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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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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).
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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).

Mentions: To investigate the specificity of FEZ1 for PKC isoforms and also to identify the region(s) in the PKC molecules involved in the association with FEZ1, the regulatory domains of various PKC isoforms were used as a bait in the yeast two-hybrid assay. As shown in Fig. 5, FEZ1 interacted strongly with the regulatory domain of PKCζ (ζ-R, residues 1–250) and moderately with that of PKCε (ε-R, residues 1–406) but not with those of other PKC isoforms. Based on the sequence comparison of PKC isoforms, the regulatory domains of PKCζ and PKCε are further divided into a conserved region (C1) and two variable regions (V1 and V3) with a considerable sequence diversity (Nishizuka, 1988). Therefore, various deletion mutants containing a part(s) of these regions of PKCζ and PKCε were then constructed to map the region interacting with FEZ1. The yeast two-hybrid assays indicated that the V1 regions of PKCζ and PKCε were essential for interaction with FEZ1 (Fig. 5). However, in the case of full-length PKC isoforms, only PKCζ interacted with FEZ1 in the two-hybrid assay (data not shown), indicating that PKCζ but not PKCε could form a stable complex with FEZ1.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

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).
Mentions: To investigate the specificity of FEZ1 for PKC isoforms and also to identify the region(s) in the PKC molecules involved in the association with FEZ1, the regulatory domains of various PKC isoforms were used as a bait in the yeast two-hybrid assay. As shown in Fig. 5, FEZ1 interacted strongly with the regulatory domain of PKCζ (ζ-R, residues 1–250) and moderately with that of PKCε (ε-R, residues 1–406) but not with those of other PKC isoforms. Based on the sequence comparison of PKC isoforms, the regulatory domains of PKCζ and PKCε are further divided into a conserved region (C1) and two variable regions (V1 and V3) with a considerable sequence diversity (Nishizuka, 1988). Therefore, various deletion mutants containing a part(s) of these regions of PKCζ and PKCε were then constructed to map the region interacting with FEZ1. The yeast two-hybrid assays indicated that the V1 regions of PKCζ and PKCε were essential for interaction with FEZ1 (Fig. 5). However, in the case of full-length PKC isoforms, only PKCζ interacted with FEZ1 in the two-hybrid assay (data not shown), indicating that PKCζ but not PKCε could form a stable complex with FEZ1.

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