Splicing kinase SRPK1 conforms to the landscape of its SR protein substrate.
The splicing function of SR proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine-serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although this process is expected to operate on lengthy arginine-serine repeats (≥8), many SR proteins contain smaller repeats of only 1-4 dipeptides, raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2β that contains a C-terminal RS domain with short arginine-serine repeats [Tra2β(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2β(ΔN) where the rate-limiting step is the dissociation of the protein substrate rather than nucleotide exchange as in the case of SRSF1. Although a functioning docking groove is required for efficient SRSF1 phosphorylation, this conserved structural element is dispensable for Tra2β(ΔN) phosphorylation. These large shifts in mechanism are likely to account for the slower net turnover rate of Tra2β(ΔN) compared to SRSF1 and may signal fundamental differences in phosphorylation among SR proteins with distinctive arginine-serine profiles. Overall, these data indicate that SRPK1 conforms to changes in RS domain architecture using a flexible kinetic mechanism and selective usage of a conserved docking groove.
Department of Pharmacology, University of California-San Diego , La Jolla, California 92093-0636, United States.
Brandon E
BE
Aubol
Michael A
MA
Jamros
Maria L
ML
McGlone
Joseph A
JA
Adams
D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D013379:Substrate Specificity
D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C097083:TRA2B protein, human; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D001120:Arginine; C066780:Clk dual-specificity kinases; C087728:SRPK1 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases