Different protein kinase C isoforms determine growth factor specificity in neuronal cells

Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cdelta (PKCdelta), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCdelta in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCzeta-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCzeta, like PKCdelta, acts upstream of MEK, and PKCzeta can potentiate Raf-1 activation by EGF. Inhibition of PKCzeta also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCzeta or PKCdelta suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity.     

Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKCzeta, leading to phosphorylation of the PKB-PH domain on Thr(34). This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP(3) even in the presence of a ceramide-activated PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.     

The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.     

Regulation of caspase 9 through phosphorylation by protein kinase C zeta in response to hyperosmotic stress

Caspase 9 is a critical component of the mitochondrial or intrinsic apoptotic pathway and is activated by Apaf-1 following release of cytochrome c from mitochondria in response to a variety of stimuli. Caspase 9 cleaves and activates effector caspases, mainly caspase 3, leading to the demise of the cell. Survival signaling pathways can impinge on this pathway to restrain apoptosis. Here, we have identified Ser144 of human caspase 9as an inhibitory site that is phosphorylated in a cell-free system and in cells in response to the protein phosphatase inhibitor okadaic acid. Inhibitor sensitivity and interactions with caspase 9 indicate that the predominant kinase that targets Ser144 is the atypical protein kinase C isoform zeta (PKCzeta). Prevention of Ser144 phosphorylation by inhibition of PKCzeta or mutation of caspase 9 promotes caspase 3 activation. Phosphorylation of serine 144 in cells is also induced by hyperosmotic stress, which activates PKCzeta and regulates its interaction with caspase 9, but not by growth factors, phorbol ester, or other cellular stresses. These results indicate that phosphorylation and inhibition of caspase 9 by PKCzeta restrain the intrinsic apoptotic pathway during hyperosmotic stress. This work provides further evidence that caspase 9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis.     

Novel functions of the phospholipase D2-Phox homology domain in protein kinase Czeta activation

It has been established that protein kinase Czeta (PKCzeta) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKCzeta remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKCzeta activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKCzeta-kinase domain also induces the activation loop phosphorylation of PKCzeta and downstream signal stimulation, as measured by p70 S6 kinase phosphorylation. Furthermore, only the PLD2-PX domain directly stimulates PKCzeta activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKCzeta. The mutant that substitutes the triple lysine residues (Lys101, Lys102, and Lys103) within the PLD2-PX domain with alanine abolishes interaction with the PKCzeta-kinase domain and activation of PKCzeta. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKCzeta activation is induced by its PX domain performing both direct activation of PKCzeta and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells.     

Ceramide directly activates protein kinase C zeta to regulate a stress-activated protein kinase signaling complex

We have previously shown that interleukin 1 (IL-1)-receptor-generated ceramide induces growth arrest in smooth muscle pericytes by activating an upstream kinase in the stress-activated protein kinase (SAPK) cascade. We now report the mechanism by which ceramide activates the SAPK signaling pathway in human embryonic kidney cells (HEK-293). We demonstrate that ceramide activation of protein kinase C zeta (PKCzeta) mediates SAPK signal complex formation and subsequent growth suppression. Ceramide directly activates both immunoprecipitated and recombinant human PKCzeta in vitro. Additionally, ceramide activates SAPK activity, which is blocked with a dominant-negative mutant of PKCzeta. Co-immunoprecipitation studies reveal that ceramide induces the association of SAPK with PKCzeta, but not with PKCepsilon. In addition, ceramide treatment induces PKCzeta association with phosphorylated SEK and MEKK1, elements of the SAPK signaling complex. The biological role of ceramide to induce cell cycle arrest is mimicked by overexpression of a constitutively active PKCzeta. Together, these studies demonstrate that ceramide induces cell cycle arrest by enhancing the ability of PKCzeta to form a signaling complex with MEKK1, SEK, and SAPK.     

Ceramide-induced inhibition of Akt is mediated through protein kinase Czeta: implications for growth arrest.

We recently demonstrated that ceramide-coated balloon catheters limit vascular smooth muscle cell (VSMC) growth after stretch injury in vivo. In that study, inhibition of VSMC growth was correlated with a decrease in phosphorylation of the cell survival kinase Akt (protein kinase B). Utilizing cultured A7r5 VSMCs, we have now examined the mechanism by which ceramide inhibits Akt phosphorylation/activation. Our initial studies showed that ceramide-induced inhibition of Akt phosphorylation was not mediated through diminution in phosphoinositide 3-kinase activity. As we have previously demonstrated that protein kinase Czeta (PKCzeta) is a target of ceramide, we proposed an alternative signaling mechanism by which ceramide induces inhibition of Akt through activation of PKCzeta. We demonstrate that C(6)-ceramide (but not the inactive analog dihydro-C(6)-ceramide) induced PKCzeta activity and also caused a selective increase in the association between Akt and PKCzeta, without affecting PKCepsilon, in A7r5 cells. In addition, the ability of ceramide to significantly decrease platelet-derived growth factor-induced Akt phosphorylation or cell proliferation was abrogated in A7r5 cells overexpressing a dominant-negative mutant of PKCzeta. Taken together, these data suggest that ceramide-mediated activation of PKCzeta leads to diminished Akt activation and consequent growth arrest in VSMCs. The therapeutic potential for ceramide to limit dysregulated VSMC growth has direct applicability to vascular diseases such as restenosis and atherosclerosis.     

