Phosphorylation of the p34(cdc2) target site on goldfish germinal vesicle lamin B3 before oocyte maturation.
The nuclear membranes surrounding fish and frog oocyte germinal vesicles (GVs) are supported by the lamina, an internal, mesh-like structure that consists of the protein lamin B3. The mechanisms by which lamin B3 is transported into GVs and is assembled to form the nuclear lamina are not well understood. In this study, we developed a heterogeneous microinjection system in which wild-type or mutated goldfish GV lamin B3 (GFLB3) was expressed in Escherichia coli, biotinylated, and microinjected into Xenopus oocytes. The localization of the biotinylated GFLB3 was visualized by fluorescence confocal microscopy. The results of these experiments indicated that the N-terminal domain plays important roles in both nuclear transport and assembly of lamin B3 to form the nuclear lamina. The N-terminal domain includes a major consensus phosphoacceptor site for the p34(cdc2) kinase at amino acid residue Ser-28. To investigate nuclear lamin phosphorylation, we generated a monoclonal antibody (C7B8D) against Ser-28-phosphorylated GFLB3. Two-dimensional (2-D) electrophoresis of GV protein revealed two major spots of lamin B3 with different isoelectric points (5.9 and 6.1). The C7B8D antibody recognized the pI-5.9 spot but not the pI-6.1 spot. The former spot disappeared when the native lamina was incubated with lambda phage protein phosphatase (lambda-PP), indicating that a portion of the lamin protein was already phosphorylated in the goldfish GV-stage oocytes. GFLB3 that had been microinjected into Xenopus oocytes was also phosphorylated in Xenopus GV lamina, as judged by Western blotting with C7B8D. Thus, lamin phosphorylation appears to occur prior to oocyte maturation in vivo in both these species. Taken together, our results suggest that the balance between phosphorylation by interphase lamin kinases and dephosphorylation by phosphatases regulates the conformational changes in the lamin B3 N-terminal head domain that in turn regulates the continual in vivo rearrangement and remodeling of the oocyte lamina.
Yamaguchi
Akihiko
A
Laboratory of Marine Biology, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-Ku, Fukuoka 812-8581, Japan. akihikoy@agr.kyushu-u.ac.jp
Katsu
Yoshinao
Y
Matsuyama
Michiya
M
Yoshikuni
Michiyasu
M
Nagahama
Yoshitaka
Y
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006054:Goldfish / Q000378:metabolism*; D034921:Lamin Type B / Q000737:chemistry / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D034881:Nuclear Lamina / Q000378:metabolism; D009865:Oocytes / Q000254:growth & development* / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
D000911:Antibodies, Monoclonal; D034921:Lamin Type B; D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D001710:Biotin; D016203:CDC2 Protein Kinase