Structural properties and mechanisms that govern association of C kinase adapter 1 with protein kinase C3 and the cell periphery

Association of an atypical protein kinase C (aPKC) with an adapter protein can affect the location, activity, substrate specificity, and physiological role of the phosphotransferase. Knowledge of mechanisms that govern formation and intracellular targeting of aPKC.adapter protein complexes is limited. Caenorhabditis elegans protein kinase C adapter proteins (CKA1 and CKA1S) bind and target aPKCs and provide prototypes for mechanistic analysis. CKA1 binds an aPKC (PKC3) via a phosphotyrosine binding (PTB) domain. A distinct, Arg/Lys-rich N-terminal region targets CKA1 to the cell periphery. We discovered that a short segment ((212)GGIDNGAFHEHEI(224)) of the V(2) (linker) region of PKC3 creates a binding surface that interacts with the PTB domain of CKA1/CKA1S. The docking domain of PKC3 differs from classical PTB ligands by the absence of Tyr and Pro. Substitution of Ile(214), Asn(216), or Phe(219) with Ala abrogates binding of PKC3 with CKA1; these residues cooperatively configure a docking site that complements an apolar surface of the CKA1 PTB domain. Phosphorylation site domains (PSD1, residues 11-25; PSD2, residues 61-77) in CKA1 route the adapter (and tethered PKC3) to the cell periphery. Phosphorylation of Ser(17) and Ser(65) in PSDs 1 and 2 elicits translocation of CKA1 from the cell surface to cytoplasm. Activities of DAG-stimulated PKCs and opposing protein Ser/Thr phosphatases can dynamically regulate the distribution of adapter protein between the cell periphery and cytoplasm.     

Positioning atypical protein kinase C isoforms in the UV-induced apoptotic signaling cascade.

Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzyme-like protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade.     

MEK5, a new target of the atypical protein kinase C isoforms in mitogenic signaling

The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that zetaPKC and lambda/iotaPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.     

Expression of the atypical protein kinase C (aPKC) isoforms iota/lambda and zeta during mouse embryogenesis

The atypical C-type protein kinases (aPKCs) comprise the third subclass of the PKC family functionally defined by insensitivity to phorbol esters, diacylgylcerol and calcium. aPKCs have been implicated in numerous biological processes including cell proliferation and survival, cell polarity, migration and inflammation. However, only insufficient data exist with regard to aPKC isoform specificity, since both mammalian aPKCs, PKC iota/lambda and PKC zeta, exhibit a high structural homology and very similar biochemical properties. In this study, we therefore used isoform-specific riboprobes and antibodies to define the characteristic expression profile of each aPKC isoform during mouse embryogenesis. Both, PKC iota/lambda and zeta show highly specific temporal and spatial patterns of expression which may help in distinguishing physiological functions of these isoforms.     

Nerve growth factor activates kinases that phosphorylate atypical protein kinase C

Activation of atypical protein kinase C by nerve growth factor (NGF) involves phosphorylation. In order to identify kinases that regulate atypical PKC (aPKC), we surveyed PC12 cell lysates for protein kinases that are activated by NGF and which could phosphorylate aPKC. Employing an in-gel kinase assay where aPKC-zeta was copolymerized within the gel matrix as a substrate, three kinases, pp175, pp87 and pp60, were identified as enzymes that phosphorylated aPKC. Phosphorylation of aPKC by these three kinases coincided with NGF-induced activation of the enzyme. Each kinase possessed a unique subcellular distribution pattern and could be activated by either ceramide or H(2)0(2), second messengers that mimic NGF signaling events. Upstream, pp175 and pp60 lie in a ras pathway, whereas pp87 lies in a pathway dependent upon src. Altogether, these findings reveal that the aPKCs are subject to regulation by a novel group of kinases.     

