In vitro characterization of the RS motif in N-terminal head domain of goldfish germinal vesicle lamin B3 necessary for phosphorylation of the p34cdc2 target serine by SRPK1

The nuclear envelopes surrounding the oocyte germinal vesicles of lower vertebrates (fish and frog) are supported by the lamina, which consists of the protein lamin B3 encoded by a gene found also in birds but lost in the lineage leading to mammals. Like other members of the lamin family, goldfish lamin B3 (gfLB3) contains two putative consensus phosphoacceptor p34cdc2 sites (Ser-28 and Ser-398) for the M-phase kinase to regulate lamin polymerization on the N- and C-terminal regions flanking a central rod domain. Partial phosphorylation of gfLB3 occurs on Ser-28 in the N-terminal head domain in immature oocytes prior to germinal vesicle breakdown, which suggests continual rearrangement of lamins by a novel lamin kinase in fish oocytes. We applied the expression-screening method to isolate lamin kinases by using phosphorylation site Ser-28-specific monoclonal antibody and a vector encoding substrate peptides from a goldfish ovarian cDNA library. As a result, SRPK1 was screened as a prominent lamin kinase candidate. The gfLB3 has a short stretch of the RS repeats (9-SRASTVRSSRRS-20) upstream of the Ser-28, within the N-terminal head. This stretch of repeats is conserved among fish lamin B3 but is not found in other lamins. In vitro phosphorylation studies and GST-pull down assay revealed that SRPK1 bound to the region of sequential RS repeats (9-20) with affinity and recruited serine into the active site by a grab-and-pull manner. These results indicate SRPK1 may phosphorylate the p34cdc2 site in the N-terminal head of GV-Iamin B3 at the RS motifs, which have the general property of aggregation. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.