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

Abstract

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.

Introduction

RNA splicing is an essential posttranscriptional modification that increases proteome diversity and regulates cell development and growth. The transesterification reactions involved in the removal of introns from precursor mRNA occur at the spliceosome, a macromolecular assembly of several small nuclear ribonucleoproteins and over 100 polypeptides.¹ With regard to the latter group, the SR proteins (splicing factors containing arginine–serine repeats) are recognized as an essential family of splicing factors that play a role in spliceosome development and many additional post-splicing events.² The SR proteins are composed of one RNA recognition motif (RRM) or two RRMs that bind short stretches of precursor mRNA known as exonic splicing enhancers and help establish the 5′ and 3′ splice sites. SR proteins derive their name from a C-terminal domain that can vary in length from 50 to over 300 residues and is composed of numerous arginine–serine dipeptide repeats (RS domain). The RS domains are polyphosphorylated by two important protein kinase families—SR-specific protein kinases (SRPKs) and Cdc2-like kinases.³ The SRPKs provide a basal level of phosphorylation that permits interaction with transportin SR and localization of the SR proteins in nuclear speckles.4, 5 The Cdc2-like kinase family then further phosphorylates the RS domains, leading to a more widespread nuclear distribution of the SR proteins.6, 7 Beyond controlling the subcellular localization of the SR proteins and their proximity to the spliceosome, there is some evidence that RS domain phosphorylation could play a role in the selection of alternative splice sites. Splice variants of the caspase-9, Bcl-x, and PKCβ (protein kinase C beta) genes have been shown to correlate with the phosphorylation status of SR proteins.8, 9

Given the essential role of RS domain phosphorylation for cytoplasmic–nuclear partitioning, investigations into the mechanism of SRPK-dependent phosphorylation are important for understanding how SR proteins ultimately engage the spliceosome and impact alternative gene splicing. Using protease footprinting techniques, we previously showed that SRPK1 rapidly phosphorylates about 10–12 serines in the N-terminal portion of the RS domain (RS1) of the prototype SR protein SRSF1 (human alternative splicing factor or ASF/SF2).7, 10 By following the reaction progress, we showed that the phosphates are placed in a highly sequential manner, with SRPK1 moving in a preferred C-terminal-to-N-terminal direction along the RS domain.¹¹ Crystallographic studies indicate that this reaction is facilitated by a docking groove in the large lobe of the kinase domain that initially binds the N-terminal portion of RS1, allowing the C-terminal region access to the active site for initial phosphorylation.¹² For processing of the lengthy RS1 segment, the enzyme–substrate complex undergoes a shift whereby the RS sequence, originally in the docking groove, slides along a channel into the active site and the secondary structure from RRM2 (β4) unfolds and occupies the docking groove.¹² While an electronegatively charged docking groove and channel bind the long Arg-Ser repeats, a small electropositive pocket (P + 2 pocket) outside the active site stabilizes the growing phosphorylated RS domain as it is expelled from the active site.¹³ Although the RRMs in SRSF1 are not essential for initial binding affinity, they play an important role in later phosphorylation steps, maintaining a processive interplay between enzyme and substrate.¹³

Our understanding of the structure–function relationship governing SRPK1 regulation is currently incomplete since approximately 40% of the primary structure of the kinase is not present in any of the X-ray models.³ Specifically, the current structure of SRPK1 lacks most of the N-terminus (70 aa) and a large spacer insert domain (SID; approximately 250 aa) that bifurcates the small N-terminal lobe and the large C-terminal lobe of the kinase (Fig. 1a). We previously showed that the SID interacts with co-chaperones (e.g., Aha1) sequestering the kinase in the cytoplasm. Removal of the SID or detachment of the chaperone complex through osmotic stress causes SRPK1 to increase its presence in the nucleus, hyper-phosphorylate SR proteins, and change the alternative splicing pattern of the E1a splicing reporter.14, 15 At this time, whether the SID adopts any regular folded structure and how chaperones engage this large segment for cytoplasmic localization of the kinase are unclear. Less is known about the N-terminal extension, although recent studies show that it is essential for induced binding of one of the RRMs of SRSF1 (RRM2) upon engagement of the docking groove with an Arg-Ser peptide.¹⁶ While most protein kinases interact weakly with their substrates,¹⁷ SRPK1 forms a very stable, high-affinity complex with SRSF1 (K_d   ∼ 20–50 nM).15, 18 The formation of the tight enzyme–substrate complex is likely important for sequential phosphorylation, which is possibly mediated by the presence of the N-terminus.

To investigate the structure of the full-length enzyme, we studied the catalytic and conformational roles of the N-terminus and SID of SRPK1. While both segments equally augment phosphorylation turnover rates, the N-terminus specifically enhances SRSF1 binding. Using hydrogen–deuterium (H–D) exchange methods, we demonstrate that while the SID is largely flexible and lacks significant protection, the N-terminus possesses regions with intermediate-to-high protection consistent with predicted areas of secondary structure. Although both segments stabilize regions important for the phosphoryl transfer reaction (the glycine-rich and activation loops), the N-terminus stabilizes the docking groove in the large lobe of the kinase domain. The latter may explain why deletion of the N-terminus reduces SR protein binding affinity. In contrast, the H–D exchange data indicate that the presence of the SID destabilizes helix αC in the N-terminal lobe and stabilizes the N-terminus. These studies demonstrate that SRPK1 uses a combination of structured and intrinsically disordered regions for maintaining efficient SR protein binding and phosphorylation.