|Home | About | Journals | Submit | Contact Us | Français|
The splicing of mRNA requires a group of essential factors known as SR proteins that participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition domains (RRMs) and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SRPK family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus and may also play important roles in alternative splicing, mRNA export and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. SRSF1, a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a posttranslational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity and rapidly modifies about 10–12 serines in the N-terminal portion of the RS domain (RS1) using a mechanism that incorporates sequential, C-to-N phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site and a β strand in RRM2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain and a highly directional processing mechanism.
The complexity of the human proteome is regulated through the alternative splicing of large precursor mRNAs (pre-mRNA).1 While this process plays a significant role in normal cellular development, changes or defects in alternative splicing have also been linked to human disease (1–3). Splicing reactions occur at the spliceosome, a macromolecular complex composed of five snRNPs (small nuclear ribonucleoproteins) (U1, U2, U4, U5, & U6) and over one hundred auxiliary proteins (4). Among these many proteins, one family of splicing factors known as SR proteins is essential for controlling numerous aspects of mRNA splicing as well as other RNA processing events. SR proteins interact with splicing components (U1-70K & U2AF35) early during spliceosome development and help establish the 5’ and 3’ splice sites (5, 6). Later, they recruit the U4/U6•U5 tri-snRNP (7) and also enhance the second catalytic step in splicing (8). Splicing is tightly coupled to transcription and SR proteins have been shown to play a role by binding the C-terminal domain of RNA polymerase II and regulating CDK9 (9). SR proteins serve roles in many post-splicing events including mRNA export (10, 11), translation regulation (12, 13) and genomic stability (14, 15). More than a decade ago it was discovered that two protein kinase families (SRPK and CLK) phosphorylate SR proteins altering their cellular distribution and activities (16–18). In the last several years, great strides have been made in understanding how the SRPKs recognize and phosphorylate the SR proteins. In this review, we will highlight how a highly dynamic and distinct interplay between kinase and substrate is necessary for the modification of the SR protein SRSF1, a prototypical SR protein involved both in constitutive and alternative splicing (19). These studies have shown that SRPK1 undergoes an interesting feeding mechanism where multiple contacts in the SR protein are utilized to catalyze a lengthy polyphosphorylation reaction.
SR proteins derive their name from a lengthy (50–300 residue) C-terminal tail rich in Arg-Ser repeats known as the RS domain (Figure 1). SRSF1 (aka ASF/SF2) represents a typical arrangement for an RS domain where Arg-Ser repeats are bracketed by smaller repeats and some isolated Arg-Ser pairs. In addition to RS domains, SR proteins also contain one or two N-terminal RNA recognition motifs (RRMs) that modulate SR protein interactions in the spliceosome by binding short mRNA sequences (splicing enhancers) (20, 21). Numerous screening procedures have revealed that the observed determinants are somewhat nonspecific raising the possibility that members of the SR protein family serve redundant functions in mRNA splicing (22). While all SR proteins can complement splicing-deficient S100 cytoplasmic extracts of HeLa cells (20) in support of this idea, there are other studies showing that certain SR proteins serve tissue-specific roles at various developmental stages (23, 24) arguing for a specialized role for some SR proteins. While there is no X-ray structure for an SR protein in either a phosphorylated or unphosphorylated state, a recent NMR structure shows that one of the RRMs of SRSF1 (RRM2) adopts a typical RNA binding fold (25). Sequence analyses suggest that SR proteins may have properties consistent with intrinsically disordered proteins owing to an RS domain that is expected to be largely unstructured (26). On the other hand, all atom calculations of an eight dipeptide repeat [(Arg-Ser)8] suggest that the unphosphorylated sequence adopts a helical form with the arginines pointing out into solution for charge minimization and a compact, ‘claw-like’ structure upon phosphorylation (27). Appreciable helical content is not detected in circular dichroism experiments for SRSF1 or its RS domain in either the phosphorylated or unphosphorylated forms (28) suggesting that if the Arg-Ser repeats possess helical structure it may not be highly stable in solution. Recent studies showed that the phosphorylated RS domain is protected from dephosphorylation by the neighboring RRMs suggesting that the RS domain may not be disordered and could pack onto other domains in the SR protein (29). Nonetheless, while the RRMs adopt a classic RNA binding fold, it is still not fully clear how the RS domain folds by itself or in context of the SR protein or how phosphorylation modifies SR protein conformation.
