SELEX approaches have been applied to determine specific SR-binding RNA sequences

el of the triple mutant after 15 min of incubation with CLK1 is reduced to about 13 sites, a value close to that for the wild-type SR protein after phosphorylation with SRPK1. The minor difference between this mutant and SRPK1-phosphorylated SRSF1 could reflect a small effect of Ser-Pro mutations on Arg-Ser phosphorylation. Overall, none PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19843565 of the mutations affected the rate constant for multisite phosphorylation. These findings indicate that CLK1 not only phosphorylates Ser-Pro dipeptides as previously suggested6 but also does not require these modifications for multisite phosphorylation of other serines in the RS domain of SRSF1. To determine whether any of the mutations in the Ser-Pro dipeptides affect the gel-shift phenomenon, we performed SDS-PAGE analyses. As shown in Fig. 6d, CLK1 readily induced a gel shift in SRSF1 that occurs within the first 2 min after approximately 510 serines are modified. In comparison, the triple mutant does not undergo a gel shift even after 10 min when the reaction has reached an apparent endpoint. To determine whether phosphorylation at all three serines is necessary to attain the gel shift, we SB 1317 price compared the mobilities of the single mutants to that for wild-type SR protein and the triple mutant at the end of the phosphorylation reaction. In general, the single mutants display intermediate mobilities on SDS-PAGE compared to SRSF1 and the triple mutant. These findings suggest that phosphorylation of all three serines in this region may be required for the observed gel shift. To assess whether this deficiency is due to poor interactions of the SR with CLK1, we performed competition studies and found that the triple mutant observed no defects in binding compared to SRSF1. Overall, these data indicate that the gel-shifted form of SRSF1 is induced by CLK1-dependent phosphorylation of three Ser-Pro dipeptides in the RS domain. Also, it appears that SRPK1 and CLK1 have overlapping substrate specificities with CLK1 being distinctive for its ability to uniquely modify Ser-Pro dipeptides. J Mol Biol. Author manuscript; available in PMC 2014 August 23. Aubol et al. Page 8 Discussion CLK1 and SRPK1 use different strategies for SR protein phosphorylation NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Through detailed kinetic and structural studies, the mechanism of SRSF1 phosphorylation by SRPK1 is now well understood.2 An electronegative docking groove in the large lobe of this kinase firmly orients a long Arg-Ser repeat so that the C-terminal end of the stretch initially resides in the active site. As the reaction proceeds, phosphoserines are expelled and new dipeptide repeats are fed from the docking groove into the active site. Thus, the juxtaposition of a docking groove and active site along with neighboring RRMs enforce a highly ordered mechanism for RS domain processing. In comparison, CLK1 lacks a similar docking groove and has been shown to catalyze a random phosphorylation mechanism.27 Despite this apparent handicap, CLK1 binds SRSF1 with better affinity than SRPK1, a result of a combination of enhanced RS domain as well as RRM2 contacts. While SRPK1 only requires N-terminal Arg-Ser repeats for high-affinity binding, CLK1 utilizes these along with additional contacts in the short, C-terminal Arg-Ser stretch to initially bind the RS domain. Given these new discoveries, we propose that CLK1, lacking the constraints of a docking groove, is likely to initially bind the RS domain in sev