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PubMed2002

Phosphatidylinositol 4,5-biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2.

Wiskott-Aldrich syndrome protein (WASP)/Scar family proteins promote actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. While Scar/WAVE proteins are thought to be involved in lamellipodia protrusion, the hematopoietic WASP has been implicated in various actin-based processes such as chemotaxis, podosome formation, and phagocytosis. Here we show that the ubiquitously expressed N-WASP is essential for actin assembly at the surface of endomembranes induced as a consequence of increased phosphatidylinositol 4,5-biphosphate (PIP2) levels. This process resulting in the motility of intracellular vesicles at the tips of actin comets involved the recruitment of the Src homology 3 (SH3)-SH2 adaptor proteins Nck and Grb2 as well as of WASP interacting protein (WIP). Reconstitution of vesicle movement in N-WASP-defective cells by expression of various N-WASP mutant proteins revealed three independent domains capable of interaction with the vesicle surface, of which both the WH1 and the polyproline domains contributed significantly to N-WASP recruitment and/or activation. In contrast, the direct interaction of N-WASP with the Rho-GTPase Cdc42 was not required for reconstitution of vesicle motility. Our data reveal a distinct cellular phenotype for N-WASP loss of function, which adds to accumulating evidence that the proposed link between actin and membrane dynamics may, at least partially, be reflected by the actin-based movement of vesicles through the cytoplasm.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Cloning and characterization of Cbl-associated protein splicing isoforms.

Cbl-associated protein (CAP) is an adaptor protein that plays important roles in both signal transduction and cytoskeleton rearrangement. Alternative splicing of the gene SORBS1 results in multiple isoforms of CAP protein. We report here the cloning of 3 new CAP isoforms, CAP2, CAP3, and CAP4, from mouse adipose tissue. RT-PCR analyses reveal that the isoform mRNAs are differentially expressed. CAP2, CAP3, and CAP4 contain a coiled-coil domain. In addition, CAP4 contains a proline-rich region, part of which exists in CAP3. Coimmunoprecipitation experiments show that CAP4 forms a homodimeric complex. While these new isoforms similarly interact with Cbl, they exhibit varied binding specificity toward vinculin. In contrast to CAP1 and CAP2, CAP4 does not interact with vinculin, and CAP3 binds with low affinity. Immunofluorescence analysis demonstrates differential subcellular localization of Myc-tagged CAP isoforms in 3T3-L1 adipocytes. These results suggest that these new isoforms of CAP might play different signaling roles.

3T3 Cells↗
PubMed2001

Integrin tyrosine phosphorylation in platelet signaling.

The beta 3 integrin cytoplasmic tyrosine (ICY) motif of alpha IIb beta 3 becomes tyrosine phosphorylated during platelet aggregation, causing Shc and myosin to interact with the beta-integrin cytoplasmic domain. Platelets from mice lacking beta 3 ICY motif tyrosines formed defective aggregates and poorly retracted clots, establishing integrin tyrosine phosphorylation as a key mediator of beta 3-integrin signals.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton.

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.

Actin-Related Protein 2↗
PubMed2002

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin.

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposi's sarcoma-associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin-DeltaEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin-DeltaEx3 contain a mitochondrial-targeting sequence, an N-terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl-2 homology)-like domain. These suggested that K7 is a new viral anti-apoptotic protein and survivin-DeltaEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl-2 resides. K7 does not associate with Bax, but does bind to Bcl-2 via its putative BH2 domain. In addition, K7 binds to active caspase-3 via its BIR domain and thus inhibits the activity of caspase-3. The BH2 domain of K7 is crucial for the inhibition of caspase-3 activity and is therefore essential for its anti-apoptotic function. Furthermore, K7 bridges Bcl-2 and activated caspase-3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti-apoptotic complex that presents effector caspases to Bcl-2, enabling Bcl-2 to inhibit caspase activity. These data also suggest that survivin-DeltaEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor-of-apoptosis protein).

Alternative Splicing↗
PubMed2003

Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization.

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (K(d)) of 880 nM. Although HS1 is a modest F-actin-binding protein with a K(d) of 400 nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott-Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

Actin-Related Protein 2↗
PubMed2002

Identification of a novel Ras-regulated proapoptotic pathway.

