Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells.

The phosphorylation of epithelial-specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2-fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8-Br-cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [gamma-32P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8-Br-cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase.     

Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Protein kinase C (PKC) has been implicated in a variety of cellular responses such as proliferation, differentiation, and secretion. We assessed the role of PKC in the mitogenic effects of gastrin-releasing peptide (in a small cell lung cancer (SCLC) cell line. Using antisera that specifically recognize the PKC isoforms alpha, beta, gamma, delta, and epsilon, we determined that PKC epsilon is the major isoform in the SCLC cell line NCI-N417, followed by PKC alpha and delta. In addition to the 90-kDa PKC epsilon, our anti-PKC epsilon antiserum specifically detected a 40-kDa immunoreactive protein. Treatment of the cells with either 20 nM phorbol myristate acetate or 50 nM GRP enhanced significantly the level of the 40-kDa protein in a time-dependent (1-8 h), cycloheximide-sensitive fashion. Subcellular fractionation revealed that 90% of PKC epsilon was in particulate form, while the 40-kDa immunoreactive protein was cytosolic. To test the hypothesis that the 40-kDa soluble protein represented a catalytically independent PKC epsilon fragment, cytosolic extracts were assayed for kinase activity. 45-50% of the activity was apparent in the absence of the PKC activators phosphatidylserine and diacylglycerol. This effector-independent kinase activity was further purified by affinity chromatography using a synthetic peptide corresponding to the pseudosubstrate region of PKC epsilon (ERMRPRKRQGAVRRRV) coupled to Sepharose. The partially purified protein, recognized by the anti-PKC epsilon antiserum, exhibited histone kinase activity with kinetics similar to those of the tryptically generated catalytic fragment of brain PKC epsilon. This activity was inhibited by staurosporine (IC50 = 1 x 10(-8) M) and by the pseudosubstrate inhibitor peptide (IC50 = 7.7 x 10(-8) M). The SCLC kinase and the brain PKC epsilon catalytic fragment were similar as indicated by the relative sizes of the PKC epsilon immunoreactive peptides generated with protease V8 from Staphylococcus aureus (Mr approximately 37,000, 34,000, 28,000, 26,000, and 25,000). Taken together, we conclude that a variant SCLC cell line expresses a constitutively active catalytic fragment of PKC epsilon. Regulation by 12-O-tetradecanoyl-13-acetate or GRP via de novo protein synthesis suggests a novel mechanism of control of PKC diversity with implications for small cell lung cancer and possibly other malignancies.     

Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR- complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down- modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.     

Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes.

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M_r, respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vitro substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.     

PKC epsilon is associated with myosin IIA and actin in fibroblasts

Proteins coimmunoprecipitating with protein kinase C (PKC) epsilon in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC epsilon immunoprecipitates, as well as known PKC epsilon binding proteins, actin, beta'Cop and cytokeratin. Myosin is not a substrate for PKC epsilon. Immunofluorescence analysis showed that PKC epsilon is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC epsilon-actin-myosin complex in cell spreading.     

Protein Kinase C-Epsilon Mediates Phorbol Ester-Induced Phosphorylation of Connexin-43

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKC?) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (≥45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKKI did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKC? significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of ≥45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKC?.     

Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor

Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC epsilon was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC epsilon interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC epsilon does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR. In vitro binding assays demonstrate dose-dependent binding of PKC epsilon to RACK1 which is inhibited by an 8-amino acid peptide based on the sequence of the sixth Trp-Asp repeat in RACK1 or by an 8-amino acid sequence in the V1 region of PKC epsilon, epsilon V1-2. A 4-amino acid sequence INAL (70-73) expressed in CFTR shares 50% homology to the RACK1 inhibitory peptide, but it does not bind PKC epsilon. NHERF1 and RACK1 bind in a dose-dependent manner. Immunofluorescence and confocal microscopy of RACK1 and CFTR revealed colocalization of the proteins to the apical and lateral regions of Calu-3 cells. The results indicate the RACK1 binds PKC epsilon and NHERF1, thus serving as a scaffold protein to anchor the enzyme in proximity to CFTR.     

Casein Kinase II Phosphorylates the Neural Cell Adhesion Molecule L1

Abstract: L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144–1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173–1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177–1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser^1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser^1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.     

