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

Abstract

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

Figure 1

Sequence alignment of rat FEZ1 and C. elegans UNC-76 proteins. The two sequences were aligned by using a BLOSUM 62–amino acid substitution matrix. Insertions (indicated by dots) are introduced to optimize the alignment. Identical and similar residues are indicated by vertical bars and plus marks, respectively, and the other residues by minus signs. The regions predicted to form amphipathic helices are shaded. Five conserved regions (1–5) are boxed. Potential sites for N-glycosylation and phosphorylation by PKC are indicated by ! and *, respectively. Amino acid residues are numbered on the right margin. The nucleotide sequence encoding rat FEZ1 protein has been submitted to the GenBank/EBI/DDBJ data bank with accession number U48249.

Figure 2

Northern blot analysis of FEZ1 mRNA. (A) Detection of FEZ1 mRNA in adult rat tissues. Northern blots containing 2 μg of poly(A)⁺ RNA from various adult rat tissues per lane were incubated with the full-length FEZ1 cDNA fragment labeled with [α-³²P]dCTP. (B) Detection of FEZ1 mRNA in developing mouse embryos. Northern blots containing 2 μg of poly(A)⁺ RNA from mouse embryos in different developmental stages (7, 11, 15, and 17 dpc) per lane were used. The positions of FEZ1 mRNA are indicated by arrows with their approximate sizes (knt, kilonucleotides) on the left margin of each blot.

Figure 3

Expression of FEZ1 protein. (A) Subcellular localization of FEZ1-FLAG protein in COS-7 cells. The lysates of COS-7 cells expressing FEZ1-FLAG were separated into the cytoplasmic (Cyt.) and membrane fractions (Mem.) and analyzed by Western blotting with an anti-FLAG mAb. As a control, untransfected COS-7 cells were used (None). Each lane contained the sample derived from ∼5.0 × 10⁵ cells. (B) In vitro synthesis of FEZ1-FLAG protein. FEZ1-FLAG protein labeled with [³⁵S]Met was synthesized as described in Materials and Methods. The reaction mixture was subjected to SDS-PAGE (12.5%) and then autoradiographed. The molecular mass of FEZ1-FLAG protein is indicated on the left margin of each gel with an arrow (in kD).

Figure 4

In vivo association of FEZ1 and PKCζ. Either PKCζ-HA or its kinase-negative mutant K281M PKCζ-HA was coexpressed with FEZ1-FLAG in COS-7 cells. The lysates were immunoprecipitated (IP) and then blotted (Blot) with either an anti-FLAG or anti-HA mAb. (A) Detection of PKCζ-HA and K281M PKCζ-HA (open arrow) in the anti-FLAG immunoprecipitates with an anti-HA mAb. (B) Detection of FEZ1-FLAG protein (closed arrow) in the anti-HA immunoprecipitates with an anti-FLAG mAb. (C) In vitro phosphorylation assay of the anti-HA immunoprecipitates. Phosphorylated FEZ1-FLAG protein is indicated by a closed arrow. IgG heavy chains derived from the mAb used for immunoprecipitation are indicated by asterisks.

Figure 5

Delineated structures of the regulatory domains of PKC isoforms and interaction with FEZ1 in the yeast two-hybrid system. β-Gal activity of yeast transformants was assayed by the plate method as described in Materials and Methods (+++, strongly positive; ++, moderately positive; +, weakly positive; and −, negative). Based on the sequence comparison, the primary structures of the regulatory domains of PKC isoforms are divided into conserved regions (C1 and C2) (boxes) and variable regions (V1–V3) (lines).

Figure 6

Intracellular localization of FEZ1 protein in COS-7 cells expressing various PKCζ. Either the wild-type PKCζ-HA, a constitutively active mutant (caPKCζ-HA), or a kinase-negative mutant (K281M PKCζ-HA) was coexpressed with FEZ1-FLAG protein in COS-7 cells. The cells were untreated (panels 1, 3, and 5) or treated with 0.1 μM staurosporin for 2 h (panels 2, 4, and 6), stained with an anti-FLAG mAb, and observed under a confocal laser scanning microscope.