Calcium- and diacylglycerol-independent serine/threonine-protein kinase that functions in phosphatidylinositol 3-kinase (PI3K) pathway and mitogen-activated protein (MAP) kinase cascade, and is involved in NF-kappa-B activation, mitogenic signaling, cell proliferation, cell polarity, inflammatory response and maintenance of long-term potentiation (LTP). Upon lipopolysaccharide (LPS) treatment in macrophages, or following mitogenic stimuli, functions downstream of PI3K to activate MAP2K1/MEK1-MAPK1/ERK2 signaling cascade independently of RAF1 activation. Required for insulin-dependent activation of AKT3, but may function as an adapter rather than a direct activator. Upon insulin treatment may act as a downstream effector of PI3K and contribute to the activation of translocation of the glucose transporter SLC2A4/GLUT4 and subsequent glucose transport in adipocytes. In EGF-induced cells, binds and activates MAP2K5/MEK5-MAPK7/ERK5 independently of its kinase activity and can activate JUN promoter through MEF2C. Through binding with SQSTM1/p62, functions in interleukin-1 signaling and activation of NF-kappa-B with the specific adapters RIPK1 and TRAF6. Participates in TNF-dependent transactivation of NF-kappa-B by phosphorylating and activating IKBKB kinase, which in turn leads to the degradation of NF-kappa-B inhibitors. In migrating astrocytes, forms a cytoplasmic complex with PARD6A and is recruited by CDC42 to function in the establishment of cell polarity along with the microtubule motor and dynein. In association with FEZ1, stimulates neuronal differentiation in PC12 cells. In inflammatory response, is required for the T-helper 2 (Th2) differentiation process, including interleukines production, efficient activation of JAK1 and the subsequent phosphorylation and nuclear translocation of STAT6. May be involved in development of allergic airway inflammation (asthma), a process dependent on Th2 immune response. In NF-kappa-B-mediated inflammatory response, can relieve the SETD6-dependent repression of NF-kappa-B target genes by phosphorylating the RELA subunit at 'Ser-311'. Is necessary and sufficient for LTP maintenance in hippocampal CA1 pyramidal cells.
BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.
J. Immunol. 165, 4632-4639 (2000)[PubMed:11035106]
Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC zeta partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.
The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Here, we demonstrate that human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells.
Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-alpha inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (alpha, beta, delta, epsilon, eta, and zeta) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes alpha and eta) are potent activators of c-Raf1, atypical PKC-zeta cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-alpha and PKC-eta was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-alpha, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-O-tetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.
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.
Interacting selectively and non-covalently with a 14-3-3 protein. A 14-3-3 protein is any of a large family of approximately 30kDa acidic proteins which exist primarily as homo- and heterodimers within all eukaryotic cells, and have been implicated in the modulation of distinct biological processes by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. Each 14-3-3 protein sequence can be roughly divided into three sections: a divergent amino terminus, the conserved core region and a divergent carboxyl terminus. The conserved middle core region of the 14-3-3s encodes an amphipathic groove that forms the main functional domain, a cradle for interacting with client proteins.
Interacting selectively and non-covalently with any of the insulin receptor substrate (IRS) proteins, adaptor proteins that bind to the transphosphorylated insulin and insulin-like growth factor receptors, are themselves phosphorylated and in turn recruit SH2 domain-containing signaling molecules to form a productive signaling complex.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
BACKGROUND: Asymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1-6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells. However, little is known about a link between PAR proteins and the GTPases in cell polarization. RESULTS: Here we have cloned cDNAs for three human homologues of PAR6, designated PAR6alpha, beta and gamma, comprising 345, 372 and 376 amino acids, respectively. The PAR6 proteins harbour a PDZ domain and a CRIB-like motif, and directly interact with GTP-bound Rac and Cdc42 via this motif and with the aPKC isoforms PKCiota/lambda and PKCzeta via the N-terminal head-to-head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell movement. CONCLUSION: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.
Evidence
2:
Inferred from Physical InteractionUniProtKB
It has been reported that prostate apoptosis response-4 (PAR-4) binds to and inhibits protein kinase Czeta (PKCzeta) which phosphorylates IkappaB kinase beta (IKKbeta) for nuclear factor kappaB (NFkappaB) activation, while p62 binds to and recruits PKCzeta to the NFkappaB signaling complex. Thus, a mechanism to coordinate the two binding proteins for the regulation of PKCzeta is expected to exist. The present data show that p62 and PAR-4 do not compete for PKCzeta binding but directly interact each other and form a ternary complex with PKCzeta. Furthermore, p62 not only enhances the catalytic activity of PKCzeta but also reactivates catalytically inactive PAR-4-bound PKCzeta. As the result, over-expression of p62 protects cells from PAR-4-mediated inactivation of NFkappaB and apoptotic death. Thus, the regulatory role of p62 for free and PAR-4-bound PKCzeta is important in activation of NFkappaB.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Mammary gland development is coupled to reproductive events by hormonal cues of ovarian and pituitary origin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. In this study KIBRA was identified as a transcript showing decreased expression associated with failed mammary gland development in Prlr knockout mammary epithelium. It is strongly up-regulated during pregnancy, falls during lactation and is again up-regulated during involution of the gland at weaning. A bioinformatic approach was undertaken to identify potential binding partners which interact with the WW domains of KIBRA. We show that KIBRA binds to a WW domain binding motif, PPxY, in the tyrosine kinase receptor DDR1, and dissociates upon treatment with the DDR1 ligands collagen type I or IV. In addition we show that KIBRA and DDR1 also interact with PKCz to form a trimeric complex. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodeling events of mammary gland development.