Early studies showed that SR proteins undergo multiple rounds of phosphorylation and dephosphorylation in route to spliceosome assembly (30–32). Phosphorylation was shown to occur in the RS domain and alter how the SR protein functions in the spliceosome. For example, the SR proteins SRSF1 and SRSF2 (aka SC35) and interact with the 70 kDa subunit of U1 (U1-70K) and the 35 kDa subunit of U2AF (U2AF35) in a phosphorylation-dependent manner (5, 6) establishing the appropriate splice sites. More than a decade ago, it was discovered that the SRPK & CLK families of protein kinases can polyphosphorylate RS domains and alter SR protein cellular distribution and splicing function (16, 17, 33). However, the role of RS domain phosphorylation in alternative splicing is not well understood. Although some studies suggest that the RRMs are the principal driving element for alternative splicing of some pre-mRNA (34), other studies have shown that the phosphoryl content of the RS domain is important. For example, phosphorylation of SRSF1 controls the alternative splicing of the caspase-9 and Bcl-x genes and induction of a pro-apoptotic phenotype (35). While further investigations are needed to provide a more forceful link between RS domain modification and splicing, it has become abundantly clear that phosphorylation is directly linked to the nuclear entry of SR proteins. It has been shown that phosphorylation of the RS domain leads to enhanced interactions with the nuclear import receptor, transportin SR, and nuclear entry of the SR proteins where they largely reside in speckles (36–38). While SRPKs play a direct role in nuclear import, the CLK family controls the nuclear distribution of SRSF1 and other SR proteins. Thus, through interactions with two families of protein kinases, the cellular location and presumably splicing function of SR proteins can be precisely controlled. Kinetic studies on SRPK1 and SRSF1, together with crystal structures of SRPK1 bound to peptide and protein substrates and the recent structures of CLK1 and CLK3 suggest an elegant mechanism of recognition and phosphorylation by these two kinases which regulate biological function of SR proteins in the cell.
Most of our knowledge regarding SRPKs comes from studies on the human SRPK, SRPK1, and the yeast analog, Sky1p. SRPKs contain a well-conserved kinase domain that is bifurcated by a large nonconserved insert domain (approx. 250 amino acids). The insert domain in SRPKs regulate sub-cellular localization, as its deletion changes the distribution pattern of the kinase from nuclear-cytoplasmic to exclusively nuclear (39). In addition to this important regulatory domain, SRPKs contain N-terminal and/or C-terminal extensions, which are not conserved. Deletion of the insert and N-terminal extension does not inactivate the catalytic activity of SRPK1 suggesting that these elements play auxiliary roles (40). The X-ray structure for SRPK1 lacking its N-terminus and most of the insert domain reveals the signature bi-lobal fold found in all eukaryotic protein kinases (Figure 2A). The small lobe is composed mostly of β strands and binds the nucleotide (ADP in the SRPK1 structure). The larger lobe is composed mostly of α helices and provides residues important for substrate binding. A short segment of the insert domain connecting the two major kinase lobes is present and adopts short helical conformations. Like other members of the CMGC group of protein kinases, SRPK1 contains a small insert within the kinase domain known as the MAPK insert which connects helices αG and αH (Figure 2A). While the X-ray structure of SRPK1 was solved with a short substrate peptide, this peptide binds unexpectedly outside the active site in a groove generated by the MAPK insert and a loop connecting helices αF and αG (Figure 2A). Later, we will discuss how this docking groove binds SRSF1 and feeds the RS domain into the active site for sequential phosphorylation.
X-ray structures of the kinase domains of CLK1 and CLK3 have been reported recently (41) and are worth noting here given their overlapping substrate specificities with the SRPKs. While the CLK family of protein kinases is capable of widespread RS domain phosphorylation, their structures are distinct from those of the SRPKs in several ways. Most significantly, the CLK enzymes lack a large insert domain dividing the kinase core and, unlike the SRPKs, are autophosphorylated on both serine and tyrosine (42). The CLK kinases have large N-termini as do the SRPKs but, unlike the SRPKs, these extensions are rich in isolated Arg-Ser dipeptides. Although the CLK family also belongs to the CMGC group of kinases, changes in the sequence of the MAPK insert and positions of helices αG and αH result in the loss of the deep substrate docking groove observed in SRPK1. In addition to the MAPK insert, CLK1 and 3 contain another small insert between stands β6 and β9 in the kinase core that interacts with a hydrophobic pocket near the hinge region connecting the kinase lobes.
