he equivalent amounts of proteins were separated on polyacrylamide gels, except one-50th quantity of His-gfSRPK1 protein was loaded for Western blotting. Fig. 8. The correlation of the length of RS repeats, with the distance to the target phosphorylation site. The gfLB3 has a SRPK1 phosphorylation site three repeats away from the RS motif, whereas tetraodon has a target Ser 13 aa away from the RS motif. Constructs of two INK-128 biological activity mutant SR proteins that have 5 additional consecutive Ala downstream of the RS repeats in wild-type A and mutant J, respectively. Red characters indicate RS repeats. Blue characters indicate amino acids appended to wild-type gfLB3. Purified gfSRPK1a was diluted 50-fold with DB, and then used in a kinase assay. Reactions were started by mixing the reaction buffer, incubated at 25 C in a water bath, and terminated by mixing with SDS sample buffer PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19841886 at each time. Samples were separated on polyacrylamide gels. Lamin-antibody complexes were visualized with chemiluminescence reagents on an X-ray film for 30 s. Relative intensity was plotted on the graph. Subscripts of the bracket indicate the number of the repetition of RS. Bars on the right indicate molecular masses. other lamins, except fish LB3, which indicates that the N-terminus of GV-lamin B3 differs from that of other lamins that have the same conserved cdc2 target site in front of the rod domain. The gfLB3 supports the nuclear membrane, but B1 and B2 are constituents of internal filamentous structure. Different nuclear distribution of lamins might be dependent on having the RS motif or not. In contrast, Xenopus LB3 has a well-characterized GV-lamin in the absence of RS repeats, but XLB1 has RS-like motif that possibly interacts with SRPK1. This suggests that Xenopus lamins are controlled differently from those of fishes. SR proteins tend to form aggregates because they have properties of intrinsically disordered proteins that likely lack a folded structure under physiological conditions. Phosphorylation on the RS domain by multiple kinases has 
shown to be critical for RS domainmediated protein interactions. Thus, reversible molecular aggregation and dissociation seem to be regulated by phosphorylation/ dephosphorylation in the RS domain. Unphosphorylated RS repeats of LBR adopt short, transient helical conformation, whereas phosphorylation of Ser induces Arg claw-like structures. The lamin B receptor is the most thoroughly studied protein of the RS domain-containing proteins. The zebrafish LBR is the only characterized LBR in the fish group that contains three RS repeats Fig. 10. Grab-and-pull model of substrate binding and phosphorylation of the p34cdc2 target site of gfLB3 by SRPK1. SRPK1 binds with affinity towards the N-terminal RS motif of gfLB3 to pull the target serine into the active site for phosphorylation. Pulling activity is dependent on the binding affinity, which correlates with the number of consecutive RS repeats of gfLB3. neighboring an SP motif in exon 2, but the total number of RS/SR motifs is less than that in human LBRs. LBR self-associates through its N-terminal domain and forms oligomers that are suggested to bind heterochromatin. The RS domain of LBR is phosphorylated A. Yamaguchi et al. / FEBS Open Bio 3 165176 175 by multiple kinases at the beginning of mitosis by nuclear translocated SRPK1 and potentially by Akt and Clk kinases. Furthermore, Ser-71, which is located just upstream of the RS repeats, is phosphorylated by cdc2 kinase during the M