Association of immature hypophosphorylated protein kinase cepsilon with an anchoring protein CG-NAP

Protein kinase C (PKC) family requires phosphorylation of itself to become competent for responding to second messengers. Much attention has been focused on elucidating the role of phosphorylation in PKC activity; however, it remains unknown where this modification takes place in the cells. This study examines whether anchoring protein is involved in the regulation of PKC phosphorylation. A certain population of PKC epsilon in rat brain extracts as well as that expressed in COS7 cells was associated with an endogenous anchoring protein CG-NAP (centrosome and Golgi localized PKN- associated protein). Pulse chase experiments revealed that the associated PKC epsilon was an immature species at the hypophosphorylated state. In vitro binding studies confirmed that non- or hypophosphorylated PKC epsilon directly bound to CG-NAP via its catalytic domain, whereas sufficiently phosphorylated PKC epsilon did not. PKC epsilon mutant at a potential phosphorylation site of Thr-566 or Ser-729 to Ala, possessing almost no catalytic activity, was associated and co-localized with CG-NAP at Golgi/centrosome area. On the other hand, wild type and a phosphorylation-mimicking mutant at Thr-566 were mainly distributed in cytosol and represented second messenger-dependent catalytic activation. These results suggest that CG-NAP anchors hypophosphorylated PKCepsilon at the Golgi/centrosome area during maturation and serves as a scaffold for the phosphorylation reaction.     

Activation of the Dunaliella acidophila plasma membrane H(+)-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site.

Trypsin treatment of purified H(+)-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H(+)-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H(+)-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.     

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.     

Purification and characterization of protein kinase C epsilon from rabbit brain.

Protein kinase C epsilon was chromatographically purified from rabbit brain to electrophoretic homogeneity. We identified the enzyme as the epsilon species of novel-type protein kinase C (nPKC epsilon), originally discovered and defined by cDNA cloning [Ohno, S., et al. (1988) Cell 53, 731-741], on the basis of the following observations: (i) the enzyme reacts specifically with an antipeptidic antiserum to nPKC epsilon but not with antisera to any of the other molecular species of PKC thus far known; (ii) it exhibits enzymatic behavior essentially identical to that of a recombinant nPKC epsilon purified from transfected COS cells [Konno, Y., et al. (1989) J. Biochem. 106, 673-678] and distinct from that of conventional PKC (alpha, beta I/II, and gamma) in its dependence on magnesium concentration and cofactors such as phospholipids, calcium, and phorbol ester; and (iii) it has an apparent molecular weight of 95.7K +/- 0.4K on SDS-PAGE, significantly greater than the other conventional and novel PKCs thus far identified. Notably, calcium exhibits a complex effect, both positive and negative, on the kinase activity of epsilon depending on the kind of substrate and the coexisting phospholipid, calling for a modification of the current notion that epsilon is a kinase unresponsive to calcium. The amount of epsilon species in the brain was estimated to be comparable to that of each conventional species, indicating that epsilon stands as one of the major PKC family members in brain. Furthermore, the enzyme shows a broader substrate spectrum than conventional PKC when examined with endogenous substrates, implying that it may cover a wider or different range of physiological functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles

Several peripheral membrane proteins associated with clathrin-coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV-associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (μ2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G-associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, μ2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the 'stripped' CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.     

An antibody to a synthetic peptide recognizes polyomavirus middle-T antigen and reveals multiple in vitro tyrosine phosphorylation sites.

Antibodies were raised against three synthetic peptides corresponding to sequences surrounding tyrosine 315, a putative in vitro phosphorylation site in polyomavirus middle-T antigen. Only one of the peptides (called C and corresponding to residues 311 to 330) elicited antibodies that recognized middle-T efficiently. Middle-T present in immunoprecipitates formed with purified anti-C serum still accepted phosphate on tyrosine in an in vitro kinase reaction. This implies that tyrosines other than 315 and 322 that lie within the antibody binding region are phosphorylated under these conditions. This conclusion was supported by the altered partial V8 proteolysis fingerprint of the labeled middle-T. Two-dimensional tryptic fingerprint analysis of 32P-labeled middle-T showed that several tryptic peptides identified as including tyrosine 315 and 322 were missing from middle-T labeled in anti-C immunoprecipitates compared with middle-T labeled in immunoprecipitates made by using anti-tumor cell serum. However, one major labeled peptide remained. This peptide was also present in fingerprints of 32P-labeled middle-T coded by M45, dl23, pAS131, and dl1013, but a peptide with altered mobility was present in dl8 middle-T. This identified the peptide as including tyrosine 250. We deduce from these data that (i) the presence of the antibody against peptide C inhibits phosphorylation of tyrosines 315 and 322; (ii) middle-T labeled in the kinase reaction after immunoprecipitation with anti-C serum is phosphorylated on tyrosine 250; and (iii) when anti-tumor cell serum is used in the in vitro kinase reaction, middle-T is phosphorylated at multiple sites, including residues 250, 315, and 322.     