While many protein kinases are highly regulated through diverse mechanisms, SRPK family members are constitutively active and require no posttranslational modifications or additional protein subunits for optimal activity. Several key structural elements are essential in the maintenance of this highly active form of SRPK1. In some protein kinases, the activation loop plays a regulatory role by controlling access to the active site and only adopts an open configuration upon phosphorylation by other protein kinases (43, 44). The activation loop of SRPK1 is comparatively short and, lacking a reversible phosphorylation site, adopts a stable conformation that allows ready access of substrates to the active site (Figure 2B). Extensive biochemical analyses have shown that the activation loop in SRPK1 is highly malleable (46). Molecular dynamics simulations showed that alternative residues can mediate contacts that are lost upon mutation of some residues in the activation segment and maintain the structural integrity of the activation segment. Thus, SRPK1 is resilient to inactivation and exhibits robust phosphorylation activity. The extensive phosphorylation that SRPK1 must execute for each SR protein is a likely explanation for evolution of such robust activity. In addition to activation loops, all protein kinases possess a catalytic loop with a conserved aspartic acid that forms a hydrogen bond with the hydroxyl serine/tyrosine of the substrate. In the case of SRPK1, the catalytic loop aspartate is ideally poised to abstract the hydroxyl hydrogen from the substrate serine, a necessary step for protein phosphorylation (45). Several short-range interactions within the small lobe and between the large and small lobes around the active site participate in maintaining the catalytically active conformation. Two conserved interactions in all active protein kinases are also present in SRPK1: an ion pair between an invariant Glu in helix αC and an invariant Lys in strand β3 in the small lobe, and a hydrogen bond between the activation loop and helix αC (Figure 2B).
While both the SRPK and CLK families catalyze multi-site phosphorylation of SR proteins, the structural data suggest that differences in critical regions such as the MAPK insert and N-terminus may impart distinct regiospecificities. To address this issue, the mechanism of phosphorylation of SRSF1 by both enzymes was investigated using mass spectrometric methods. As shown in Figure 1, the RS domain of SRSF1 contains many serines throughout and it is not clear whether these two kinases show preferences for specific residues. The mapping of phosphorylation sites in the RS domain is a vexing problem owing to the redundancy of the Arg-Ser repeats and difficulties in separating/identifying the closely related polybasic fragments in traditional mapping studies. This problem has been circumvented using a modified form of SRSF1 that contains four Arg-to-Lys substitutions in the RS domain. Upon phosphorylation and cleavage with the endoprotease LysC, five fragments encompassing the complete RS domain of SRSF1 could be identified by MALDI-TOF mass spectrometry (47). These studies detected about 8 phosphoserines in the N-terminal portion of the RS domain. To define further the phosphorylation segment in SRSF1, a wide series of truncation derivatives were made and their phosphoryl contents were assessed using mass spectrometry (48). These studies showed definitively that SRPK1 is a regiospecific protein kinase preferring to phosphorylate up to 12 serines in the N-terminal region of the RS domain of SRSF1 (Figure 1). Single turnover kinetic studies reveal that this N-terminal region (RS1) is phosphorylated very efficiently within 1–2 minutes. In comparison, CLK1 does not show this regiospecificity and instead can phosphorylate all 20 serines in the RS domain of SRSF1 (47). Furthermore, CLK1 appears to be able to phosphorylate completely the RS domain of SRSF1 even if the RS1 segment is pre-phosphorylated by SRPK1. This sequential phosphorylation of the RS1 (by SRPK1) and RS2 (by CLK1) segments is biologically relevant as it has been demonstrated that SRSF1 lacking RS2 sequences translocates to the nucleus but is neither additionally phosphorylated nor dispersed in the nucleus by CLK1 (40). These studies provide a model where SRPK1 phosphorylates RS1 leading to translocation of SRSF1 from the cytoplasm to nuclear speckles while CLK1 phosphorylates RS2 leading to broad nuclear dispersion of the SR protein.
