Fission Yeast Mitotic Regulator Dsk1 Is an SR Protein-specific Kinase

Abstract

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known to bind the in vivo phosphoepitope shared by SR proteins, indicating that the phosphorylation sites resided in the RS domain. Moreover, the fission yeast U2AF65 homolog, Prp2/Mis11 protein, was phosphorylated more efficiently by Dsk1 than by a human SR protein-specific kinase, SRPK1. Thus, these in vitro results suggest that Dsk1 is a fission yeast SR protein-specific kinase, and Prp2/Mis11 is likely an in vivo target for Dsk1. Together with previous genetic data, the studies support the notion that Dsk1 may play a role in coordinating pre-mRNA splicing and the cell division cycle.Many cellular processes are regulated coordinately with the progression of the cell division cycle (e.g. Refs. 1 and 2), and RNA splicing is likely to be included in this regulation. For example, RNA splicing may be down-regulated in the cell when transcription is repressed during mitosis and up-regulated when RNA precursors are produced actively during cell growth (3). Indeed, evidence indicates that the pre-mRNA splicing apparatus is disassembled during mitosis and has to be reassembled when cells exit mitosis (4). Because protein phosphorylation is involved in the regulation of virtually every aspect of cellular processes (5, 6), we decided to look for protein kinases in fission yeast which are involved in regulating splicing during the cell cycle. The Dsk1 kinase in Schizosaccharomyces pombe is an excellent candidate because it has a role in cell cycle progression (7), and it is homologous to a human SR protein kinase (8). Compared with budding yeast, fission yeast (S. pombe) has the advantage of being more similar to higher eukaryotes especially with regard to the appearance of introns in protein-encoding genes (9 -11).The dsk1 ϩ gene was originally identified as a multicopy suppressor of cold-sensitive dis1 mutants (7). dis1 mutants are defective in sister chromatid separation at the restrictive temperature, and mitosis never reaches completion in these mutants (12). The Dis1 protein is associated with microtubules and the spindle pole body and probably is phosphorylated by Cdc2 kinase (13). dsk1 ϩ gene is not essential for viability, probably because of a redundancy in its function in fission yeast, but overexpression of dsk1 ϩ results in a delay at the G 2 /M phase transition (7). dsk1 ϩ encodes a 61-kDa protein kinase, but its in vivo substrate ha...

Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint

“…For this purpose, a large number of crosses were done between mis3-224 and various mutants implicated in DNA replication, RNA metabolism, cell cycle control and other cellular aspects (see Experimental procedures). We found that mis3-224 was synthetic lethal with two mutations involved in RNA metabolism, namely, dsk1 null (Takeuchi & Yanagida 1993;Tang et al 1998) and cold-sensitive (cs) dis3-54 mutant (Kinoshita et al 1991). Tetrad dissection indicated that the mis3-224 dsk1 null double mutant failed to grow at any temperature.…”

Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation

“…Srp2 contains two short SR\RS repeat elements (SR1 and SR2) flanking an arginine-rich region (Figure 1), which is necessary for targeting the protein to the nucleus. The srp2 + gene is essential for cell viability, and its second RBD and the two SR elements are required for its function in i o. SR proteinspecific kinases are conserved in fission yeast [26,29]. Dis1-suppression kinase (Dsk1) protein, originally described as a mitotic regulator [30], displays high activity in phosphorylating S. pombe Srp1 and Srp2 proteins in itro and is a functional homologue of human SRPK1 based on complementation assay in i o.…”

“…Plasmids pET-28bGST, pET-28adsk1 + , pET-28bGST-prp2 + , pET-28asrp1 + , pET-28bGST-srp1 + , pET-28bsrp2 + and pET28bGST-srp2 + have been described previously [26,29]. Recombinant proteins, GST-Srp1, Srp1, GST-Srp2, Srp2, GST-pre-RNA processing 2 (Prp2) and Dsk1, were expressed in the Escherichia coli strain BL21(DE3)pLysS, and the relative amounts of recombinant proteins in lysates were estimated as described previously [26,29].…”

“…Recombinant proteins, GST-Srp1, Srp1, GST-Srp2, Srp2, GST-pre-RNA processing 2 (Prp2) and Dsk1, were expressed in the Escherichia coli strain BL21(DE3)pLysS, and the relative amounts of recombinant proteins in lysates were estimated as described previously [26,29].…”

“…The samples were processed for SDS\PAGE and Western-blot analysis. Immunoblotting in most experiments was performed as described previously [26,29]. When using 3C5 monoclonal antibody, 25 mM NaF and 1 mM NaVO $ were present as phosphatase inhibitors to prevent dephosphorylation.…”