Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regu latory domain of protein kinase C zeta (PKC zeta) as a bait, we have cloned a gene coding for a novel PKC zeta-interacting protein homologous to the C aenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasci culation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northe rn blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adu lt rat brain and throughout the developmental stages of mouse embryo. By th e yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was sho wn to interact with the NH2-terminal variable region (V1) of PKC zeta and w eakly with that of PKC epsilon. In the COS-7 cells coexpressing FEZ1 and PK C zeta, FEZ1 was present mainly in the plasma membrane, associating with PK C zeta and being phosphorylated. These results indicate that FEZ1 is a nove l substrate of PKC zeta;. When the constitutively active mutant of PKC zeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment o f the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic trans location of FEZ1 is directly regulated by the PKC zeta activity. Although e xpression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 a nd constitutively active PKC zeta stimulated the neuronal differentiation o f PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bund ling and elongation within axon bundles in the nematode, these results sugg est that FEZ1 plays a crucial role in the axon guidance machinery in mammal s by interacting with PKC zeta.