Chen-Ting Ma

A Sliding Docking Interaction Is Essential for Sequential and Processive Phosphorylation of an SR Protein by SRPK1

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.

Mass spectrometric and kinetic analysis of ASF/SF2 phosphorylation by SRPK1 and Clk/Sty.

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.

Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1.

The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-to-N-terminal) and processive mechanism--a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single-turnover and multiturnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible protein-protein interactions to facilitate both early and late phases of the processive phosphorylation of SR proteins.

Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases.

SR proteins are essential splicing factors whose biological function is regulated through phosphorylation of their C-terminal RS domains. Prior studies have shown that cytoplasmic-nuclear translocalization of the SR protein SRSF1 is regulated by multisite phosphorylation of a long Arg-Ser repeat in the N-terminus of the RS domain while subnuclear localization is controlled by phosphorylation of a shorter Arg-Ser repeat along with several Ser-Pro dipeptides in the C-terminus of the RS domain. To better understand how these two kinases partition Arg-Ser versus Ser-Pro specificities, we monitored the phosphorylation of SRSF1 by CLK1 and SRPK1. Although SRPK1 initially binds at the center of the RS domain phosphorylating in an orderly, N-terminal direction, CLK1 makes widespread contacts in the RS domain and generates multiple enzyme-substrate complexes that induce a random addition mechanism. While SRPK1 rapidly phosphorylates N-terminal serines, SRPK1 and CLK1 display similar activities toward Arg-Ser repeats in the C-terminus, suggesting that these kinases may not separate function in a strict linear manner along the RS domain. CLK1 induces a unique gel shift in SRSF1 that is not the result of enhanced Arg-Ser phosphorylation but rather is the direct result of the phosphorylation of several Ser-Pro dipeptides. These prolines are important for binding and phosphorylation of the SR protein by CLK1 but not for the SRPK1-dependent reaction. The data establish a new view of SR protein regulation in which SRPK1 and CLK1 partition activities based on Ser-Pro versus Arg-Ser placement rather than on N- and C-terminal preferences along the RS domain.

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs protects against dephosphorylation, suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.

Regiospecific phosphorylation control of the SR protein ASF/SF2 by SRPK1.

SR proteins (splicing factors containing arginine-serine repeats) are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 (human alternative splicing factor) contains two N-terminal RNA recognition motifs (RRMs) (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine-rich) domain that can be phosphorylated at numerous serines by the protein kinase SR-specific protein kinase (SRPK) 1. The RS domain [C-terminal domain that is rich in arginine-serine repeats (residues 198-248)] is further divided into N-terminal [RS1: N-terminal portion of the RS domain (residues 198-227)] and C-terminal [RS2: C-terminal portion of the RS domain (residues 228-248)] segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regiospecific and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, we performed engineered footprinting and single-turnover experiments to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.

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

ASF/SF2, a member of the serine-arginine (SR) protein family, has two RRM domains (RRM1 and RRM2) and a C-terminal domain rich in RS dipeptides. SR protein kinase 1 (SRPK1) phosphorylates approximately 12 of these serines using a semiprocessive mechanism. The X-ray structure of the ASF/SF2-SRPK1 complex revealed several features of the complex that raised intriguing questions about how the substrate is phosphorylated by the kinase. The part of the RS domain destined to be phosphorylated at later stages of the reaction docks to a kinase groove distal to the active site while the neighboring RRM2 binds near the active site [Ngo, J. C., et al. (2008) Mol. Cell 29, 563-576]. In this study, we investigate the interplay between the RS domain and RRM2 for stable association and phosphorylation of ASF/SF2. Despite several contacts in the enzyme-substrate complex, free RRM2 does not bind efficiently to SRPK1 unless the docking groove is occupied by the RS domain. This domain cross-talk enhances the processive phosphorylation of the RS domain. The RRM-SRPK1 contact residues control the folding of a critical beta-strand in RRM2. Unfolding of this structural element may force the N-terminal serines of the RS domain into the active site for sequential phosphorylation. Thus, ASF/SF2 represents a new class of substrates that use unique primary sequence to induce allosteric binding, processive phosphorylation, and product release.

Regulating SR Protein Phosphorylation through Regions Outside the Kinase Domain of SRPK1

SR proteins (splicing factors containing arginine-serine repeats) are essential splicing factors whose phosphorylation by the SR-specific protein kinase (SRPK) family regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, nonhomologous spacer insert domain (SID) that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, we performed hydrogen-deuterium exchange and steady-state kinetic experiments on SRPK1. Unlike the kinase core, the large SID lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts a stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and SID equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to a stable structure to facilitate high-affinity SR protein interactions and phosphorylation rates.

Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif

Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.