The pivotal role of protein kinase C zeta (PKCzeta) in insulin- and AMP-activated protein kinase (AMPK)-mediated glucose uptake in muscle cells

Insulin and AMP-activated protein kinase (AMPK) signal pathways are involved in the regulation of glucose uptake. The integration of signals between these two pathways to maintain glucose homeostasis remains elusive. In this work, stimulation of insulin and berberine conferred a glucose uptake or surface glucose transporter 4 (GLUT4) translocation that was less than simple summation of their effects in insulin-sensitive muscle cells. Using specific inhibitors to key kinases of both pathways and PKCzeta small interference RNA, protein kinase C zeta (PKCzeta) was found to regulate insulin-stimulated protein kinase B (PKB) activation and inhibit AMPK activity on dorsal cell surface. In the presence of berberine, PKCzeta controlled AMPK activation and AMPK blocked PKB activity in perinuclear region. The inhibition effect of PKCzeta on AMPK activation or the arrestment of PKB activity by AMPK still existed in basal condition. These results suggest that there is antagonistic regulation between insulin and AMPK signal pathways, which is mediated by the switch roles of PKCzeta.     

Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.     

Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C.

We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or alpha2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gbeta gamma stimulation of phosphoinositide 3-kinase gamma (PI-3Kgamma), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCzeta at concentrations as low as 1 nM. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of beta gamma binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling.     

Role of atypical protein kinase C in estradiol-triggered G1/S progression of MCF-7 cells

Expression of a dominant negative atypical protein kinase C (aPKC), PKCzeta, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERalpha), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCzeta by Thr410 phosphorylation. Serine phosphorylation of PKCzeta is simultaneously induced. PKCzeta activation leads to recruitment of Ras to a multimolecular complex that also includes hERalpha, Src, PI 3-kinase, and aPKC. We propose that PKCzeta pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.     

Cross-talk between protein kinases Czeta and B in cyclic AMP-mediated sodium taurocholate co-transporting polypeptide translocation in hepatocytes

Cyclic AMP stimulates taurocholate (TC) uptake and sodium taurocholate co-transporting polypeptide (Ntcp) translocation in hepatocytes via the phosphoinositide-3 kinase (PI3K) signaling pathway. The aim of the present study was to determine whether protein kinase (PK) Czeta, one of the downstream mediators of the PI3K signaling pathway, is involved in cAMP-mediated stimulation of TC uptake. Studies were conducted in isolated rat hepatocytes and in HuH-7 cells stably transfected with rat liver Ntcp (HuH-Ntcp cells). Studies in hepatocytes showed that cAMP activates PKCzeta in a PI3K-dependent manner without inducing translocation of PKCzeta to the plasma membrane. Inhibition of cAMP-induced PKCzeta activity by myristoylated PKC (zeta/lambda) pseudosubstrate, a specific inhibitor of PKCzeta, and G? 6850, a PKC inhibitor, resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. Studies in HuH-Ntcp cells showed that inhibition of cAMP-induced PKCzeta activation by dominant-negative (DN) PKCzeta resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. DN PKCzeta also inhibited wild-type PKCzeta-induced increases in PKCzeta activity, TC uptake, and Ntcp translocation. Myristoylated PKC (zeta/lambda) pseudosubstrate and DN PKCzeta also inhibited cAMP-induced activation of PKB in hepatocytes and HuH-Ntcp cells, respectively. Neither DN PKB nor constitutively active PKB affected cAMP-induced activation of PKCzeta, and wild-type PKCzeta did not activate PKB. Taken together, these results suggest that cAMP-induced activation of PKB is dependent on cAMP-induced stimulation of PKCzeta. It is proposed that cAMP-induced Ntcp translocation involves the activation of the PI3K/PKCzeta signaling pathway followed by the activation of the PI3K/PKB signaling pathway.     

