dependent manner. This is supported by the inhibition of MAPK phosphorylation by another blocker of phosphoinositide 3-kinase, LY294002. An upstream kinase of MAPK, MEK1/2, is significantly phosphorylated 15 min after reoxygenation and observed mainly in the nucleus, whereas it is present in the cytoplasm in serum stimulation. The phosphorylation of MEK is blocked by PKC inhibitors and phosphoinositide 3-kinase inhibitors, as observed in the case of MAPK phosphorylation. These observations indicate that PKCzeta, which is activated by phosphoinositide 3-kinase, induces MAPK activation through MEK in the nucleus during reoxygenation after ischemic hypoxia.     

p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex

p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCzeta as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCzeta mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCzeta mutant (myristoylated PKCzeta [myr-PKCzeta]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCzeta. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCzeta in vivo in a growth factor-independent manner, while PDK-1 and PKCzeta can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

Background: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop.Results: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC βII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC α and βII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme.Conclusions: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.     

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

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.     

The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1

The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.     

Synergy of epidermal growth factor and 12(S)-hydroxyeicosatetraenoate on protein kinase C activation in lens epithelial cells

12(S)-hydroxyeicosatetraenoic acid (12(S)HETE) is a bioactive metabolite of arachidonic acid synthesized by 12-lipoxygenase. The 12-lipoxygenase blocker, baicalein, prevents epidermal growth factor (EGF)-induced activation of protein kinase C (PKC) alpha and beta in lens epithelial cells, whereas supplementation with 12(S)HETE reverses this effect, suggesting that EGF and 12(S)HETE may work together to activate PKC. This study investigates the mechanism of PKCbeta activation by EGF and 12(S)HETE. 12(S)HETE alone directed translocation of PKCbeta through the C1 rather than the C2 domain, without activating phosphoinositide 3-kinase (PI3K) or MAPK signaling or increasing intracellular calcium concentration. In the presence of baicalein, EGF triggered an asymmetric phosphorylation of the EGF receptor initiating signaling through PI3K and MAPK, but not PLCgamma. Together, 12(S)HETE and EGF synergistically increased phosphorylation of PKCbeta in the activation loop and C terminus as well as PKCbeta-specific activity. PI3K inhibitors blocked phosphorylation, but MEK1 inhibitors did not. Microvesicles containing phosphatidylinositol 3,4,5-trisphosphate mimicked the action of EGF on PKCbeta activity in the presence of 12(S)HETE. Kinase-inactive PKCbeta mutations in either activation loop or C terminus were effectively translocated by 12(S)HETE, as was PKCbeta in the presence of chelerythrine or G?-6983. These findings indicate that unphosphorylated PKCbeta is translocated to the membrane by 12(S)HETE and phosphorylated by EGF-dependent PI3K signaling, to generate catalytically competent PKCbeta.     

Membrane localization of 3-phosphoinositide-dependent protein kinase-1 stimulates activities of Akt and atypical protein kinase C but does not stimulate glucose transport and glycogen synthesis in 3T3-L1 adipocytes

It is reported that 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is activated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner and phosphorylates Akt, p70S6 kinase, and atypical protein kinase C (PKC), but its function on insulin signaling is still unclear. We cloned a full-length pdk-1 cDNA from a human brain cDNA library, and the adenovirus to overexpress wild type PDK-1 (PDK-1WT) or membrane-targeted PDK-1 (PDK-1CAAX) was constructed. Overexpressed PDK-1WT existed mainly at cytosol, and PDK-1CAAX was located at the plasma membrane. In 3T3-L1 adipocytes, insulin induced mobility shift of PDK-1 protein, but overexpressed PDK-1WT and CAAX were shifted at the basal state. Insulin stimulated tyrosine phosphorylation of PDK-1WT, but PDK-1CAAX was already tyrosine-phosphorylated at the basal state. Overexpression of PDK-1WT led to a full activation of PKC zeta/lambda without insulin stimulation but showed only the minimum effects to stimulate phosphorylation of Akt and GSK-3. In contrast, the overexpression of PDK-1CAAX caused phosphorylation of Akt and GSK-3 more strongly without insulin stimulation. However, PDK-1CAAX did not affect 2-deoxyglucose uptake and inhibited glycogen synthesis, surprisingly. Finally, PDK-1CAAX expression inhibited insulin-induced ERK1/2 phosphorylation in a dose-dependent manner. Taken together, the translocation of PDK-1 from cytosol to the plasma membrane is critical for Akt and GSK-3 activation. On the other hand, only atypical PKC and Akt activation was insufficient for stimulation of glucose transport, and constitutive activation of Akt-GSK-3 pathway may inhibit glycogen synthesis and MAPK cascade in 3T3-L1 adipocytes.     

Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.     

Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.     

Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase

General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338.     

Phosphoinositide-dependent kinase phosphorylation of protein kinase C Apl II increases during intermediate facilitation in aplysia

Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine mollusk Aplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation.     

Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1

A putative Akt kinase phosphorylation site ((64)ydRIRplSYp(73)) was found in Rac1/CDC42 and Rho family proteins (RhoA, RhoB, RhoC, and RhoG). Phosphorylation of Rac1 by Akt kinase was assayed with recombinant Rac1 protein and the fluorescein-labeled Rac1 peptide. It was shown that the Rac1 peptide and the recombinant protein were phosphorylated by the activated recombinant Akt kinase and the lysate of SK-MEL28 cells, a human melanoma cell line. The phosphorylation of Rac1 inhibited its GTP-binding activity without any significant change in GTPase activity. Both the GTP-binding and GTPase activities of Rac1 S71A protein (with the serine residue to be phosphorylated replaced with alanine) were abolished regardless of the treatment of Akt kinase. Akt kinase activity and Rac1 peptide phosphorylation were down-regulated by the treatment of SK-MEL28 cells with wortmannin or LY294002 (a phosphoinositide 3-kinase inhibitor), but JNK/SAPK kinase activity was up-regulated. Thus, the results suggest that Akt kinase of the phosphoinositide 3-kinase signal transduction pathway phosphorylates serine 71 of Rac1 as one of its authentic substrates and modulates the Rac1 signal transduction pathway through phosphorylation.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.     

Protein kinase C-zeta and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes.

The mechanisms used by insulin to activate the multifunctional intracellular effectors, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are only partly understood and appear to vary in different cell types. Presently, in rat adipocytes, we found that insulin-induced activation of ERK was blocked (a) by chemical inhibitors of both phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)-zeta, and, moreover, (b) by transient expression of both dominant-negative Deltap85 PI3K subunit and kinase-inactive PKC-zeta. Further, insulin effects on ERK were inhibited by kinase-inactive 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and by mutation of Thr-410 in the activation loop of PKC-zeta, which is the target of PDK-1 and is essential for PI3K/PDK-1-dependent activation of PKC-zeta. In addition to requirements for PI3K, PDK-1, and PKC-zeta, we found that a tyrosine kinase (presumably the insulin receptor), the SH2 domain of GRB2, SOS, RAS, RAF, and MEK1 were required for insulin effects on ERK in the rat adipocyte. Our findings therefore suggested that PDK-1 and PKC-zeta serve as a downstream effectors of PI3K, and act in conjunction with GRB2, SOS, RAS, and RAF, to activate MEK and ERK during insulin action in rat adipocytes.     

A novel protein kinase C target site in protein kinase D is phosphorylated in response to signals for cardiac hypertrophy

Protein kinase D (PKD) regulates cardiac myocyte growth and contractility through phosphorylation of proteins such as class IIa histone deacetylases (HDACs) and troponin I (TnI). In response to agonists that activate G-protein-coupled receptors (GPCRs), PKD is phosphorylated by protein kinase C (PKC) on two serine residues (Ser-738 and Ser-742 in human PKD1) within an activation loop of the catalytic domain, resulting in stimulation of PKD activity. Here, we identify a novel PKC target site located adjacent to the auto-inhibitory pleckstrin homology (PH) domain in PKD. This site (Ser-412 in human PKD1) is conserved in each of the three PKD family members and is efficiently phosphorylated by multiple PKC isozymes in vitro. Employing a novel anti-phospho-Ser-412-specific antibody, we demonstrate that this site in PKD is rapidly phosphorylated in primary cardiac myocytes exposed to hypertrophic agonists, including norepinephrine (NE) and endothelin-1 (ET-1). Differential sensitivity of this event to pharmacological inhibitors of PKC, and data from in vitro enzymatic assays, suggest a predominant role for PKCδ in the control of PKD Ser-412 phosphorylation. Together, these data suggest a novel, signal-dependent mechanism for controlling PKD function in cardiac myocytes.     

Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction

Protein kinase B (PKB or Akt) is a mitogen-regulated protein kinase involved in the protection of cells from apoptosis, the promotion of cell proliferation and diverse metabolic responses [1]. Its activation is initiated by the binding of 3' phosphorylated phosphoinositide lipids to its pleckstrin homology (PH) domain, resulting in the induction of activating phosphorylation at residues Thr308 and Ser473 by upstream kinases such as phosphoinositide-dependent protein kinase-1 (PDK1) [2]. Adhesion of epithelial cells to extracellular matrix leads to protection from apoptosis via the activation of phosphoinositide (PI) 3-kinase and Akt/PKB through an unknown mechanism [3] [4]. Here, we use the localisation of Akt/PKB within the cell to probe the sites of induction of PI 3-kinase activity. In fibroblasts, immunofluorescence microscopy showed that endogenous Akt/PKB localised to membrane ruffles at the outer edge of the cell following mitogen treatment as did green fluorescent protein (GFP) fusions with full-length Akt/PKB or its PH domain alone. In epithelial cells, the PH domain of Akt/PKB localised to sites of cell-cell and cell-matrix contact, distinct from focal contacts, even in the absence of serum. As this localisation was disrupted by PI 3-kinase inhibitory drugs and by mutations that inhibit interaction with phosphoinositides, it is likely to represent the sites of constitutive 3' phosphoinositide generation that provide a cellular survival signal. We propose that the attachment-induced, PI-3-kinase-mediated survival signal in epithelial cells is generated not only by cell-matrix interaction but also by cell-cell interaction.