Cross-regulation of novel protein kinase C (PKC) isoform function in cardiomyocytes. Role of PKC epsilon in activation loop phosphorylations and PKC delta in hydrophobic motif phosphorylations

Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKC epsilon (at the C-terminal hydrophobic motif) and PKC delta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC delta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC delta kinase activity is attenuated when PKC delta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC delta and PKC epsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC delta and WT-PKC epsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC delta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC epsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC epsilon in nPKC activation loop phosphorylations and PKC delta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC delta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

The presence in a mouse T cell line of a 97-kDa protein kinase C (PKC) with characteristics similar to known members of the novel PKC subgroup and its possible role in lymphocyte gene expression.

The receptor for tumor-promoting phorbol esters has been shown to be the Ca+2/phospholipid dependent enzyme protein kinase C (PKC). There are two major groups of PKC, the conventional PKC isotypes alpha, beta I, beta II, gamma) and the novel Ca+2-independent PKC (delta, epsilon, zeta, eta). Phorbol esters previously have been demonstrated to increase human IFN-gamma gene expression after treatment of a murine T cell line (Cl 9) that has been transfected with human IFN-gamma genomic DNA. In contrast, treatment with Ca+2 ionophore alone or in combination with phorbol ester did not enhance IFN-gamma production in a synergistic manner above the level obtained with phorbol ester treatment alone. To determine whether the lack of effect of Ca+2 ionophore is due to a defect in PKC, we compared the level of PKC autophosphorylation in the mouse T cell line (Cl 9), a mouse epidermal cell line (JB6), and purified rat brain PKC by in vitro kinase assays. The results demonstrate that instead of the expected 80-kDa autophosphorylated PKC band seen in purified rat brain PKC or mouse JB6 cell lysates, only a novel 97-kDa Ca+2-independent phosphoprotein was observed in Cl 9 cells. To ascertain if there was any nucleic acid sequence similarity to PKC epsilon, we hybridized Cl 9 poly(A+) RNA with a cloned fragment of the PKC epsilon gene and observed two hybridizing RNA bands (4.4 and 4.0 kb). Our results suggest that the 97-kDa phosphoprotein is similar to, but not identical with, PKC epsilon and is the major PKC expressed in the Cl 9 murine T cell line. These data suggested that the 97-kDa PKC may be responsible for the induction of both the transfected human IFN-gamma gene and the endogenous murine IL-2R alpha-chain.     

Purification of CK1 by affinity chromatography on immobilised axin

Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.     

Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon)

The protein kinase C (PKC)-related enzyme PKC(mu)/PKD (protein kinase D) is activated by activation loop phosphorylation through PKC(eta). Here we demonstrate that PKC(mu) is activated by the direct phosphorylation of PKC(epsilon). PKC(mu) colocalizes with PKC(epsilon) in HEK293 and MCF7 cells as shown by confocal immunofluorescence analyses. PDK1, known as the upstream kinase for several PKC isozymes, associates intracellularly with PKC(epsilon) and PKC(eta). PKC(eta) is phosphorylated by PDK1 in vitro, leading to kinase activation as similarly reported for PKC(epsilon) activation by PDK1. Coexpression of PDK1, PKC(epsilon) and PKC(mu) in HEK293 cells results in PKC(mu) activation. In contrast, the coexpression of PDK1 and PKC(eta) with PKC(mu) does not activate PKC(eta) or consequently PKC(mu). PDK1/PKC(epsilon)-triggered activation of PKC(mu) inhibits JNK, a downstream effector of PKC(mu), whereas upon transient expression of PDK1, PKC(eta), and PKC(mu), JNK is not affected. These data implicate PKC(epsilon) as the biologically important upstream kinase for PKC(mu) in HEK293 cells, regulating downstream effectors. Our results further indicate a PDK1/PKC(eta)/PKC(mu) controlled negative regulation of PKC(eta) kinase activity. In this study, we show that differentially activated kinase cascades involving PDK1 and novel PKC isotypes are responsible for the regulation of PKC(mu) activity and consequently inhibit the JNK pathway.