The MARCKS protein plays a critical role in phosphatidylinositol 4,5-bisphosphate metabolism and directed cell movement in vascular endothelial cells

The MARCKS protein (myristoylated alanine-rich C kinase substrate) is an actin- and calmodulin-binding protein that is expressed in many mammalian tissues. The role of MARCKS in endothelial signaling responses is incompletely understood. We found that siRNA-mediated knockdown of MARCKS in cultured endothelial cells abrogated directed cell movement in a wound healing assay. We used biochemical and cell imaging approaches to explore the role of MARCKS in endothelial signal transduction pathways activated by insulin. Insulin treatment of vascular endothelial cells promoted the dose- and time-dependent phosphorylation of MARCKS. Cell imaging and hydrodynamic approaches revealed that MARCKS is targeted to plasmalemmal caveolae and undergoes subcellular translocation in response to insulin. Insulin treatment promoted an increase in levels of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) in plasmalemmal caveolae. The insulin-stimulated increase in caveolar PIP(2) was blocked by siRNA-mediated knockdown of MARCKS, as determined using both biochemical assays and imaging studies using FRET-based PIP(2) biosensors. The critical role of PIP(2) in MARCKS responses was explored by examining the PIP(2)- and actin-binding proteins Arp2/3 and N-WASP. Insulin promoted the rapid and robust phosphorylation of both N-WASP and Arp2/3, but these phosphorylation responses were markedly attenuated by siRNA-mediated MARCKS knockdown. Moreover, MARCKS knockdown effectively abrogated N-WASP activation in response to insulin, as determined using a FRET-based N-WASP activity biosensor. Taken together, these studies show that MARCKS plays a key role in insulin-dependent endothelial signaling to PIP(2) and is a critical determinant of actin assembly and directed cell movement in the vascular endothelium.     

Characterization of PDK2 activity against protein kinase B gamma

Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase controlled by insulin, various growth factors, and phosphatidylinositol 3-kinase. Full activation of the PKB enzyme requires phosphorylation of a threonine in the activation loop and a serine in the C-terminal tail. PDK1 has clearly been shown to phosphorylate the threonine, but the mechanism leading to phosphorylation of the serine, the PDK2 site, is unclear. A yeast two-hybrid screen using full-length human PKBgamma identified protein kinase C (PKC) zeta, an atypical PKC, as an interactor with PKBgamma, an association requiring the pleckstrin homology domain of PKBgamma. Endogenous PKBgamma was shown to associate with endogenous PKCzeta both in cos-1 cells and in 3T3-L1 adipocytes, demonstrating a physiological interaction. Immunoprecipitates of PKCzeta, whether endogenous PKCzeta from insulin-stimulated 3T3-L1 adipocytes or overexpressed PKCzeta from cos-1 cells, phosphorylated S472 (the C-terminal serine phosphorylation site) of PKBgamma, in vitro. In vivo, overexpression of PKCzeta stimulated the phosphorylation of approximately 50% of the PKBgamma molecules, suggesting a physiologically meaningful effect. However, pure PKCzeta protein was incapable of phosphorylating S472 of PKBgamma. Antisense knockout studies and use of a PDK1 inhibitor showed that neither PKB autophosphorylation nor phosphorylation by PDK1 accounted for the S472 phosphorylation in PKCzeta immunoprecipitates. Staurosporine inhibited the PKCzeta activity but not the PDK2 activity in PKCzeta immunoprecipitates. Together these results indicate that an independent PDK2 activity exists that physically associates with PKCzeta and that PKCzeta, by binding PKBgamma, functions to deliver the PDK2 to a required location. PKCzeta thus functions as an adaptor, associating with a staurosporine-insensitive PDK2 enzyme that catalyzes the phosphorylation of S472 of PKBgamma. Because both PKCzeta and PKB have been proposed to be required for mediating a number of crucial insulin responses, formation of an active signaling complex containing PKCzeta, PKB, and PDK2 is an attractive mechanism for ensuring that all the critical sites on targets such as glycogen synthase kinase-3 are phosphorylated.     

Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling

The intracellular redox state is a key determinant of cell fate, such as cell survival, proliferation, differentiation, and apoptosis. Redox imbalance is closely linked to a variety of human diseases, so that the intracellular redox condition should be tightly regulated. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. ROS are not only toxicants to the cell, but also second messengers in intracellular signal transduction, and control the action of several signaling pathways, including mitogen-activated protein (MAP) kinases. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase of the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways, which is preferentially activated in response to various types of stress such as oxidative stress and plays pivotal roles in a wide variety of cellular responses. Recent studies have revealed that ASK1 is also required for innate immune response through ROS production. In this review, we focus on redox control of cell function by MAP kinase signaling, and provide the advanced mechanism of redox-regulated ASK1 activation and physiological roles of the ASK1-MAP kinase pathway in stress signaling.     

Insulin-sensitive phospholipid signaling systems and glucose transport. Update II

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidylcholine with subsequent increases in phosphatidic acid (PA) and diacylglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.