While it is not uncommon for protein kinases to exhibit somewhat relaxed substrate specificities and phosphorylate more than one site in their protein target [e.g.,(49–52)], SRPK1 possesses the distinct ability to efficiently insert numerous phosphates in close proximity in RS domains. In general, protein kinases recognize local charges flanking the site of phosphorylation (53). Random library searches have shown that SRPK prefers to phosphorylate serine but not threonine that is next to arginine residues (54). These studies were performed with a biased peptide library (arginine fixed in the P-3 position) and only contained a single serine for modification. In contrast, SRPKs phosphorylate many consecutive serines in a richly electropositive substrate. To accomplish this task SRPK would need to maneuver deftly through a substrate whose charge is dramatically changing after each round of phosphorylation. The question this raises is whether these splicing kinases must re-engage the RS domain after each phosphorylation reaction through a sequence of dissociation/association steps (distributive phosphorylation) or whether the RS domain can stay attached during subsequent rounds of phosphorylation simply translating through the active site (processive phosphorylation) (Figure 3A). There are examples of protein kinases that catalyze multi-site phosphorylation using either mechanism and sometimes a combination of both. For example, the nonreceptor protein tyrosine kinase Src phosphorylates up to 15 tyrosines in the protein Cas using a processive mechanism (49, 50). In contrast, the dual specific protein kinase MEK activates MAPK through a two-site phosphorylation mechanism that is fully distributive (52, 55). Finally, the yeast cyclin-CDK complex from budding yeast (Pho80–Pho85) appears to phosphorylate 5 serines in the transcription factor Pho4 using a semiprocessive mechanism (56).
The question of how a splicing kinase modifies an SR protein was originally addressed for SRPK1 and its substrate SRSF1 using a start-trap protocol (57). In this experiment, a peptide inhibitor or a kinase-dead form of SRPK1 [kdSRPK1] is added at the start of the reaction to a pre-formed enzyme-substrate complex in single turnover experiments (i.e.-[SRPK1]>[SRSF1]). If the enzyme phosphorylates the RS domain in a distributive manner then free enzyme and phospho-intermediates of the SR protein are generated during the reaction that can be trapped by the inhibitor or kdSRPK1 and lead to reaction inhibition (47, 48, 57, 58). However, if the mechanism is processive, then no free enzyme or phospho-intermediates will be released and the peptide inhibitor or kdSRPK1 cannot stop the reaction. For SRSF1 it was found that SRPK1 phosphorylates, on average, 5–8 of the 12 available serines in RS1 using a processive reaction before the enzyme dissociates and continues in a distributive manner. These findings suggest that SRPK1 may use a dual-track mechanism incorporating both processive and distributive phosphorylation steps (Figure 3A). Such a process is expected to require a stable enzyme-substrate complex. Indeed, competition and single turnover analyses indicate that the SRPK1-SRSF1 complex displays unusually high affinity with a Kd between 50–100 nM (48, 57). It is likely that this initial high affinity is diminished during subsequent phosphorylation steps, driving a shift from processive to distributive phosphorylation. Accordingly, it has been shown that SRPK1 inefficiently pulls down phosphorylated SRSF1 whereas the unphosphorylated SR protein is robustly pulled down (29, 59). While this mechanism has been established using SRSF1 that has a rather short RS domain, it remains to be seen whether processivity is a general feature of SRPKs and other SR proteins with much larger RS domains. It is interesting to note that expanding the number of Arg-Ser repeats in SRSF1 leads to enhanced processivity suggesting that other SR proteins could also be phosphorylated using this mechanism (60).
