SRPK1 and Clk/Sty protein kinases show distinct substrate specificities for serine/arginine-rich splicing factors.
Serine/arginine-rich (SR) proteins are essential for pre-mRNA splicing, and modify the choice of splice site during alternative splicing in a process apparently regulated by protein phosphorylation. Two protein kinases have been cloned that can phosphorylate SR proteins in vitro: SRPK1 and Clk/Sty. Here, we show that these two kinases phosphorylate the same SR proteins in vitro, but that SRPK1 has the higher specific activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic peptide mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosphoserine-arginine residues or phosphoserine-proline residues. In vitro, the Clk/Sty kinase phosphorylated Ser-Arg, Ser-Lys, or Ser-Pro sites, whereas SRPK1 had a strong preference for Ser-Arg sites. These results suggest that SRPK1 and Clk/Sty may play different roles in regulating SR splicing factors, and suggest that Clk/Sty has a broader substrate specificity than SRPK1.
Colwill
K
K
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Feng
L L
LL
Yeakley
J M
JM
Gish
G D
GD
Cáceres
J F
JF
Pawson
T
T
Fu
X D
XD
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012694:Serine / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D013379:Substrate Specificity
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; 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