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© The Rockefeller University Press, 0021-9525/1999//403 $5.00
The Journal of Cell Biology, Volume 144, Number 3, , 1999 403-411

Regular Articles

Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C {zeta}–interacting Protein



Shun'ichi Kuroda*,{ddagger}, Noritaka Nakagawa*,{ddagger}, Chiharu Tokunaga{ddagger}, Kenji Tatematsu*,{ddagger}, and Katsuyuki Tanizawa*

* Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan; and {ddagger} Biosignal Research Center, Kobe University, Kobe, Hyogo, 657-8501, Japan

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory 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 Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. 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. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKC{zeta} and weakly with that of PKC{varepsilon}. In the COS-7 cells coexpressing FEZ1 and PKC{zeta}, FEZ1 was present mainly in the plasma membrane, associating with PKC{zeta} and being phosphorylated. These results indicate that FEZ1 is a novel 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 of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKC{zeta} activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKC{zeta} stimulated the neuronal differentiation of 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 bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKC{zeta}.

Key Words: neuropeptides • UNC-76 protein • phosphorylation • protein binding • protein kinase C

Abbreviations used in this paper: β-Gal, β-galactosidase; caPKC{zeta}-HA, a constitutively active mutant of PKC{zeta}-HA; dpc, days postcoitum; FEZ1, fasciculation and elongation protein zeta-1; FEZ1-FLAG, NH2-terminally FLAG-tagged FEZ1 protein; GST, glutathione-S-transferase; K281M PKC{zeta}-HA, a kinase-negative mutant protein of PKC{zeta}-HA; MAPK, mitogen-activated protein kinase; nt, nucleotides; PKC, protein kinase C; PKC{zeta}-HA, NH2-terminally HA-tagged PKC{zeta}; RACE, rapid amplification of cDNA ends; X-Gal, 5-bromo-4-chloro-3-indoryl-β-D-galactoside.


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