The domain structures are denoted as shown in the lower box

tif of gfLB3 One of the well-known SR proteins is ASF/SF2, which has 20 TG-02 serines within the RS domain; of these serines, SRPK1 phosphorylates nearly a dozen serines adjacent to arginine. We next addressed whether the short RS stretch of gfLB3 is sufficient for high-affinity binding of SRPK1 for phosphorylation of Ser-28. We constructed GSTgfLB3N with various RS repeats by using site-directed mutagenesis. Purified gfSRPK1a was used for an in vitro kinase reaction. Phosphorylation activity decreased in a stepwise manner, corresponding to the repeat number of RS . Low activity was detected in the sequential RS null mutant. These results show that SRPK1 binds to the sequential RS repeats to Invertebrate lamins Drosophila C 2 Drosophila 1 Dmo Sea urchin 1 C. elegans 4 c The number of RS/SR repeats and percentage of Arg and Ser upstream of the sequence of the conserved p34cdc2 target Ser, derived from vertebrate and invertebrate lamin databases. Lamin protein sequences were from the current version of Genbank or ENSEMBL database. The Drosophila melanogaster lamin C,; the Drosophila lamin Dmo,; the sea urchin B-type lamin,; the Caenorhabditis elegans lamin,; the goldfish LB1,; the goldfish LB2,; the zebrafish LB1,; the zebrafish LB2; the torafugu LA1,; the torafugu LB1; the torafugu LB2,; the Tetraodon nigroviridis LA1,; the tetraodon LB1,; the tetraodon LB2,; the Xenopuslaevis LA,; the Xenopus LB1,; the Xenopus LB2,; the chicken LA,; the chicken LB1,; the chicken LB2,; the mouse LA,; the mouse LB1,; the mouse LB2,. As for LB3, the sequences of Fig. 4 were used for analysis. The LB3 gene was lost in the mammalian lineage. a Pufferfish LB3s have RS repeats bracketed by single Ala, Arg, or Ser. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19840854 b Xenopus LB1 has an RS-rich region that possibly binds to SRPK1. c C. elegans lamin does not contain the conserved p34cdc2 target Ser; therefore, the number of RS and the percentage of Arg and Ser were counted and calculated in the sequence region, respectively. Asterisks indicate lamins that contain more than two consecutive RS/SR repeats. ND indicates not done because of the absence of cDNA information. A. Yamaguchi et al. / FEBS Open Bio 3 165176 173 phosphorylate Ser-28. In contrast, intense phosphorylation was detected on Ser-28 with extended RS repeats. The highest activity was detected in mutant J, which has the longest consecutive RS, aside from mutant M. One amino acid substitution of Ser to Ala slightly decreased the intensity. The intensity of mutant L, which had shorter RS repeats divided by additional tetrapeptides, decreased abruptly. This finding indicates that the length of consecutive RS repeats is important for SRPK1 binding, whereas insertion of a neutral amino acid has a lesser effect. The first RS is also involved in gfSRPK1 binding because mutant E had a significantly low intensity, in spite of the presence of three repeats of RS; an additional repeat compensated for the missing RS of mutant E. These results indicate that gfLB3 has a functional stretch of RS repeats, where SRPK1 binds to the region with RS repeats to catalyze the phosphorylation of Ser-28 eight amino acids downstream of the RS repeat at the active site. Similar substrate specificity was detected by recombinant human SRPK1. The mutant E, which deleted the first RS repeat seems to have different substrate specificity between goldfish and human SRPK1 on the histogram. A significant difference is that the first RS was not necessary for hsSRPK1 but was necessary for gfSRP