Although SRPK1 can processively phosphorylate several serines in SRSF1, it is not clear how this enzyme attaches phosphates in close succession to a highly charged substrate. DNA polymerase, a classic processive enzyme, adds nucleotide triphosphates in a rigid 5’→3’ direction and initiates strictly at a DNA primer (61). To address whether SRPK1 is likewise directional an engineered protease footprinting technique was employed (58). In these experiments, a lysine is placed in the center of the RS1 segment of SRSF1 and then several additional Lys-to-Arg mutations in RRM2 are inserted. When the resulting substrate is cleaved with LysC, two fragments easily identified on a gel can be attained that correspond to the N- and C-terminal halves of RS1. This method permits a fast and quantitative method for sorting phosphates placed on either the N- or C-terminal end of RS1. By monitoring the phosphorylation reaction in single turnover mode and converting the substrate into N- and C-terminal fragments with LysC at various reaction stages it can be shown that SRPK1 phosphorylates the RS1 segment in a C-to-N-terminal direction (Figure 3B). Furthermore, by altering the position of the cleavage site in the RS domain, the initiation region at the C-terminal end of the RS1 segment (initiation box) can also be identified. Interestingly, while SRPK1 prefers to start phosphorylation in the initiation box (Ser221–Ser225), mutations in this region do not halt catalysis indicating that the enzyme possesses the flexibility to move to other sites (58). This adaptability is likely to be an important feature of SRPK1 function as the RS domains in other SR proteins are larger and more diverse (Figure 1). In addition to rapid RS1 phosphorylation, SRPK1 is capable of phosphorylating about 3 serines in RS2 although about 100-fold slower than the serines in RS1 (60). This overwhelming specificity for RS1 over RS2 is due to SRPK1’s preference for long Arg-Ser repeats since adding such repeats greatly increases phosphorylation rates in RS2. These findings suggest that SRPK1 scans RS domains in search of long Arg-Ser stretches and is clearly capable of docking at additional sites based on local sequence factors. Overall, SRPK1 moves in a well-defined C-to-N direction along the RS domain of SRSF1 and possibly could use a similar mechanism for other SR proteins although it may be capable of recognizing different and possibly multiple initiation boxes.
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 (Figure 2A) or the core region of SRSF1 (RRM2-RS1) (Figure 4A). 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 (R191VKVDGPR) 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 (Figure 4A). This was surprising as this RS segment (N’-RS1; S201YGRSRSRSR), binds to a pocket far from the active site (Figure 4A), 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).
Since prior mapping studies showed that SRPK1 moves along the RS domain in a C-to-N direction (Figure 3B), 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 (Figure 4B). 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, 62). 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 (Figure 4B). 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 (Figure 5). 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 (Figure 4A). Moreover, the corresponding segment that would constitute the SRPK1 docking groove is shallow and dispersed with both acidic and basic charge patches (Figure 5). 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 (Figure 5). 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.
Recent structural and mechanistic studies on the splicing kinase SRPK1 has uncovered a novel phosphorylation mechanism in which a long section of the substrates’s RS domain is fed into the active site through a docking groove in the large lobe (Figure 4). This mechanism bears similarities to polymerase-type chain reactions where the enzyme binds in a defined region and then proceeds in a directional manner. SRPK1 starts in a narrow initiation box that is defined by the length of a greater binding channel that encompasses the docking groove and the active site, a total distance that can accommodate the RS1 segment of the SR protein SRSF1. After initiation, the driving force for the directional reaction is likely to involve a combination of repulsive interactions between the phosphoserines and the electronegative channel and attractive electrostatic interactions between the phosphoserines and an electropositive P+2 pocket. Whether discrete initiation and extension reactions as those found in SRSF1 are common within the SR protein family await further investigations. While a prototype for the family and the first to be investigated at a refined mechanistic level, SRSF1 possesses a relatively small RS domain compared to others in the SR protein family. It will be interesting to learn how the catalytic principles uncovered for SRSF1 apply to SR proteins with considerably larger RS domains with multiple, lengthy Arg-Ser repeats.
This work was supported by NIH grants to J.A. (GM67969) and G.G. (GM084277).
1Abbreviations: SRSF1, human alternative splicing factor; CLK, cdc2-like kinase; LysC, lysyl endoproteinase; MAPK, mitogen activated protein kinase; RS domain, domain rich in Arg-Ser repeats; RS1, N-terminal portion of SRSF1 RS domain; RS2, C-terminal portion of SRSF1 RS domain; pre-mRNA, precursor mRNA; RRM, RNA recognition motif; snRNP, small nuclear ribonucleoprotein; SR protein: splicing factor containing C-terminal Arg-Ser repeats; SRPK, SR-specific protein kinase.