title: Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, USA.
authors.forename: Brandon E
authors.initials: BE
authors.lastname: Aubol
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Plocinik
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Hagopian
authors.forename: Chen-Ting
authors.initials: CT
authors.lastname: Ma
authors.forename: Maria L
authors.initials: ML
authors.lastname: McGlone
authors.forename: Reeti
authors.initials: R
authors.lastname: Bandyopadhyay
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Fu
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; C066780:Clk dual-specificity kinases; C087728:SRPK1 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases
keywords: BSA; CLK; Cdc2-like kinase; NIH; National Institutes of Health; RNA recognition motif; RRM; SR protein; SR-specific protein kinase; SRPK; bovine serum albumin; kinase; kinetics; phosphorylation; splicing