PKCδ survival signaling in cells containing an activated p21Ras protein requires PDK1

Protein kinase C δ (PKCδ) modulates cell survival and apoptosis in diverse cellular systems. We recently reported that PKCδ functions as a critical anti-apoptotic signal transducer in cells containing activated p21^Ras and results in the activation of AKT, thereby promoting cell survival. How PKCδ is regulated by p21^Ras, however, remains incompletely understood. In this study, we show that PKCδ, as a transducer of anti-apoptotic signals, is activated by phosphotidylinositol 3′ kinase/phosphoinositide-dependent kinase 1 (PI₃K–PDK1) to deliver the survival signal to Akt in the environment of activated p21^Ras. PDK1 is upregulated in cells containing an activated p21Ras. Knock-down of PDK1, PKCδ, or AKT forces cells containing activated p21^Ras to undergo apoptosis. PDK1 regulates PKCδ activity, and constitutive expression of PDK1 increases PKCδ activity in different cell types. Conversely, expression of a kinase-dead (dominant-negative) PDK1 significantly suppresses PKCδ activity. p21^Ras-mediated survival signaling is therefore regulated by via a PI₃K–AKT pathway, which is dependent upon both PDK1 and PKCδ, and PDK1 activates and regulates PKCδ to determine the fate of cells containing a mutated, activated p21^Ras.     

Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt pathway in epidermal growth factor signaling

The phosphoinositide 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathway regulates various cellular functions, especially cell survival and cell cycle progression. In contrast to other survival pathways, there have been few reports of scaffold proteins that regulate signaling cascade specificity in this pathway. Here we identify a 5′ repressor element under dual-repression binding protein 1 (Freud-1)/Akt kinase-interacting protein 1 (Aki1) as a novel scaffold for the PDK1/Akt pathway. Freud-1/Aki1 (also known as CC2D1A) expression induced formation of a PDK1/Akt complex and regulated Akt activation in a concentration-dependent biphasic manner. Freud-1/Aki1 also associated with epidermal growth factor (EGF) receptor in response to EGF stimulation and was required for Akt activation induced by EGF, but not by insulin-like growth factor 1. Freud-1/Aki1 gene silencing decreased Akt kinase activity, resulting in induction of apoptosis and increased sensitivity toward chemotherapeutic agents. Our results suggest that Freud-1/Aki1 is a novel receptor-selective scaffold protein for the PDK1/Akt pathway and present a new activation mechanism of Akt.     

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC?? of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.     

Sustained Akt/PKB activation and transient attenuation of c-jun N-Terminal kinase in the inhibition of apoptosis by IGF-1 in vascular smooth muscle cells

Characteristics of hVSMC apoptosis and its inhibition by insulin-like growth factor-1 (IGF-1) remain unclear. Also unclear is whether a balance in hVSMCs exists whereby c-Jun N-terminal stress kinases (JNK) promote apoptosis while extracellular signal-regulated (ERK1/2) MAP kinases inhibit cell death. In this study, we examined the involvement of Akt/PKB and its upstream kinase, PDK1 and whether JNK activation correlated with human and rat VSMC apoptosis induced by staurosporine and by c-myc, respectively. We observed a strong, sustained JNK activation (and c-Jun phosphorylation), which correlated with VSMC apoptosis. IGF-1 (13.3 nM), during apoptosis inhibition, transiently inhibited JNK activity at 1 h in a phosphatidylinositol 3-kinase (PI3-K)- and MEK-ERK-dependent manner, as wortmannin (100 nM) or PD98059 (30 M) partially attenuated the IGF-1 effect. PKC down-regulation had no effect on JNK inhibition by IGF-1. While IGF-1 alone produced a strong phosphorylation of Akt/PKB in hVSMCs up to 6 h, it was notably stronger and more sustained during ratmyc and hVSMCs apoptosis inhibition. Further, whereas transient expression of phosphorylated Akt protected VSMCs from apoptosis by nearly 50%, expression of dominant interfering alleles of Akt or PDK1 strongly inhibited IGF-1-mediated VSMC survival. These results demonstrate for the first time that transient inhibition of a pro-apoptotic stimulus in VSMCs may be sufficient to inhibit a programmed cell death and that sustained anti-apoptotic signals (Akt) elicited by IGF-1 are augmented during a death stimulus. Furthermore, PI3-K and ERK-MAPK pathways may cooperate to protect VSMCs from cell death.This work was supported by a grant from the Nebraska cancer and Smoking Related Disease Program, Department of Health, Nebraska, and National Institutes of Health Grants R01HL070885 (D.K.A.) and R01HL073349 (D.K.A.).     

Inhibition of insulin-induced activation of Akt by a kinase-deficient mutant of the epsilon isozyme of protein kinase C

Akt, also known as protein kinase B, is a protein-serine/threonine kinase that is activated by growth factors in a phosphoinositide (PI) 3-kinase-dependent manner. Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. The potential role of the epsilon isozyme of protein kinase C (PKC) in the activation of Akt induced by insulin has now been examined. Expression of a kinase-deficient mutant of PKCepsilon (epsilonKD), but not that of wild-type PKCepsilon or of kinase-deficient mutants of PKCalpha or PKClambda, with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin in Chinese hamster ovary cells or L6 myotubes. Whereas the epsilonKD mutant did not affect insulin stimulation of PI 3-kinase activity, the phosphorylation and activation of Akt induced by a constitutively active mutant of PI 3-kinase were inhibited by epsilonKD, suggesting that epsilonKD affects insulin signaling downstream of PI 3-kinase. PDK1 (3'-phosphoinositide-dependent kinase 1) is thought to participate in Akt activation. Overexpression of PDK1 with the use of an adenovirus vector induced the phosphorylation and activation of Akt; epsilonKD inhibited, whereas wild-type PKCepsilon had no effect on, these actions of PDK1. These results suggest that epsilonKD inhibits the insulin-induced phosphorylation and activation of Akt by interfering with the ability of PDK1 to phosphorylate Akt.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1.

Serine/threonine kinase Akt is thought to mediate many biological actions toward anti-apoptotic responses. Screening of drugs that could interfere with the Akt signaling pathway revealed that Hsp90 inhibitors (e.g. geldanamycin, radicicol, and its analogues) induced Akt dephosphorylation, which resulted in Akt inactivation and apoptosis of the cells. Hsp90 inhibitors did not directly affect Akt kinase activity in vitro. Thus, we examined the effects of Hsp90 inhibitors on upstream Akt kinases, phosphatidylinositide-3-OH kinase (PI3K) and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Hsp90 inhibitors had no effect on PI3K protein expression. In contrast, treatment of the cells with Hsp90 inhibitors decreased the amount of PDK1 without directly inhibiting PDK1 kinase activity. We found that the kinase domain of PDK1 was essential for complex formation with Hsp90 and that Hsp90 inhibitors suppressed PDK1 binding to Hsp90. PDK1 degradation mechanisms revealed that inhibition of PDK1 binding to Hsp90 caused proteasome-dependent degradation of PDK1. Treatment of proteasome inhibitors increased the amount of detergent-insoluble PDK1 in Hsp90 inhibitor-treated cells. Therefore, the association of PDK1 with Hsp90 regulates its stability, solubility, and signaling. Because Akt binding to Hsp90 is also involved in the maintenance of Akt kinase activity, Hsp90 plays an important role in PDK1-Akt survival signaling pathway.     

Deletion of PDK1 Causes Cardiac Sodium Current Reduction in Mice

Background

The AGC protein kinase family regulates multiple cellular functions. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is involved in the pathogenesis of arrhythmia, and its downstream factor, Forkhead box O1 (Foxo1), negatively regulates the expression of the cardiac sodium channel, Nav1.5. Mice are known to die suddenly after PDK1 deletion within 11 weeks, but the underlying electrophysiological bases are unclear. Thus, the aim of this study was to investigate the potential mechanisms between PDK1 signaling pathway and cardiac sodium current.

Methods and Results

Using patch clamp and western blotting techniques, we investigated the role of the PDK1-Foxo1 pathway in PDK1 knockout mice and cultured cardiomyocytes. We found that PDK1 knockout mice undergo slower heart rate, prolonged QRS and QTc intervals and abnormal conduction within the first few weeks of birth. Furthermore, the peak sodium current is decreased by 33% in cells lacking PDK1. The phosphorylation of Akt (308T) and Foxo1 (24T) and the expression of Nav1.5 in the myocardium of PDK1-knockout mice are decreased, while the nuclear localization of Foxo1 is increased. The role of the PDK1-Foxo1 pathway in regulating Nav1.5 levels and sodium current density was verified using selective PDK1, Akt and Foxo1 inhibitors and isolated neonatal rat cardiomyocytes.

Conclusion

These results indicate that PDK1 participates in the dysregulation of electrophysiological basis by regulating the PDK1-Foxo1 pathway, which in turn regulates the expression of Nav1.5 and cardiac sodium channel function.     

Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon)

The protein kinase C (PKC)-related enzyme PKC(mu)/PKD (protein kinase D) is activated by activation loop phosphorylation through PKC(eta). Here we demonstrate that PKC(mu) is activated by the direct phosphorylation of PKC(epsilon). PKC(mu) colocalizes with PKC(epsilon) in HEK293 and MCF7 cells as shown by confocal immunofluorescence analyses. PDK1, known as the upstream kinase for several PKC isozymes, associates intracellularly with PKC(epsilon) and PKC(eta). PKC(eta) is phosphorylated by PDK1 in vitro, leading to kinase activation as similarly reported for PKC(epsilon) activation by PDK1. Coexpression of PDK1, PKC(epsilon) and PKC(mu) in HEK293 cells results in PKC(mu) activation. In contrast, the coexpression of PDK1 and PKC(eta) with PKC(mu) does not activate PKC(eta) or consequently PKC(mu). PDK1/PKC(epsilon)-triggered activation of PKC(mu) inhibits JNK, a downstream effector of PKC(mu), whereas upon transient expression of PDK1, PKC(eta), and PKC(mu), JNK is not affected. These data implicate PKC(epsilon) as the biologically important upstream kinase for PKC(mu) in HEK293 cells, regulating downstream effectors. Our results further indicate a PDK1/PKC(eta)/PKC(mu) controlled negative regulation of PKC(eta) kinase activity. In this study, we show that differentially activated kinase cascades involving PDK1 and novel PKC isotypes are responsible for the regulation of PKC(mu) activity and consequently inhibit the JNK pathway.     

Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.     

3-磷酸肌醇依赖性蛋白激酶1结构和功能

PDK1可调节AGC激酶家族中一些重要蛋白激酶。这些激酶包括蛋白激酶B（PKB／Akt）、p70核小体S6激酶（p70 ribosomal S6 kinase,S6K）、血清和糖皮质激素诱导激酶（SGK）和蛋白激酶C（PKC）等,它们在细胞代谢、生长、增殖和存活等生理过程中具有重要作用。因此,了解PDK1生物学特性可能对其调节的AGC激酶持续活化的癌症治疗具有一定推动作用。本文对PDK1的结构、遗传和生化特点进行了综述。     

Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255-10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCmicro, or protein kinase D (PKD), but not PKCalpha/beta, PKCzeta/lambda, or PKCdelta/. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling.