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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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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.
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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.

Mentions: A confocal laser scanning microscope was used to observe the intracellular localization of FEZ1 expressed in COS-7 cells. When the cells coexpressing FEZ1-FLAG and PKCζ-HA were examined, the plasma membrane was stained strongly with an anti-FLAG mAb and its cytoplasmic peripheries were also stained weakly (Fig. 6, panel 1). This indicates that FEZ1-FLAG is present predominantly in the plasma membrane of COS-7 cells, in which the expressed PKCζ is usually in an inactive form. On the other hand, in the cells expressing caPKCζ-HA (constitutively active mutant) (Schonwasser et al., 1998) instead of PKCζ-HA, FEZ1-FLAG was detected uniformly in the cytoplasm (Fig. 6, panel 3). In the cells expressing K281M PKCζ-HA (kinase-negative mutant), however, FEZ1-FLAG was again localized mostly in the plasma membrane (Fig. 6, panel 5). When these cells were treated with staurosporin, a PKC inhibitor common to all isoforms (Tamaoki et al., 1986), FEZ1-FLAG was present in the plasma membrane (Fig. 6, panels 2, 4, and 6). Particularly, it should be noted that FEZ1-FLAG present in the cytoplasm of the caPKCζ-HA–expressing cells was dynamically translocated into the plasma membrane by the staurosporin treatment (Fig. 6, panel 4). These results demonstrate that the translocation of FEZ1 from the plasma membrane, where it is normally localized, to the cytoplasm is regulated directly by the PKCζ activity.


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)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132904&req=5

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.
Mentions: A confocal laser scanning microscope was used to observe the intracellular localization of FEZ1 expressed in COS-7 cells. When the cells coexpressing FEZ1-FLAG and PKCζ-HA were examined, the plasma membrane was stained strongly with an anti-FLAG mAb and its cytoplasmic peripheries were also stained weakly (Fig. 6, panel 1). This indicates that FEZ1-FLAG is present predominantly in the plasma membrane of COS-7 cells, in which the expressed PKCζ is usually in an inactive form. On the other hand, in the cells expressing caPKCζ-HA (constitutively active mutant) (Schonwasser et al., 1998) instead of PKCζ-HA, FEZ1-FLAG was detected uniformly in the cytoplasm (Fig. 6, panel 3). In the cells expressing K281M PKCζ-HA (kinase-negative mutant), however, FEZ1-FLAG was again localized mostly in the plasma membrane (Fig. 6, panel 5). When these cells were treated with staurosporin, a PKC inhibitor common to all isoforms (Tamaoki et al., 1986), FEZ1-FLAG was present in the plasma membrane (Fig. 6, panels 2, 4, and 6). Particularly, it should be noted that FEZ1-FLAG present in the cytoplasm of the caPKCζ-HA–expressing cells was dynamically translocated into the plasma membrane by the staurosporin treatment (Fig. 6, panel 4). These results demonstrate that the translocation of FEZ1 from the plasma membrane, where it is normally localized, to the cytoplasm is regulated directly by the PKCζ activity.

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