BACKGROUND: The Ras-GTPase controls cell fate decisions through the binding of an array of effector molecules, such as Raf and PI 3-kinase, in a GTP-dependent manner. NORE1, a noncatalytic polypeptide, binds specifically to Ras-GTP and to several other Ras-like GTPases. NORE is homologous to the putative tumor suppressor RASSF1 and to the Caenorhabditis elegans polypeptide T24F1.3. RESULTS: We find that all three NORE-related polypeptides bind selectively to the proapoptotic protein kinase MST1, a member of the Group II GC kinases. Endogenous NORE and MST1 occur in a constitutive complex in vivo that associates with endogenous Ras after serum stimulation. Targeting recombinant MST1 to the membrane, either through NORE or myristoylation, augments the apoptotic efficacy of MST1. Overexpression of constitutively active Ki-RasG12V promotes apoptosis in a variety of cell lines; Ha-RasG12V is a much less potent proapoptotic agent; however, a Ha-RasG12V effector loop mutant (E37G) that binds NORE, but not Raf or PI 3-kinase, exhibits proapoptotic efficacy approaching that of Ki-RasG12V. The apoptotic action of both Ki-RasG12V and Ha-RasG12V, E37G is suppressed by overexpression of the MST1 carboxy-terminal noncatalytic segment or by the NORE segment that binds MST1. CONCLUSIONS: MST1 is a phylogenetically conserved partner of the NORE/RASSF polypeptide family, and the NORE-MST1 complex is a novel Ras effector unit that mediates the apoptotic effect of Ki-RasG12V.

3T3 Cells↗
PubMed2002

DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines.

The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10-related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines.

Actins↗
PubMed2003

A highly conserved NTRK3 C-terminal sequence in the ETV6-NTRK3 oncoprotein binds the phosphotyrosine binding domain of insulin receptor substrate-1: an essential interaction for transformation.

Receptor tyrosine kinases are integral components of cellular signaling pathways and are frequently deregulated in malignancies. The NTRK family of neurotrophin receptors mediate neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in oncogenesis. The ETV6-NTRK3 (EN) gene fusion occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma, and encodes the oligomerization domain of the ETV6 transcription factor fused to the protein-tyrosine kinase domain of NTRK3. The EN protein functions as a constitutively active protein-tyrosine kinase with potent transforming activity in multiple cell lineages, and EN constitutively activates both the Ras-MAPK and phosphatidylinositol 3-kinase-Akt pathways. EN transformation is associated with constitutive tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Further, IRS-1 functions as the adaptor protein linking EN to downstream signaling pathways. However, the exact nature of the EN-IRS-1 interaction remains unknown. We now demonstrate that EN specifically binds the phosphotyrosine binding domain of IRS-1 via an interaction at the C terminus of EN. An EN mutant lacking the C-terminal 19 amino acids does not bind IRS-1 and lacks transforming ability. Moreover, expression of an IRS-1 polypeptide containing the phosphotyrosine binding domain acts in a dominant negative manner to inhibit EN transformation, and overexpression of IRS-1 potentiates EN transforming activity. These findings indicate that EN.IRS-1 complex formation through the NTRK3 C terminus is essential for EN transformation.

Agar↗
PubMed2002

Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: roles of protein kinase Cdelta, Src/proline-rich tyrosine kinase 2, and epidermal growth receptor trans-activation.

Agonist activation of endogenous angiotensin II (Ang II) AT(1) receptors expressed in hepatic C9 cells markedly stimulated inositol phosphate production, phosphorylation of the proline-rich tyrosine kinase PyK-2, and ERK activation. Ang II caused activation of protein kinase C delta (PKCdelta) in C9 cells, and its stimulatory actions on Pyk2 and extracellularly regulated kinase (ERK) phosphorylation were abolished by PKC depletion and selective inhibition of PKCdelta by rottlerin, but not by Ca(2+)-chelators. These effects, and the similar actions of the Src kinase inhibitor PP2 indicate the involvement of PKCdelta and Src kinase in ERK activation. In C9 cells, phorbol-12-myristate-13-acetate (PMA) caused much greater phosphorylation of Pyk2 and ERK than the Ca(2+) ionophore ionomycin, and the effects of PMA and Ang II were abolished in PKC-depleted cells. Ang II increased the association of Pyk2 with Src and with the epidermal growth factor receptor (EGF-R). EGF caused much greater tyrosine phosphorylation of the EGF-R than Ang II and PMA. Ang II-induced activation of ERK, but not Pyk2, was prevented by inhibition of EGF receptor phosphorylation by AG 1478 and of Src kinase by PP1. Ang II also increased the association of the adaptor protein Grb2 with the EGF-R. These findings indicate that Src and Pyk2 act upstream of the EGF-R and that the majority of Ang II-induced ERK phosphorylation is dependent on trans-activation of the EGF-R. Ang II-induced ERK activation in C9 cells is initiated by a PKCdelta-dependent but Ca(2+)-independent mechanism and is mediated by the Src/Pyk2 complex through trans-activation of the EGF-R.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways.

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.

