Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors.
The U1 snRNP-specific 70K protein is one of the few snRNP proteins from higher eukaryotic cells that is phosphorylated in vivo (1,2). Immunoaffinity purified spliceosomal snRNPs (U1, U2, U5, and U4/U6) were tested for their ability to phosphorylate in vitro the U1-specific 70K protein. An snRNP-associated kinase activity which phosphorylates all U1-70K isoelectric variants was identified. Like its in vivo counterpart, this snRNP-associated enzyme phosphorylates solely serine residues of the 70K protein, preferentially utilizing ATP as a phosphodonor. Tryptic phosphopeptide analysis revealed an overlapping set of at least four radiolabeled peptides in the in vivo and in vitro phosphorylated protein, suggesting that the snRNP-associated serine kinase is responsible, at least in part, for the 70K protein phosphorylation observed in vivo. Chymotryptic digestion of in vitro, 32P-labeled 70K protein and in vitro phosphorylation studies with a synthetic peptide, indicated that the multiple 70K phosphorylation sites are limited to a highly charged, C-terminal domain of the protein. In vitro phosphorylation studies with the splicing factor ASF/SF2 and several deletion mutants demonstrated that, similar to the U1-70K protein, the snRNP-associated serine kinase phosphorylates the carboxy terminal RS-rich domain of ASF/SF2. A potential general role for this enzyme in the phosphorylation of splicing factors and its consequences for pre-mRNA splicing regulation are discussed.
Woppmann
A
A
Institut für Molekularbiologie und Tumorforschung, Marburg, Germany.
Will
C L
CL
Kornstädt
U
U
Zuo
P
P
Manley
J L
JL
Lührmann
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
D010750:Phosphoproteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012694:Serine; D000255:Adenosine Triphosphate; D001120:Arginine; D011494:Protein Kinases; D002918:Chymotrypsin