The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site

BMC Biochemistry - Tập 7 Số 1 - 2006
Katrin de Graaf1, Hanna Czajkowska1, Sabine Rottmann2,3, Len C. Packman4, Richard Lilischkis2, Bernhard Lüscher2, Walter Becker1
1Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Aachen, Germany
2Division of Biochemistry and Molecular Biology, Medical Faculty of the RWTH Aachen University, Aachen, Germany
3Genomics Institute of the Novartis Research Foundation, San Diego, USA
4Department of Biochemistry, University of Cambridge, Cambridge, UK

Tóm tắt

Abstract 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. Conclusion 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.

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Tập 7 Số 1

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