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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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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).
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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).

Mentions: The lysate of COS-7 cells expressing FEZ1-FLAG was separated into the cytoplasmic and membrane fractions. Western blotting with an anti-FLAG mAb indicated that FEZ1-FLAG (∼55 kD) was equally present in both the cytoplasmic and membrane fractions (Fig. 3 A). The molecular mass of FEZ1-FLAG produced in COS-7 cells was ∼10 kD larger than that of the protein synthesized by in vitro transcription and translation (Fig. 3 B), which agreed well with the value calculated from the deduced amino acid sequence of FEZ1 (45,207). As described above, FEZ1 contains four potential sites for N-glycosylation. In addition, FEZ1-FLAG was found to be phosphorylated by in vivo labeling of COS-7 cells with [32P]H3PO4 (data not shown), which could also be a cause for the retarded migration on SDS-PAGE. These results strongly suggest that FEZ1 undergoes posttranslational modification (N-glycosylation and/or phosphorylation) in the mammalian cells.


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)

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).
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Related In: Results  -  Collection

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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).
Mentions: The lysate of COS-7 cells expressing FEZ1-FLAG was separated into the cytoplasmic and membrane fractions. Western blotting with an anti-FLAG mAb indicated that FEZ1-FLAG (∼55 kD) was equally present in both the cytoplasmic and membrane fractions (Fig. 3 A). The molecular mass of FEZ1-FLAG produced in COS-7 cells was ∼10 kD larger than that of the protein synthesized by in vitro transcription and translation (Fig. 3 B), which agreed well with the value calculated from the deduced amino acid sequence of FEZ1 (45,207). As described above, FEZ1 contains four potential sites for N-glycosylation. In addition, FEZ1-FLAG was found to be phosphorylated by in vivo labeling of COS-7 cells with [32P]H3PO4 (data not shown), which could also be a cause for the retarded migration on SDS-PAGE. These results strongly suggest that FEZ1 undergoes posttranslational modification (N-glycosylation and/or phosphorylation) in the mammalian cells.

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