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.
Velazquez-Dones
Adolfo
A
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093, USA.
Hagopian
Jonathan C
JC
Ma
Chen-Ting
CT
Zhong
Xiang-Yang
XY
Zhou
Huilin
H
Ghosh
Gourisankar
G
Fu
Xiang-Dong
XD
Adams
Joseph A
JA
D000255:Adenosine Triphosphate / Q000737:chemistry; D017398:Alternative Splicing; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry; D008956:Models, Chemical; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D000068103:Serine-Arginine Splicing Factors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors
D009687:Nuclear Proteins; D010710:Phosphates; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000068103:Serine-Arginine Splicing Factors; D012313:RNA; D000255:Adenosine Triphosphate; C066780:Clk dual-specificity kinases; C087728:SRPK1 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase