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

Abstract

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-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 PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ 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 PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ 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 PKCζ.

A novel DISC1-interacting partner DISC1-Binding Zinc-finger protein: implication in the modulation of DISC1-dependent neurite outgrowth

“…The interaction of DISC1 with FEZ1 in PC12 cells was markedly enhanced by exposure to NGF, along with neurite extension, 11,14 whereas PACAP had no effect (our unpublished observation). On the other hand, PACAP caused a marked, but transient, decrease of association between DISC1 and DBZ in PC12 cells ( Figure 3c) and NGF had a similar effect (data not shown).…”

“…11 The interaction of DISC1 with FEZ1 was markedly enhanced along with neurite extension in PC12 cells by nerve growth factor (NGF) stimulation. 11,14 An association between singlenucleotide polymorphisms of the FEZ1 gene and schizophrenia has also been suggested in a Japanese population. 15 Kendrin, also termed pericentrin-B, is a calmodulin-binding protein that is specifically localized to the centrosome, and we have previously demonstrated that Kendrin is involved in the centrosomal localization of DISC1.…”

Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite outgrowth

“…FEZ1 is reported to be involved in axonal outgrowth and fasciculation, 21,22 and we have shown that FEZ1 colocalizes with F-actin. Reorganization of the actinbased cytoskeletal structure is required for the neurite outgrowth of neurons.…”

“…We raised an antibody against FEZ1 which detected a protein of (Figure 4p), the size revealed by in vitro synthesis. 22 DISC1 exhibited a punctate distribution in the cytosol of SK-N-SH cells with the perinuclear high-density region (Figure 4a). DISC1 was also located on some filamentous structures which overlapped with F-actin as stress fibers detected by phalloidin staining (Figure 4a-c).…”

“…21 Coexpression of FEZ1 and the constitutively active mutant of PKCz induced PC12 cells to neuronal differentiation. 22 From these observations, FEZ1 is assumed to play a crucial role in the axon guidance machinery in mammals, although the molecular mechanism involving FEZ1 is still unclear. To determine the regions in FEZ1 and DISC1 involved in their interaction, a yeast two-hybrid assay was performed using various shorter fragments of human FEZ1 and human DISC1 (Figure 3a).…”