No abstract

The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis‐specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2‐specific tyrosine kinase. This phosphorylation occurs in the N‐terminal region of the protein that lies outside the C‐terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1‐inhibitory kinase and a Wee1‐stimulatory phosphatase, which controls the phosphorylation of the N‐terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C‐terminal domain by the Nim1 protein and the other involving phosphorylation of its N‐terminal region by a different kinase.

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...

Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore, dsk1؉ gene determines the phenotype of prp2 ؉ overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the doubledeletion mutant. In conjunction with the resemblance of dsk1 ؉ and SRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms.Arginine/serine-rich (RS) domain-containing proteins are among the best-characterized non-snRNP proteins participating in pre-mRNA splicing (for reviews, see references 8 and 19). Members of the protein superfamily are involved in constitutive splicing and are specific modulators of alternative splicing (15,19). Mammalian serine/arginine-rich (SR) proteins are featured by one or more RNA recognition motifs at the NH 2 terminus and by an RS domain at the COOH terminus. Other RS domain-containing proteins are relatively less defined with respect to the arrangement of the two structural elements in a protein (8,11,19,35).SR proteins are heavily phosphorylated, predominantly in the RS domain (4,5,12,41). Several kinases have been reported to phosphorylate RS domain-containing splicing factors (5,12,30,39,50,53), including SR protein-specific kinase (SRPK) and Cdc28/Cdc2-like kinase (Clk/Sty). Based on studies in mammalian nuclear extracts, both phosphorylation and dephosphorylation of SR proteins are required for pre-mRNA splicing. Phosphorylation of SR proteins may promote spliceosome assembly by facilitating specific protein interactions while preventing SR proteins from binding randomly to RNA (54). Once a functional spliceosome has formed, dephosphorylation of SR proteins is necessary to allow the transesterification reaction to occur (3, 23). Recently, human type 2C Ser/Thr phosphatase PP2C␥ was reported to be...

The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine\arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid