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

Kuroda S, Nakagawa N, Tokunaga C, Tatematsu K, Tanizawa K - J. Cell Biol. (1999)

Bottom Line: When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan. skuroda@sanken.osaka-u.ac.jp

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.

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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.
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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.

Mentions: Using the regulatory domain of rat PKCζ (residues 1–250) fused with the yeast GAL4 DNA-binding domain as a bait, we screened a rat brain cDNA library by the two-hybrid method in yeast. Among ∼1.0 × 107 yeast transformants (Leu+, Trp+) expressing rat cDNA-derived proteins fused with the GAL4 activation domain, one positive clone that exhibited both β-Gal activity and His+ phenotype only in the presence of the bait plasmid was obtained. Since the clone was found to harbor a 5′-terminal truncated form of a cDNA fragment upon nucleotide sequencing (data not shown), RACE was performed with another rat brain cDNA library to obtain a full-length cDNA. Finally, a cDNA consisting of 1662 bp and encoding a polypeptide of 393 amino acid residues was isolated and named temporarily as zeta-1. By computer search of the protein sequences registered in the GenBank/EBI/DDBJ data bank, we have come across the C. elegans UNC-76 protein reported recently (Bloom and Horvitz, 1997), showing significant sequence homology with the rat zeta-1 protein (identity, 31%; similarity, 63%) (Fig. 1). The nematode gene unc-76 (unc, uncoordinated) is necessary for normal axonal bundling and elongation within fascicles in the nematode (Hedgecock et al., 1985; Desai et al., 1988; McIntire et al., 1992). A human gene coding for a protein homologous to the nematode UNC-76 protein has also been cloned and shown to rescue locomotory defects caused by unc-76 mutations in the nematode (Bloom and Horvitz, 1997). The rat zeta-1 protein shows a 96% sequence identity with the reported human homologue of UNC-76. Hence, these mammalian homologues of the nematode UNC-76 protein (human and rat zeta-1 proteins) have been designated FEZ1 on the basis of the presumed roles in axonal fasciculation and elongation in mammals (Bloom and Horvitz, 1997).


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

Kuroda S, Nakagawa N, Tokunaga C, Tatematsu K, Tanizawa K - J. Cell Biol. (1999)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132904&req=5

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.
Mentions: Using the regulatory domain of rat PKCζ (residues 1–250) fused with the yeast GAL4 DNA-binding domain as a bait, we screened a rat brain cDNA library by the two-hybrid method in yeast. Among ∼1.0 × 107 yeast transformants (Leu+, Trp+) expressing rat cDNA-derived proteins fused with the GAL4 activation domain, one positive clone that exhibited both β-Gal activity and His+ phenotype only in the presence of the bait plasmid was obtained. Since the clone was found to harbor a 5′-terminal truncated form of a cDNA fragment upon nucleotide sequencing (data not shown), RACE was performed with another rat brain cDNA library to obtain a full-length cDNA. Finally, a cDNA consisting of 1662 bp and encoding a polypeptide of 393 amino acid residues was isolated and named temporarily as zeta-1. By computer search of the protein sequences registered in the GenBank/EBI/DDBJ data bank, we have come across the C. elegans UNC-76 protein reported recently (Bloom and Horvitz, 1997), showing significant sequence homology with the rat zeta-1 protein (identity, 31%; similarity, 63%) (Fig. 1). The nematode gene unc-76 (unc, uncoordinated) is necessary for normal axonal bundling and elongation within fascicles in the nematode (Hedgecock et al., 1985; Desai et al., 1988; McIntire et al., 1992). A human gene coding for a protein homologous to the nematode UNC-76 protein has also been cloned and shown to rescue locomotory defects caused by unc-76 mutations in the nematode (Bloom and Horvitz, 1997). The rat zeta-1 protein shows a 96% sequence identity with the reported human homologue of UNC-76. Hence, these mammalian homologues of the nematode UNC-76 protein (human and rat zeta-1 proteins) have been designated FEZ1 on the basis of the presumed roles in axonal fasciculation and elongation in mammals (Bloom and Horvitz, 1997).

Bottom Line: When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan. skuroda@sanken.osaka-u.ac.jp

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.

Show MeSH
Related in: MedlinePlus