Studies on the SRPK1-dependent phosphorylation of SRSF1 have uncovered a remarkable catalytic mechanism displaying very unique features. How SRPK1 achieves multi-site and directional phosphorylation at the molecular level has recently been revealed through the X-ray structures of SRPK1 bound to either a short peptide substrate () or the core region of SRSF1 (RRM2-RS1) (). These two structures show that SRPK1 possesses a docking region in the large lobe that can accept a portion of the RS domain. This acidic docking groove in the kinase accommodates basic peptides about 6–7 residues in length. Mutation of several acidic residues within the docking groove (e.g., D564, E571, D548) eliminates processive phosphorylation and strong directional preferences within the RS domain (48
). The peptide-bound form of SRPK1 allowed identification of a small segment preceding the RS domain of SRSF1 (R191
VKVDGPR) as the cognate substrate site that specifically interacts with the docking groove. Mutations of two basic residues in this segment (R191A & K193A) altered the catalytic mechanism suggesting the importance of this region in SR protein phosphorylation (40
). However, a subsequent structure co-crystallized with a truncated form of SRSF1 (RRM2-RS1) revealed that the N-terminal part of the RS domain rather than residues 191–196 was bound to the docking groove (). This was surprising as this RS segment (N’-RS1; S201
YGRSRSRSR), binds to a pocket far from the active site (), yet eventually undergoes phosphorylation based on mapping studies (58
). These two kinase structures appeared to offer differing perspectives on which regions outside the RRMs bind in the docking groove. In the RRM2-RS1-bound structure, the docking groove binds an N-terminal segment of RS1 (residues 201–210) whereas in the peptide-bound structure, the docking groove binds sequences further N-terminal from N’-RS1 (residues 191–198).
Model Describing How the RS domain of SRSF1 Is Threaded Into the Active Site of SRPK1
Since prior mapping studies showed that SRPK1 moves along the RS domain in a C-to-N direction (), it is possible that the structure of the SRPK1-SRSF1 complex changes as a function of phosphorylation, and that the two X-ray structures present two distinct states along the catalytic pathway. This model was tested using mutant forms of SRPK1 and SRSF1 that differentially cross-link as a function of ATP. A cysteine placed in the docking groove of SRPK1 (K604C) cross-links with a cysteine substituted in the segment preceding the RS domain (K193C) only in the presence of ATP. In comparison, a second mutant form of SRSF1 where a cysteine is inserted in N’-RS1 (R204C) cross-links with the docking groove cysteine in the absence of ATP. When considered in light of the directional phosphorylation mechanism, these structural observations can be used to propose a model for substrate phosphorylation in which the Arg-Ser repeat motif constitutes a mobile docking element, where part of RS1 which is to be phosphorylated (N’-RS1; residues 204–210) first serves as a docking sequence placing a C-terminal serine from the initiation box at the active site (). As each serine undergoes phosphorylation, the docking motif moves by two residue increments towards the N-terminus. Each Arg-Ser tract from the docking groove is sequentially displaced by an N-terminal tract with the originally identified docking motif in the docking groove at the end of the reaction. In essence, the entire RS1 motif is fed through the active site of the kinase until the furthest N-terminal docking motif (residues 191–196) ‘hits’ the kinase docking groove. Interestingly, residues 191–196 lie in βstrand 4 of RRM2 so that it must unfold in order to occupy the docking groove, a result supported by circular dichroism and mutagenesis experiments (28
). Although the C-terminal residues of RS1 are poorly defined in the structure, a single phosphoserine resulting form a small impurity in the co-crystallized nucleotide analog (AMPPNP) was found in the basic P+2 pocket of the kinase (). Mutations in this pocket (R515,518,561A) reduce the rate of phosphate incorporation in the N-terminal portion of RS1 (48
) suggesting that the P+2 pocket stabilizes the growing phosphorylated RS domain.
Although structural studies on SRPK1 are the most advanced at this time, it is likely that other SR-directed protein kinases will use aspects of the above “feeding” mechanism. For example, the yeast SRPK, Sky1p, also contains a similar charged docking groove akin to SRPK1, which plays a role in recognition of its cognate substrate Npl3 (). Although Npl3 lacks a classic RS domain, it has a single RS dipeptide at the very C-terminus of its RGG (Arg-Gly-Gly rich domain). In vitro studies on Sky1p and Npl3 show that the RGG domain contains multiple docking motifs, at least one of which is essential for the interaction of Npl3 with Sky1p (63
). Although Sky1p modifies a rather distinct substrate compared to SRSF1, it appears that the mobile docking element may be a conserved feature in SR and SR-like proteins and their kinases. In comparison to SRPK1, the X-ray structures of the CLK kinases revealed no deep groove that would fit a peptide with geometric complementarity (). Moreover, the corresponding segment that would constitute the SRPK1 docking groove is shallow and dispersed with both acidic and basic charge patches (). This compares to the highly acidic nature of the SRPK1 docking groove. This charge distribution suggests that the hypo-phosphorylated RS domain with alternate positive and negative charge could interact with CLK with high efficiency compared to unphosphorylated RS domain. That is, the product of SRPK1 phosphorylation might be the substrate of CLK. We showed that CLK1 will readily phosphorylate approximately 7 serines in the RS2 segment in SRSF1 when it is pre-phosphorylated in RS1 by SRPK1 (47
). In comparison, SRPK1 can phosphorylate about 3 serines in RS2 but very inefficiently (60
). The differences between SRPK1 and CLK1 are likely to be rooted in differences in docking elements and charge dispersal (). While SRPK1 catalyzes a very strict, directional mechanism owing to its electronegative docking groove, CLK1 lacking such a groove randomly phosphorylates the RS domain of SRSF1 (29
). Understanding how CLK kinases modify RS domains will be greatly advanced with the generation of a CLK:RS domain structure and further investigations into its substrate specificity.
Surface Electrostatic Properties of SRPK and CLK Kinases