Activation of the transcription factor NF-kappaB after engagement of the T cell receptor (TCR) is important for T cell proliferation and activation during the adaptive immune response. Recent reports have elucidated a signaling pathway that involves the protein kinase C (PKC), the scaffold protein CARD11 (also called CARMA-1), the caspase recruitment domain (CARD)-containing protein Bcl10, and the paracaspase (protease related to caspases) MALT1 as critical intermediates linking the TCR to the IkappaB kinase (IKK) complex. However, the events proximal to the TCR that initiate the activation of this signaling pathway remain poorly defined. We demonstrate that 3-phosphoinositide-dependent kinase 1 (PDK1) has an essential role in this pathway by regulating the activation of PKC and through signal-dependent recruiting of both PKC and CARD11 to lipid rafts. PDK1-associated PKC recruits the IKK complex, whereas PDK1-associated CARD11 recruits the Bcl10-MALT1 complex, thereby allowing activation of the IKK complex through Bcl10-MALT1-dependent ubiquitination of the IKK complex subunit known as NEMO (NF-kappaB essential modifier). Hence, PDK1 plays a critical role by nucleating the TCR-induced NF-kappaB activation pathway in T cells.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappaB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Phosphoregulation of Arp2/3-dependent actin assembly during receptor-mediated endocytosis.

In both yeast and mammals, endocytic internalization is accompanied by a transient burst of actin polymerization. The yeast protein kinases Prk1p and Ark1p, which are related to the mammalian proteins GAK and AAK1, are key regulators of this process. However, the molecular mechanism(s) by which they regulate actin assembly at endocytic sites have not yet been determined. The Eps15-like yeast protein Pan1p is a Prk1p substrate that is essential for endocytic internalization and for proper actin organization. Pan1p is an Arp2/3 activator and here we show that this activity is dependent on F-actin binding. Mutation of all 15 Prk1p-targeted threonines in Pan1p to alanines mimicked the ark1Delta prk1Delta phenotype, demonstrating that Pan1p is a key Prk1p target in vivo. Moreover, phosphorylation by Prk1p inhibited the ability of Pan1p to bind to F-actin and to activate the Arp2/3 complex, thereby identifying the endocytic phosphoregulation mechanism of Prk1p. We conclude that Prk1p phosphorylation of Pan1p shuts off Arp2/3-mediated actin polymerization on endocytic vesicles, allowing them to fuse with endosomes.

Actin-Related Protein 2↗
PubMed2005

Screening for genomic fragments that are methylated specifically in colorectal carcinoma with a methylated MLH1 promoter.

A subset of colorectal carcinomas (CRCs) is associated with microsatellite instability (MSI) of the genome. Although extensive methylation of CpG islands within the promoter regions of DNA mismatch repair genes such as MLH1 is thought to play a central role in tumorigenesis for MSI-positive sporadic CRCs, it has been obscure whether such aberrant epigenetic regulation occurs more widely and affects other cancer-related genes in vivo. Here, by using methylated CpG island amplification coupled with representational difference analysis (MCA-RDA), we screened genomic fragments that are selectively methylated in CRCs positive for MLH1 methylation, resulting in the identification of hundreds of CpG islands containing genomic fragments. Methylation status of such CpG islands was verified for 28 genomic clones in 8 CRC specimens positive for MLH1 methylation and the corresponding paired normal colon tissue as well as in 8 CRC specimens negative for methylation. Many of the CpG islands were preferentially methylated in the MLH1 methylation-positive CRC specimens, although methylation of some of them was more widespread. These data provide insights into the complex regulation of the methylation status of CpG islands in CRCs positive for MSI and MLH1 methylation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

CAPON expression in skeletal muscle is regulated by position, repair, NOS activity, and dystrophy.

In skeletal muscle, the localization of nNOS is destabilized in the absence of dystrophin, which impacts muscle function and satellite cell activation. In neurons, the adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS), which produces the key signaling molecule nitric oxide (NO). While a CAPON-like gene is known to compensate functionally for a dystrophic phenotype in muscle of Caenorhabditis elegans, CAPON expression has not been reported for mammalian muscle. Here, CAPON expression was identified in mouse muscle using Northern and Western blotting and in situ hybridization in combination with immunostaining for laminin. CAPON RNA was expressed in developing normal and dystrophic muscles near fiber junctions with tendons, and levels increased from 1 to 3 weeks. In regenerating normal muscle and also in dystrophic muscles in the mdx mouse, CAPON transcripts were prominent in satellite cells and new myotubes. Expression of CAPON RNA increased in diaphragm muscle of normal and mdx mice after treatment with L-arginine, the NOS substrate. Both CAPON and utrophin protein levels increased in dystrophic quadriceps muscle after treatment with the steroid deflazacort plus L-arginine, known to reduce the dystrophic phenotype. The identification of CAPON transcripts and protein in mammalian muscle and responses to L-arginine suggest CAPON may have a functional role in stabilizing neuronal NOS in skeletal muscle in the cytoskeletal complex associated with dystrophin/utrophin, with possible applications to therapy for human muscular dystrophy.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes.

ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3.Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5'-O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.

ADP-Ribosylation Factor 1↗
PubMed2002

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.

3-Phosphoinositide-Dependent Protein Kinases↗