The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site.
BACKGROUND
The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells.


RESULTS
Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1.


CONCLUSIONS
The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.
de Graaf
Katrin
K
Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany. degraaf@gmx.de
Czajkowska
Hanna
H
Rottmann
Sabine
S
Packman
Len C
LC
Lilischkis
Richard
R
Lüscher
Bernhard
B
Becker
Walter
W
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D006801:Humans; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D034622:RNA Interference; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D000097603:Dyrk Kinases
D051357:Cyclin-Dependent Kinase 2; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; D000097603:Dyrk Kinases; C112491:SF3B1 protein, human