Protein kinase C mu is located at the Golgi compartment

Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes.     

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.     

Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes.

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.     

Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) mu) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.     

Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop

Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.     

Dimerization-induced activation of soluble insulin/IGF-1 receptor kinases: an alternative mechanism of activation.

To study the role of kinase dimerization in the activation of the insulin receptor (IR) and the insulin-like growth factor receptor-1 (IGF-1R), we have cloned, expressed, and purified monomeric and dimeric forms of the corresponding soluble kinase domains via the baculovirus expression system. Dimerization of the kinases was achieved by fusion of the kinase domains to the homodimeric glutathione S-transferase (GST). Kinetic analyses revealed that kinase dimerization results in substantial increases (10-100-fold) in the phosphotransferase activity in both the auto- and substrate phosphorylation reactions. Furthermore, kinase dimerization rendered the autophosphorylation reaction concentration-independent. However, whereas dimerization was required for the rapid autophosphorylation of the kinases, it was not essential for the enhanced kinase activity in substrate phosphorylation reactions. Comparison of HPLC-phosphopeptide maps of the monomeric and dimeric kinases revealed that dimerization leads to an increased phosphorylation of the regulatory activation loop of the kinases, strongly suggesting that bis- and trisphosphorylation of the activation loop are mediated by transphosphorylation within the kinase dimers. Most strikingly, limited proteolysis revealed that GST-mediated dimerization by itself had a major impact on the conformation of the activation loop by stabilizing a conformation that corresponds to the active, phosphorylated form of the kinase. Thus, in analogy to the insulin/IGF-1-ligated holoreceptors, the dimeric GST-kinases are primed to rapid autophosphorylation by an increase in the local concentration of both phosphoryl donor and phosphoryl acceptor sites and by a dimerization-induced conformational change of the activation loop that leads to an efficient transphosphorylation of the regulatory tyrosine residues.     

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.     

Phosphorylation of EEA1 by p38 MAP kinase regulates mu opioid receptor endocytosis

Morphine analgesic properties and side effects such as tolerance are mediated by the mu opioid receptor (MOR) whose endocytosis is considered of primary importance for opioid pharmacological effects. Here, we show that p38 mitogen-activated protein kinase (MAPK) activation is required for MOR endocytosis and sufficient to trigger its constitutive internalization in the absence of agonist. Further studies established a functional link between p38 MAPK and the small GTPase Rab5, a key regulator of endocytosis. Expression of an activated mutant of Rab5 stimulated endocytosis of MOR ligand-independently in wild-type but not in p38alpha-/- cells. We found that p38alpha can phosphorylate the Rab5 effectors EEA1 and Rabenosyn-5 on Thr-1392 and Ser-215, respectively, and these phosphorylation events regulate the recruitment of EEA1 and Rabenosyn-5 to membranes. Moreover, phosphomimetic mutation of Thr-1392 in EEA1 can bypass the requirement for p38alpha in MOR endocytosis. Our results highlight a novel mechanism whereby p38 MAPK regulates receptor endocytosis under physiological conditions via phosphorylation of Rab5 effectors.     

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.     

PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.     

Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.     

Regulation of Chk2 ubiquitination and signaling through autophosphorylation of serine 379

The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.     

Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex

Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.     

cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes

In addition to the classical role of protein kinase C (PKC) as a mediator of transmembrane signals initiated at the plasma membrane, there is also significant evidence to suggest that a more sustained PKC activity is necessary for a variety of long term cellular responses. To date, the subcellular localization of PKC during sustained activation has not been extensively studied. We report here that long term activation of PKC (1 h) leads to the selective translocation of classical PKC isoenzymes, alpha and betaII, to a juxtanuclear compartment. Juxtanuclear translocation of PKC required an intact C1 and C2 domain, and occurred in a microtubule-dependent manner. This juxtanuclear compartment was localized close to the Golgi complex but displayed no overlap with Golgi markers, and was resistant to dispersal with Golgi disrupting agents, brefeldin A and nocodazole. Further characterization revealed that PKCalpha and betaII translocated to a compartment that colocalized with the small GTPase, rab11, which is a marker for the subset of recycling endosomes concentrated around the microtubule-organizing center/centrosome. Analysis of the functional consequence of cPKC translocation on membrane recycling demonstrated a cPKC-dependent sequestration of transferrin, a marker of membrane recycling, in the cPKC compartment. These results identify a novel site for cPKC translocation and define a novel function for the sustained activation of PKCalpha and betaII in regulation of recycling components.     

A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150(Glued) by live-cell imaging. Time-lapse analysis of p150(Glued) revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150(Glued) phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150(Glued) phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150(Glued) phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150(Glued) during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150(Glued)-labeled microtubules just prior to transport, implicating microtubules and dynactin in a search-capture mechanism for minus-end-directed organelles.     

The localization of protein kinase Cdelta in different subcellular sites affects its proapoptotic and antiapoptotic functions and the activation of distinct downstream signaling pathways

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis and survival in diverse cellular systems. PKCdelta translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKCdelta constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKCdelta affects its apoptotic and survival functions. PKCdelta-Cyto, PKCdelta-Mito, and PKCdelta-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKCdelta-ER. PKCdelta-Cyto and PKCdelta-Mito underwent cleavage, whereas no cleavage was observed in the PKCdelta-Nuc and PKCdelta-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKCdelta-Cyto and PKCdelta-Mito. In contrast to the apoptotic effects of the PKCdelta-Cyto, PKCdelta-Mito, and PKCdelta-Nuc, the PKCdelta-ER protected the cells from tumor necrosis factor-related apoptosis-inducing ligand-induced and etoposide-induced apoptosis. Moreover, overexpression of a PKCdelta kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKCdelta and increased tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. The localization of PKCdelta differentially affected the activation of downstream signaling pathways. PKCdelta-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKCdelta-Nuc increased c-Jun NH(2)-terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKCdelta-Cyto, whereas c-Jun NH(2)-terminal kinase activation mediated the apoptotic effect of PKCdelta-Nuc. Our results indicate that the subcellular localization of PKCdelta plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways.     

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.     

Integrin-mediated Activation of Focal Adhesion Kinase Is Required for Signaling to Jun NH2-terminal Kinase and Progression through the G1 Phase of the Cell Cycle

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.