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

Abstract

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-lamin B3 at the RS motifs, which have the general property of aggregation.

Regulation of lamin properties and functions: does phosphorylation do it all?

“…Moreover, fish and amphibian lamins B3 contain SR/RS dipeptide motifs specific for serine/arginine protein kinases (SRPKs). Such a motif is present in four copies in goldfish and torafugu, in three copies in zebrafish, and in one and two copies in Xenopus lamin A/B3 and B1/B2, respectively [ 54 ]. This motif is also present in invertebrates: Ce-lamin has four copies, lamin C from D. melanogaster has two copies and lamin Dm has a single copy.…”

“…This may reflect the adaptation of lamins to the biology of organisms in which oocytes are stored in the ovaries for long periods of time in prophase (diplotene). For example, in goldfish a major component of the oocyte nuclear lamina is lamin B3, and when Cdk1 kinase is not active, a still unknown SRPK kinase phosphorylates lamin B3 at the Cdk1 site [ 54 , 55 ].…”

“…This may lead to a situation where a particular phosphoacceptor amino acid residue in a lamin may be phosphorylated by an entire set of kinases. The best-known example of this effect is the N-terminal 'mitotic' consensus site and nearby sites, which can be modified not only by Cdk1, but also by other Cdks, Gsk3, ERK1-2/ MAPK, PKA and PKC, and by virus kinases both in vitro and in vivo [23][24][25][26]31,32,35,54,55,[95][96][97][98]. This means that multiple sites of phosphorylation can be modified by a wide range of stimuli (i.e.…”

Comparison of the CDC2-like kinase family across eukaryotes highlights the functional conservation of these unique biological thermometers

“…However, limited research has explored the genetic and functional diversification of this gene family. While some partial phylogenetic analyses of CLKs have been performed elsewhere 10,[12][13][14] , to date no study has demonstrated a complete eukaryotic timeline, which could offer valuable insights in predicting functional roles and diversification across species 15 . Similarly, whilst crystal structure comparisons of human CLK1-4 have been determined 1,2,16,17 , there exists a gap in evaluating the structural conservation of CLK homologs between different eukaryotes.…”

Nanobodies as new tools for studying large cargo transport and lamina organization

“…Fish and amphibian lamins contain additional SR/RS dipeptide motifs, which are targeted by serine/arginine protein kinases. This feature was hypothesized to reflect an adaptation of lamins to the biology of oocyte storage in ovaries over a long period of time (Yamaguchi et al, 2013).…”