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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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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.
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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.

Mentions: To examine in vivo association of FEZ1 and PKCζ, the lysates of COS-7 cells coexpressing FEZ1-FLAG and PKCζ-HA were analyzed by the immunoprecipitation assay. By Western blotting with the anti-HA mAb, PKCζ-HA with an approximate Mr of 72,000 was detected in the anti-FLAG immunoprecipitates (Fig. 4 A), indicating coprecipitation of PKCζ-HA with FEZ1-FLAG. Similarly, by Western blotting with an anti-FLAG mAb, FEZ1-FLAG with an approximate Mr of 55,000 was also detected in the anti-HA immunoprecipitates (Fig. 4 B), indicating coprecipitation of FEZ1-FLAG with PKCζ-HA. These results clearly show that FEZ1-FLAG associates with PKCζ-HA in vivo. Under the same conditions, FEZ1-FLAG did not associate with other HA-conjugated conventional and novel PKC isoforms (α, βI, γ, δ, and ε) (data not shown). We then analyzed the anti-HA immunoprecipitates by the phosphorylation assay as described in Materials and Methods and found that FEZ1 in the FEZ1-FLAG/PKCζ-HA complex could be phosphorylated (Fig. 4 C). When a kinase-negative mutant enzyme of PKCζ-HA (K281M PKCζ-HA) was coexpressed with FEZ1-FLAG in COS-7 cells, FEZ1-FLAG was found to be associated with K281M PKCζ-HA (Fig. 4, A and B) but not phosphorylated (Fig. 4 C). Collectively, these results indicate that FEZ1 is a novel cellular substrate of PKCζ and its association with PKCζ is independent of the activity of PKCζ. On the other hand, when GST-FEZ1 expressed in bacterial cells was incubated with the conventional PKC isoforms (mixture of α, βI, βII, and γ) purified from the rat brain in the presence of [γ-32P]ATP, ∼0.4 mol of phosphate was incorporated per mole of GST-FEZ1. The GST protein alone could not be phosphorylated under the same conditions (Kuroda et al., 1996). This result shows that FEZ1 can also be phosphorylated in vitro by the conventional PKC isoforms. The discrepancy between in vivo and in vitro phosphorylation assays is discussed later (see Discussion).


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)

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.
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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.
Mentions: To examine in vivo association of FEZ1 and PKCζ, the lysates of COS-7 cells coexpressing FEZ1-FLAG and PKCζ-HA were analyzed by the immunoprecipitation assay. By Western blotting with the anti-HA mAb, PKCζ-HA with an approximate Mr of 72,000 was detected in the anti-FLAG immunoprecipitates (Fig. 4 A), indicating coprecipitation of PKCζ-HA with FEZ1-FLAG. Similarly, by Western blotting with an anti-FLAG mAb, FEZ1-FLAG with an approximate Mr of 55,000 was also detected in the anti-HA immunoprecipitates (Fig. 4 B), indicating coprecipitation of FEZ1-FLAG with PKCζ-HA. These results clearly show that FEZ1-FLAG associates with PKCζ-HA in vivo. Under the same conditions, FEZ1-FLAG did not associate with other HA-conjugated conventional and novel PKC isoforms (α, βI, γ, δ, and ε) (data not shown). We then analyzed the anti-HA immunoprecipitates by the phosphorylation assay as described in Materials and Methods and found that FEZ1 in the FEZ1-FLAG/PKCζ-HA complex could be phosphorylated (Fig. 4 C). When a kinase-negative mutant enzyme of PKCζ-HA (K281M PKCζ-HA) was coexpressed with FEZ1-FLAG in COS-7 cells, FEZ1-FLAG was found to be associated with K281M PKCζ-HA (Fig. 4, A and B) but not phosphorylated (Fig. 4 C). Collectively, these results indicate that FEZ1 is a novel cellular substrate of PKCζ and its association with PKCζ is independent of the activity of PKCζ. On the other hand, when GST-FEZ1 expressed in bacterial cells was incubated with the conventional PKC isoforms (mixture of α, βI, βII, and γ) purified from the rat brain in the presence of [γ-32P]ATP, ∼0.4 mol of phosphate was incorporated per mole of GST-FEZ1. The GST protein alone could not be phosphorylated under the same conditions (Kuroda et al., 1996). This result shows that FEZ1 can also be phosphorylated in vitro by the conventional PKC isoforms. The discrepancy between in vivo and in vitro phosphorylation assays is discussed later (see Discussion).

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