Evidence
4:
Inferred from Physical InteractionIntAct
Using yeast two-hybrid, we isolated atypical PKCzeta as a PKCtheta-interacting kinase and demonstrated that it selectively interacted with, and was phosphorylated by, PKCtheta. Importantly, however, both atypical PKCzeta and PKCiota were functionally required in TCR/CD28-mediated activation of NF-kappaB downstream of PKCtheta in Jurkat T cells albeit, activation responses of PKCzeta-deficient CD3+ T cells were comparable with wildtype controls. This normal activation thresholds of PKCzeta-/- T cells suggested that PKCiota, the closest structural relative, might play a compensatory role in TCR/CD28-induced signalling. Consistently, both PKCzeta and PKCiota resided in the plasma membrane lipid raft microdomains of Jurkat as well as primary mouse CD3+ T cells. Thus, PKCtheta, the established constituent of the immunological synapse, physically and functionally interacted with PKCzeta and PKCiota. Together, these data demonstrate that atypical PKCzeta/iota isotypes serve as direct downstream targets of PKCtheta in the signalling pathway leading to NF-kappaB activation in T lymphocytes.
Evidence
5:
Inferred from Physical InteractionUniProtKB
Centaurin-alpha(1) is a member of the family of ADP-ribosylation factors (ARF) GTPase activating proteins (GAPs), although ARF GAP activity has not yet been demonstrated. The human homologue, centaurin-alpha(1) functionally complements the ARF GAP activity of Gcs1 in yeast. Although Gcs1 is involved in the formation of actin filaments in vivo, the function of centaurin remains elusive. We have identified a number of novel centaurin-alpha(1) binding partners; including CKIalpha and nucleolin. In this report, we have focused on the interaction of centaurin-alpha(1) with PKC. All groups of PKC associate directly through their cysteine rich domains. Centaurin-alpha(1) is also a substrate for all PKC classes and we have identified the two sites of phosphorylation. This is the first report of a kinase that phosphorylates centaurin-alpha(1).
Evidence
6:
Inferred from Physical InteractionIntAct
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
Evidence
7:
Inferred from Physical InteractionUniProtKB
PKCzeta (protein kinase Czeta) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane in stimulated cells. How PKCzeta modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCzeta identified 80K-H protein as an interactor with PKCzeta. GST (glutathione S-transferase) pull-down assays with GST-tagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKCzeta from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3-5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKCzeta, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCzeta, 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKCzeta, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCzeta-80K-H-munc18c complex that enhances GLUT4 translocation to the plasma membrane.
Evidence
8:
Inferred from Physical InteractionIntAct
The polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3zeta and eta (Par-5), Par-6a, -b, -c, and PKClambda (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex "modules" formed around MARK4 (Par-1), Par-3.Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1.Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity.
Evidence
9:
Inferred from Physical InteractionIntAct
HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.
Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.
Interacting selectively and non-covalently with a protein kinase, any enzyme that catalyzes the transfer of a phosphate group, usually from ATP, to a protein substrate.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
A process that is carried out at the cellular level which results in dynamic structural changes to the arrangement of constituent parts of cytoskeletal structures comprising actin filaments and their associated proteins.
The immediate defensive reaction (by vertebrate tissue) to infection or injury caused by chemical or physical agents. The process is characterized by local vasodilation, extravasation of plasma into intercellular spaces and accumulation of white blood cells and macrophages.
The memory process that deals with the storage, retrieval and modification of information a long time (typically weeks, months or years) after receiving that information. This type of memory is typically dependent on gene transcription regulated by second messenger activation.
A process that modulates synaptic plasticity such that synapses are changed resulting in the increase in the rate, or frequency of synaptic transmission at the synapse.
A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of cytoskeletal structures comprising microtubules and their associated proteins.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
J. Immunol. 165, 4632-4639 (2000)[PubMed:11035106]
Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC zeta partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.
Any process that enhances the establishment or increases the extent of the excitatory postsynaptic potential (EPSP) which is a temporary increase in postsynaptic potential due to the flow of positively charged ions into the postsynaptic cell. The flow of ions that causes an EPSP is an excitatory postsynaptic current (EPSC) and makes it easier for the neuron to fire an action potential.
The process of creating protein oligomers, compounds composed of a small number, usually between three and ten, of component monomers that are not all identical. Oligomers may be formed by the polymerization of a number of monomers or the depolymerization of a large protein polymer.
A series of reactions, mediated by the intracellular serine/threonine kinase protein kinase C, which occurs as a result of a single trigger reaction or compound.
Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
A 1779-bp complementary DNA (cDNA) that encodes protein kinase C-zeta (PKC-zeta) has been isolated from a human frontal cortex library using traditional plaque-screening methods and PCR screening. The deduced 592-amino-acid sequence of the human PKC-zeta clone has a 95-96% identity to those deduced from the previously described rat and mouse PKC-zeta clones.
The directed movement of a vesicle along a microtubule, mediated by motor proteins. This process begins with the attachment of a vesicle to a microtubule, and ends when the vesicle reaches its final destination.
IEAOrtholog Compara
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reaction
Atypical PKCs (PRKCI and PRKCZ) exhibit an elevated basal enzymatic activity (that may be due to the interaction with SMG1 or SQSTM1) and are not regulated by diacylglycerol, phosphatidylserine, phorbol esters or calcium ions. Two specific sites, Thr-410 (activation loop of the kinase domain) and Thr-560 (turn motif), need to be phosphorylated for its full activation. Phosphatidylinositol 3,4,5-trisphosphate might be a physiological activator (By similarity).
Protein involved in the localized protective response to tissue damage, microbial infection, or the presence of foreign matter. It is characterized by swelling, redness, heat and pain and involves a complex series of events including vascular changes and accumulation of blood cells, such as neutrophil leucocytes and mononuclear phagocytes, at the site of injury.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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