To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCα, βII, δ, and ε fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCε–EGFP induced cell processes whereas the other isoforms did not. The effect of PKCε–EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCε regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCε and neurites induced during retinoic acid- and growth factor–induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCε– PSC1V3 protein contained α-tubulin, neurofilament-160, and F-actin, but the PKCε–PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y.  These data suggest that PKCε, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.

Many proteins are S-acylated, affecting their localization and function. Dynamic S-acylation in response to various stimuli has been seen for several proteins in vivo. The regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1 appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid lipofuscinosis.

We have previously shown that protein kinase C⑀ (PKC⑀) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKC⑀-mediated neurite induction. We show an increased association of PKC⑀ to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKC⑀ is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKC⑀ actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKC⑀ overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKC⑀ independent of both serum and the actin-binding site, and PKC⑀ colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKC⑀ in a pathway leading to neurite outgrowth. Localization of PKC⑀ to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.

No abstract

No abstract

We have shown previously that protein kinase C (PKC) ⑀ 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 PKC␣ were also neuritogenic. Further mutation studies indicated that amino acids immediately Nterminal 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 PKC⑀ completely abolished the neurite-inducing capacity of PKC⑀ 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 PKC⑀, indicating that this motif may be critical for some morphological effects of PKC.The induction and elongation of neurites are cellular processes driven by cytoskeletal changes. These are under the control of different intracellular transduction pathways mediating signals from other cells or the extracellular matrix. The members of the PKC 1 family constitute one important family controlling the outgrowth of neurites. PKC isoforms have been suggested to both positively and negatively influence the outgrowth of neurites.There are 10 different PKC isoforms that are divided into three subclasses according to their structure and requirements for activation, classical PKCs (␣, ␤I, ␤II, and ␥), novel PKCs (␦, ⑀, , and ), and atypical PKCs (/ and ). Of these isoforms, particularly PKC␦ (1-3) and PKC⑀ (3-8) have been suggested to positively influence neurite outgrowth in several different cell types. However, there are also indications that PKC isoforms, for instance PKC⑀, can counteract outgrowth (9). Our group has shown previously that overexpression of PKC⑀ in neuroblastoma (7,8) and in immortalized neural precursor (3) cells leads to neurite outgrowth. A similar morphological effect of PKC⑀ has also been observed in fibroblasts (10, 11). The neurite-inducing effect of PKC⑀ is independent of its catalytic activity, and a region from the regulatory domain of the enzyme encompassing the two C1 domains with flanking structures is necessary and sufficient for the effect (7).Many proteins,...

Correspondence to: Daphne A. Haas-Kogan, dhaaskogan@radonc.ucsf.edu. Ralph P. Ermoian and Tania Kaprealian contributed equally. Results-Multiple comparison analyses revealed a significant correlation with grade for all variables examined, except phosphorylated-S6. Expression of phosphorylated-4E-BP1, phosphorylated-PKB/Akt, PTEN, TSC1, and TSC2 correlated with grade (P < 0.01 for all). We extended our analyses to ask whether decreases in TSC proteins levels were due to changes in mRNA levels, or due to changes in post-transcriptional alterations. We found significantly lower levels of TSC1 and TSC2 mRNA in GBMs than in grade II gliomas or non-tumor brain (P < 0.01). NIH Public AccessConclusions-Expression levels of critical signaling molecules upstream and downstream of mTOR differ between non-tumor brain and gliomas of any grade. The single variable whose expression did not differ between non-tumor brain and gliomas was phosphorylated-S6, suggesting that other protein kinases, in addition to mTOR, contribute significantly to S6 phosphorylation. mTOR provides a rational therapeutic target in gliomas of all grades, and clinical benefit may emerge as mTOR inhibitors are combined with additional agents.

Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCL L isoforms in this process. The PKCL Lspecific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCL L inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCL L isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells. ß