Protein kinase Cepsilon is important for migration of neuroblastoma cells.

BACKGROUND: Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. METHODS: PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. RESULTS: Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCbeta isoforms did not have an effect. Downregulation of PKCepsilon, but not of PKCalpha or PKCdelta, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCepsilon. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. CONCLUSION: PKCepsilon is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCepsilon but they may be involved in TPA-mediated migration.

Identification of acidic amino acid residues in the protein kinase C alpha V5 domain that contribute to its insensitivity to diacylglycerol.

The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC alpha indicating a conformational difference elicited by the mutations. Expression of the isolated PKC alpha V5 domain led to increased diacylglycerol sensitivity of PKC alpha. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC alpha sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC alpha that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC alpha in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC alpha.

Distinct roles of PKC isoforms in NCAM-mediated neurite outgrowth.

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)-mediated neurite outgrowth was tested using a co-culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12-E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM-stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM-mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase-deficient forms of PKCalpha, betaI and betaII blocked NCAM-mediated neurite extension, but had no effect on nerve growth factor (NGF)-mediated neurite outgrowth. Expression of two PKCepsilon constructs: (i) a fragment from PKCepsilon encompassing the pseudosubstrate, the C1a domain (including the actin-binding site, ABS), and parts of the V3 region, or (ii) the PKCepsilon-specific ABS blocked NCAM-mediated neurite extension in both cases. These two constructs also partially inhibited NGF-stimulated neuritogenesis indicating that PKCepsilon is a positive regulator of both NCAM- and NGF-mediated differentiation. We suggest that PKCepsilon is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM-stimulated neurite outgrowth.

Identification of conserved amino acids N-terminal of the PKC epsilon C1b domain crucial for protein kinase C epsilon-mediated induction of neurite outgrowth.

We have shown previously that protein kinase C (PKC) epsilon can induce neurite outgrowth independently of its catalytic activity via a region encompassing its C1 domains. In this study we aimed at identifying specific amino acids in this region crucial for induction of neurite outgrowth. Deletion studies demonstrated that only 4 amino acids N-terminal and 20 residues C-terminal of the C1 domains are necessary for neurite induction. The corresponding regions from all other novel isoforms but not from PKCalpha were also neuritogenic. Further mutation studies indicated that amino acids immediately N-terminal of the C1a domain are important for plasma membrane localization and thereby for neurite induction. Addition of phorbol ester made this construct neurite-inducing. However, mutation of amino acids flanking the C1b domain reduced the neurite-inducing capacity even in the presence of phorbol esters. Sequence alignment highlighted an 8-amino acid-long sequence N-terminal of the C1b domain that is conserved in all novel PKC isoforms. Specifically, we found that mutations of either Phe-237, Val-239, or Met-241 in PKCepsilon completely abolished the neurite-inducing capacity of PKCepsilon C1 domains. Phorbol ester treatment could not restore neurite induction but led to a plasma membrane translocation. Furthermore, if 12 amino acids were included N-terminal of the C1b domain, the C1a domain was dispensable for neurite induction. In conclusion, we have identified a highly conserved sequence N-terminal of the C1b domain that is crucial for neurite induction by PKCepsilon, indicating that this motif may be critical for some morphological effects of PKC.

Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase Calpha in response to diacylglycerol.

We have seen that protein kinase Calpha (PKCalpha) is transiently translocated to the plasma membrane by carbachol stimulation of neuroblastoma cells. This is induced by the Ca2+ increase, and PKCalpha does not respond to diacylglycerol (DAG). The unresponsiveness is dependent on structures in the catalytic domain of PKCalpha. This study was designed to investigate if and how the kinase activity and autophosphorylation are involved in regulating the translocation. PKCalpha enhanced green fluorescent protein translocation was studied in living neuroblastoma cells by confocal microscopy. Carbachol stimulation induced a transient translocation of PKCalpha to the plasma membrane and a sustained translocation of kinase-dead PKCalpha. In cells treated with the PKC inhibitor GF109203X, wild-type PKCalpha also showed a sustained translocation. The same effects were seen with PKCbetaI, PKCbetaII, and PKCdelta. Only kinase-dead and not wild-type PKCalpha translocated in response to 1,2-dioctanoylglycerol. To examine whether autophosphorylation regulates relocation to the cytosol, the autophosphorylation sites in PKCalpha were mutated to glutamate, to mimic phosphorylation, or alanine, to mimic the non-phosphorylated protein. After stimulation with carbachol, glutamate mutants behaved like wild-type PKCalpha, whereas alanine mutants behaved like kinase-dead PKCalpha. When the alanine mutants were treated with 1,2-dioctanoylglycerol, all cells showed a sustained translocation of the protein. However, neither carbachol nor GF109203X had any major effects on the level of autophosphorylation, and GF109203X potentiated the translocation of the glutamate mutants. We, therefore, hypothesize that 1) autophosphorylation of PKCalpha limits its sensitivity to DAG and 2) that kinase inhibitors augment the DAG sensitivity of PKCalpha, perhaps by destabilizing the closed conformation.