Repletion of atypical protein kinase C following RNA interference-mediated depletion restores insulin-stimulated glucose transport

The role of atypical protein kinase C (aPKC) in insulin-stimulated glucose transport in myocytes and adipocytes is controversial. Whereas studies involving the use of adenovirally mediated expression of kinase-inactive aPKC in L6 myocytes and 3T3/L1 and human adipocytes, and data from knock-out of aPKC in adipocytes derived from mouse embryonic stem cells and subsequently derived adipocytes, suggest that aPKCs are required for insulin-stimulated glucose transport, recent findings in studies of aPKC knockdown by small interfering RNA (RNAi) in 3T3/L1 adipocytes are conflicting. Moreover, there are no reports of aPKC knockdown in myocytes, wherein insulin effects on glucose transport are particularly relevant for understanding whole body glucose disposal. Presently, we exploited the fact that L6 myotubes and 3T3/L1 adipocytes have substantially different (30% nonhomology) major aPKCs, viz. PKC-zeta in L6 myotubes and PKC-lambda in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-zeta, but not PKC-lambda, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-lambda. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-lambda, but not PKC-zeta, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-zeta. These findings in knockdown and, more convincingly, rescue studies, strongly support the hypothesis that aPKCs are required for insulin-stimulated glucose transport in myocytes and adipocytes.     

Partitioning-defective protein 6 regulates insulin-dependent glycogen synthesis via atypical protein kinase C

The atypical isoforms of protein kinase C (aPKCs) play an important role in insulin signaling and are involved in insulin-stimulated glucose uptake in different cell systems. On the other hand, aPKCs also are able to negatively regulate important proteins for insulin signaling, like phosphatidylinositol 3-kinase and protein kinase B/Akt. To find aPKC-interacting proteins that may promote positive or negative activities of aPKCs, a yeast two-hybrid screen was performed. Partitioning-defective protein 6 (Par6) was detected in human cDNA libraries of different adult insulin-sensitive tissues. Although Par6 is known as an aPKC-interacting protein during development, no role for Par6 in insulin signaling has been reported so far. We therefore studied the effects of Par6 overexpression in C2C12 murine myoblasts. In these cells, Par6 associated constitutively with endogenous aPKCs, and the expression level as well as the activity of aPKCs were increased. Insulin-dependent association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate 1 was hampered and the phosphorylation of Akt/glycogen synthase kinase-3alpha/beta was significantly impaired after stimulation with insulin or with platelet-derived growth factor. Consequently, insulin-dependent glycogen synthesis was down-regulated (1.44 vs. 2.24 fold, P < 0.01). We therefore suggest that Par6 acts as a negative regulator of the insulin signal.     

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ～20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ～35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.     

Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation

Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src–aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration.     

Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase

Protein kinase C (PKC) isozymes play a central role in cellular signaling. Levels of PKC control the amplitude of agonist-induced signaling and alterations in these levels are associated with disease states, most notably cancer, yet mechanisms that control the turnover of the protein are poorly understood. Here we identify an E3 ligase that catalyzes the ubiquitin-mediated degradation of PKC. Specifically, we identified a RING finger domain-containing protein, RINCK (for RING-finger protein that interacts with C kinase) from a yeast two-hybrid screen using the amino terminus of PKCbeta as bait. RINCK encodes a protein of 581 amino acids that contains a RING finger domain, a B-box, and two coiled-coil regions, the three domains that form the signature motif of the large family of diverse TRIM (tripartite motif) proteins. Co-immunoprecipitation studies using tsA201 cells reveal that RINCK and PKC associate with each other in cells. Studies using fragments of PKCbeta reveal that this interaction is mediated by the C1A domain of PKC. RINCK induces the ubiquitination of PKC both in vitro and in cells. Overexpression of RINCK reduces the levels of PKC in cells, whereas genetic knockdown of endogenous RINCK increases the levels of PKC. This increase was observed for all PKC isozymes examined (including conventional, novel, and atypical). The RINCK-mediated degradation of PKC occurs independently of the classic phorbol ester-mediated down-regulation: genetic depletion of RINCK had no effect on the phorbol ester-mediated down-regulation and, additionally, up-regulated the levels of isozymes that cannot bind phorbol esters. Our data reveal a novel mechanism that provides amplitude control in PKC signaling through ubiquitination catalyzed by RINCK, an E3 ligase that specifically recognizes the C1 domain of PKC isoforms.     

Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells

We found in the present study that stimulation of A(2A) adenosine receptors (A(2A)-R) prevents apoptosis in PC12 cells. This A(2A)-protective effect was blocked by protein kinase A (PKA) inhibitors and was not observed in a PKA-deficient PC12 variant. Stimulation of PKA also prevented apoptosis, suggesting that PKA is required for the protective effect of A(2A)-R. A general PKC inhibitor, but not down-regulation of conventional and novel PKCs, readily blocked the protective effect of A(2A)-R stimulation and PKA activation, suggesting that atypical PKCs (aPKCs) serve a critical role downstream of PKA. Consistent with this hypothesis, stimulation of A(2A)-R or PKA enhanced nuclear aPKC activity. In addition, the A(2A)-protective effect was blocked by a specific inhibitor of one aPKC, PKCzeta, whereas overexpression of a dominant-positive PKCzeta enhanced survival. In contrast, inhibitors of MAP kinase and phosphatidylinositol 3-kinase did not modulate the A(2A)-protective effect. Dominant-negative Akt also did not alter the A(2A)-protective effect, whereas it significantly reduced the protective action of nerve growth factor. Collectively, these data suggest that aPKCs can function downstream of PKA to mediate the A(2A)-R-promoted survival of PC12 cells. Furthermore, the results indicate that different extracellular stimuli can employ distinct signaling pathways to protect against apoptosis induced by the same insult.     

蛋白激酶C同工酶分子结构及功能研究进展

蛋白激酶Ｃ（ＰＫＣ）是至少包括１１种亚型在内的丝／苏氨酸蛋白激酶家族,可分为传统型（ｃＰＫＣｓ）、新型（ｎＰＫＣｓ）、非典型（ａＰＫＣｓ）和ＰＫＣ－ｕ四大类。各ＰＫＣ亚型在ＡＴＰ结合位点、磷脂酰基转移位点、假性底物位点、佛波酯结合位点的氨基酸序列既高度保守又有变异。ＰＫＣ在机体内分布和作用十分广泛,本文主要介绍了ＰＫＣ在肿瘤形成、侵润和转移及肿瘤耐药性产生,调节造血干／祖细胞定向分化成熟,以及激素     

Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.     

Protein kinase C lambda/iota (PKClambda/iota): a PKC isotype essential for the development of multicellular organisms

PKClambda/iota belongs to the third group of the PKC family, atypical PKC (aPKC), together with PKCzeta based on its sequence divergence from conventional and novel PKCs observed not only in the N-terminal regulatory domain but also in the kinase domain. Although one of the most distinct features of aPKC is its single, unrepeated cysteine-rich domain, recent studies have revealed that the N-terminal regulatory domain has additional aPKC-specific structural motifs involved in various protein-protein interactions, which are important for the regulation and the subcellular targeting of aPKC. The identification of aPKC-specific binding proteins has significantly facilitated our understanding of the activation mechanism as well as the physiological function of aPKC at the molecular level. In particular, the finding that the mammalian homologs of the Caenorhabditis elegans proteins, PAR-3 and PAR-6, bind aPKC unexpectedly opens a new avenue for exploring a thus far completely unrecognized critical function of aPKC, that is, as a component of an evolutionarily conserved cell polarity machinery. Together with the great progress in the genome project as well as in the genetic analysis of model organisms, these advances are leading us into the new era of aPKC study in which functional divergence between PKClambda/iota and zeta can be discussed in elaborately.     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

Interaction codes within the family of mammalian Phox and Bem1p domain-containing proteins

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, lambda/iota- and zeta PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.     

Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.     

Cloning and characterization of a protein kinase C gene from the spruce budworm, Choristoneura fumiferana

Recent studies have implicated protein kinase C (PKC) in the control of 20-hydroxyecdysone (20E)-dependent gene expression during molting and metamorphosis in insects. To further understand the role of this kinase in 20E signal transduction, we cloned a homolog of mammalian PKC by RT-PCR and 5'/3'-RACE from adult of the moth Choristoneura fumiferana. The full-length cDNA of the C. fumiferana PKC (CfPKC1) is 2.3 kb with an open reading frame encoding a protein of 669 amino acids. The deduced amino acid sequence contains all the characteristic features of the classical protein kinase C subfamily. Northern and Western blot analysis showed that CfPKC1 was distributed ubiquitously in various tissues and at different developmental stages. Activation of CfPKC1 with the PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in a rapid redistribution of the protein from the cytosol to the plasma membrane. Knock-down of the CfPKC1 gene by double-stranded RNA interference or treatment of the CF-203 cells with PKC-specific inhibitors reduces the expression of the 20E-responsive genes CHR3 and E75. This data suggests that CfPKC1 is involved in the 20E-response gene expression in C. fumiferana.