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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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Northern blot analysis of FEZ1 mRNA. (A) Detection  of FEZ1 mRNA in adult rat tissues. Northern blots containing  2 μg of poly(A)+ RNA from various adult rat tissues per lane  were incubated with the full-length FEZ1 cDNA fragment labeled with [α-32P]dCTP. (B) Detection of FEZ1 mRNA in developing mouse embryos. Northern blots containing 2 μg of  poly(A)+ RNA from mouse embryos in different developmental  stages (7, 11, 15, and 17 dpc) per lane were used. The positions of  FEZ1 mRNA are indicated by arrows with their approximate  sizes (knt, kilonucleotides) on the left margin of each blot.
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Figure 2: Northern blot analysis of FEZ1 mRNA. (A) Detection of FEZ1 mRNA in adult rat tissues. Northern blots containing 2 μg of poly(A)+ RNA from various adult rat tissues per lane were incubated with the full-length FEZ1 cDNA fragment labeled with [α-32P]dCTP. (B) Detection of FEZ1 mRNA in developing mouse embryos. Northern blots containing 2 μg of poly(A)+ RNA from mouse embryos in different developmental stages (7, 11, 15, and 17 dpc) per lane were used. The positions of FEZ1 mRNA are indicated by arrows with their approximate sizes (knt, kilonucleotides) on the left margin of each blot.

Mentions: Northern blot analysis of eight tissues from adult rat has shown that FEZ1 mRNA with a size of ∼1700 nucleotides (nt) is expressed abundantly and exclusively in the brain, although faint expression of mRNA with a size of ∼4000 nt is also observed in the liver (Fig. 2 A). It is interesting to note that PKCζ mRNAs are also highly expressed in the brain (Ono et al., 1988b). The nematode UNC-76 is detected throughout the nervous system of the animals at all developmental stages from embryos (before outgrowth of the first axons) through adult worms (Bloom and Horvitz, 1997). Therefore, we investigated further the FEZ1 mRNA expression during development of mouse embryo with rat FEZ1 cDNA as a probe; the mouse FEZ1 gene deposited in the GenBank EST (Expressed Sequence Tag) database shows a very high sequence identity (>92%) with rat FEZ1. As shown in Fig. 2 B, a 6000-nt mRNA is expressed abundantly in the early to mid stage of development (from 7 d postcoitum [dpc] to 15 dpc). In the late stage of development (17 dpc), the 6000-nt mRNA disappears and instead a 1700-nt mRNA is expressed abundantly. Furthermore, a 2000-nt mRNA is expressed constantly, though slightly, in all developmental stages. It has been known that development of the nervous system in mouse embryo begins soon after 7 dpc with the neural plate formation and ends before 17 dpc. By cDNA cloning, all mRNAs observed here (1700-, 2000-, 4000-, and 6000-nt mRNAs) were confirmed to contain a 5′-untranslated sequence (∼100 nt), the same rat/mouse FEZ1 gene (∼1200 nt), a 3′-untranslated sequence of various lengths (from 340 to 4600 nt), and a poly (A)+ sequence (<100 nt).


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)

Northern blot analysis of FEZ1 mRNA. (A) Detection  of FEZ1 mRNA in adult rat tissues. Northern blots containing  2 μg of poly(A)+ RNA from various adult rat tissues per lane  were incubated with the full-length FEZ1 cDNA fragment labeled with [α-32P]dCTP. (B) Detection of FEZ1 mRNA in developing mouse embryos. Northern blots containing 2 μg of  poly(A)+ RNA from mouse embryos in different developmental  stages (7, 11, 15, and 17 dpc) per lane were used. The positions of  FEZ1 mRNA are indicated by arrows with their approximate  sizes (knt, kilonucleotides) on the left margin of each blot.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Northern blot analysis of FEZ1 mRNA. (A) Detection of FEZ1 mRNA in adult rat tissues. Northern blots containing 2 μg of poly(A)+ RNA from various adult rat tissues per lane were incubated with the full-length FEZ1 cDNA fragment labeled with [α-32P]dCTP. (B) Detection of FEZ1 mRNA in developing mouse embryos. Northern blots containing 2 μg of poly(A)+ RNA from mouse embryos in different developmental stages (7, 11, 15, and 17 dpc) per lane were used. The positions of FEZ1 mRNA are indicated by arrows with their approximate sizes (knt, kilonucleotides) on the left margin of each blot.
Mentions: Northern blot analysis of eight tissues from adult rat has shown that FEZ1 mRNA with a size of ∼1700 nucleotides (nt) is expressed abundantly and exclusively in the brain, although faint expression of mRNA with a size of ∼4000 nt is also observed in the liver (Fig. 2 A). It is interesting to note that PKCζ mRNAs are also highly expressed in the brain (Ono et al., 1988b). The nematode UNC-76 is detected throughout the nervous system of the animals at all developmental stages from embryos (before outgrowth of the first axons) through adult worms (Bloom and Horvitz, 1997). Therefore, we investigated further the FEZ1 mRNA expression during development of mouse embryo with rat FEZ1 cDNA as a probe; the mouse FEZ1 gene deposited in the GenBank EST (Expressed Sequence Tag) database shows a very high sequence identity (>92%) with rat FEZ1. As shown in Fig. 2 B, a 6000-nt mRNA is expressed abundantly in the early to mid stage of development (from 7 d postcoitum [dpc] to 15 dpc). In the late stage of development (17 dpc), the 6000-nt mRNA disappears and instead a 1700-nt mRNA is expressed abundantly. Furthermore, a 2000-nt mRNA is expressed constantly, though slightly, in all developmental stages. It has been known that development of the nervous system in mouse embryo begins soon after 7 dpc with the neural plate formation and ends before 17 dpc. By cDNA cloning, all mRNAs observed here (1700-, 2000-, 4000-, and 6000-nt mRNAs) were confirmed to contain a 5′-untranslated sequence (∼100 nt), the same rat/mouse FEZ1 gene (∼1200 nt), a 3′-untranslated sequence of various lengths (from 340 to 4600 nt), and a poly (A)+ sequence (<100 nt).

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