Allosteric interactions direct binding and phosphorylation of ASF/SF2 by SRPK1.

The SR protein family plays essential roles in pre-mRNA splicing. These proteins are thought to be involved in every step during the assembly of the spliceosome, the catalyst for splicing reactions (2, 3). SR proteins are modular and contain one or two RNA recognition motifs (RRMs) at the N-terminus and an arginine-serine dipeptide rich domain at the C-terminus. One of the most well studied members of the SR protein family, ASF/SF2, contains two N-terminal RRMs followed by a 50-residue C-terminus RS domain. The RS domain can be divided into two modules, RS1 and RS2, whose sequence and structural properties allow them to be distinctly recognized and regulated by functionally unique kinases. Of the eighteen serines in the RS domain, twelve are present as every other residue, primarily as RS dipeptides within a 24 residue-stretch, which is referred to as the RS1 motif (residues 204 to 227). The other six serines are present within the C-terminus RS2 motif (residues 228–248). Members of two kinase families, SR protein kinase (SRPK) and Cdc2-like-kinase/Serine-Threonine-Tyrosine (CLK/STY), phosphorylate the RS domain following the distribution rules of the serines: SRPK1 phosphorylates the RS1 serines in the cytoplasm generating hypo-phosphorylated ASF/SF2 (p-ASF/SF2). CLK/STY kinases are nuclear and they further phosphorylate the RS2 serines generating hyper-phosphorylated ASF/SF2 (pp-ASF/SF2) (4). Hypophosphorylation of ASF/SF2, p-ASF/SF2, is essential for the nuclear import of this SR protein (5, 6). Splicing experiments in vitro have demonstrated that ASF/SF2 undergoes cycles of phosphorylation and dephosphorylation during the assembly of the spliceosome (7, 8, 9). Several recent reports support this by showing that both SRPKs and protein phosphatases are present within the spliceosome (10, 11, 12). It is however unclear if phosphorylation and dephosphorylation of ASF/SF2 are subject to the entire RS domain (RS1 and RS2) or either one of the two modules (RS1 or RS2). The basic principles of SR protein phosphorylation by SRPK1 are emerging from recent kinetic and structural studies. The phosphorylation mechanism in the ASF/SF2:SRPK1 system defines a new paradigm for protein phosphorylation: SRPK1 has been shown to bind ASF/SF2 with high affinity (Kd = 50 nM) which promotes processive-phosphorylation of about 8 of the 12 serines in the RS1 motif. This extended sequential reaction requires sliding of the substrate along a highly charged groove in the kinase (5, 13). The x-ray crystal structure of ASF/SF2: SRPK1: AMP-PNP complex provided clues for the basis of high affinity binding between SRPK1 and ASF/SF2 (1). The complex used for crystallization used the core ASF/SF2 fragment spanning residues 105–219 while SRPK1 in the complex is devoid of a long insert, known as the spacer domain, that bifurcates the two kinase domains and the N-terminus non-homologous regions (SRPK1ΔNS3) (Figure 1A). The structure of the core ASF/SF2: SRPK1 complex showed tripartite interaction between the kinase and substrate. The most surprising of which is the N-terminus half of the RS1 motif that is destined to be phosphorylated remains bound to a groove distal to active site (Figure 1B–1C). This interaction is dominated by electrostatics where a negatively charged surface interacts with the positive charged RS domain. A second interface is between a phospho-serine and a positively charged pocket of the kinase. We showed in a previous study that the pocket surrounding this phospho-serine plays an important role in maintaining high efficient phosphorylation of the RS domain (14). Figure 1 Structural model of the ASF/SF2: SRPK1 complex The last interface is formed between the RRM2 of ASF/SF2 and the kinase where RRM2 makes a 3-point contact with both the small and large lobes at the front of the kinase (Figure 1B–1C). In particular, W134 and Q135 of ASF/SF2 are stacked against H90 and W88 of SRPK1, while R154 protrudes into a pocket formed by helices αD and αF, and is stabilized by interaction with E534 and Y549. Additionally, Q135 also contacts the backbone of the glycine-rich loop of SRPK1. In all, nearly 1100A of surface area is buried upon the complex formation. The β4 motif of RRM2, which was previously thought to be involved in binding to the kinase remained in a folded conformation in the structure (13). Based on the structural information and correlated cross-linking and secondary structure melting analysis, we showed that the N-terminal portion of the RS domain (N′-RS1) translocates from the docking groove to the active site in a sequential manner during catalysis, which ultimately requires the melting of β4 strand in RRM2 (1). It was proposed that β4 docks into the kinase groove during the phopshorylation of the last few serines. However, β4 may not interact strongly in the kinase docking groove and may facilitate dissociation of the phosphorylated ASF/SF2. The β4 motif thus, may act as a dissociative docking motif. Kinetic studies have shown that the RS domain linked to a fragment of RRM2 has an intrinsic capacity to initiate processive phosphorylation but that it is very limited in extending this reaction beyond 3 steps (14). These observations suggest a potential role for the RRM in the mechanism of ASF/SF2 phosphorylation. In this work we have investigated the role of RRM2 in kinase binding and phosphorylation. We find that the isolated RRM binds weakly to the kinase, and, alteration of all three contact residues in RRM2 enhance the kinase binding to a small extent suggesting that the binding interface is flexible. Surprisingly, binding is significantly enhanced in the presence of the RS peptide. Moreover, this RS dependent enhancement of RRM binding is observed mainly with the full length but not SRPK1ΔNS3. Finally, we show that in the presence of the RRM, SRPK1 phosphorylates the isolated RS domain in enhanced efficiency. We propose a model of substrate recognition, phosphorylation, and product release.