Research ArticleInteracting factors and cellular localization of SR protein-specific kinase Dsk1
Introduction
Posttranslational modifications (PTMs) are used as means of amplifying the complexity of the proteome. Reversible phosphorylation mediated by protein kinases is one of the most common types of PTMs [1]. Serine/arginine protein-specific kinases (SRPKs) constitute a subfamily of serine-specific kinases, the prototype of which, human SRPK1, was isolated as the activity to phosphorylate serine/arginine-rich (SR) splicing factors [2]. Dsk1 (dis1-suppressing protein kinase1) is the ortholog of human SRPK1 in fission yeast Schizosaccharomyces pombe [3], [4], [5]. Based on bioinformatics data, SRPKs are conserved throughout evolution with members present in every eukaryotic organism studied (For a review, see [6]), indicating the importance of the kinase family for cellular life.
Alternative pre-mRNA splicing is a highly regulated pathway allowing the expression of different proteins from a single gene [7]. SR proteins are a class of evolutionarily conserved regulators pivotal for spliceosome assembly and the splice selection in pre-mRNA splicing (for a review, see [8]). They are characterized by one or two NH2-terminal RNA binding domains/RNA recognition motifs (RBDs/RRMs) and a COOH-terminal arginine–serine (RS) domain. SF2/ASF, newly named SRSF1 [9], acts as an oncoprotein in many human cancers [10], illustrating the significance of SR protein functions. Reversible phosphorylation by several families of protein kinases is critical for modulating the splicing machinery and SR proteins. The best studied kinases include dual-specificity tyrosine (Y)-phosphorylation-regulated kinases (DYRKs), Cdc2-like kinases or serine/threonine/tyrosine kinases (Clk/Sty), and SRPKs [3], [11], [12], [13]. SRPKs predominantly phosphorylate the serine residues in the RS domain of SR proteins [3]. The specific phosphorylation by SRPK-like kinases is validated by phosphoproteomics analyses [14]. The prerequisite of SR protein phosphorylation for pre-mRNA splicing has been well documented [15], [16], [17]. Interestingly, SRPK1 and Clk/Sty processively phosphorylate SRSF1 in a sequential order to allow the formation of the ternary complex of ESE (exonic splicing enhancer):SRSF1 (RBD):U1-70K [18], [19], [20], providing molecular evidence for the functional requirement of the de/phosphorylation cycle during pre-mRNA splicing.
The SRPK-mediated phosphorylation modulates the functions of SR proteins beyond the splicing step. To participate in gene expression events in the nucleus, phosphorylation of SR proteins by human SRPK1 is required for their interaction with receptor hMtr10/Transportin-SR2 (TRN-SR2) for nuclear import and subnuclear localization [21]. Similarly, phosphorylation by SRPKs is necessary for the nuclear import of SR proteins in Saccharomyces cerevisiae and S. pombe [22], [23]. In the nucleus, transcription and splicing are intimately coupled [24]. SRPK-mediated phosphorylation of SR proteins releases the splicing factors from nuclear speckles to transcription sites via interaction with the C-terminal domain of the RNA polymerase II large subunit, thereby spatially linking transcription to splicing in vivo, [25]. In connecting pre-mRNA splicing with downstream events, phosphorylation of the SR-like protein, Npl3, is necessary for its release of mRNA after nuclear export and its re-import into the nucleus in S. cerevisiae [26]. On the other hand, dephosphorylation of SR proteins enables them to serve as adapters for TAP/Mex67-dependent mRNA export [27], [28]. The role of SR proteins is not limited to RNA metabolism pathways within the nucleus. Shuttling SR proteins have been found to associate with ribosomes in the cytoplasm and stimulate translation in vivo and in vitro [29]. Interestingly, reversible phosphorylation differentially affects nuclear and cytoplasmic functions of SRSF1 [30]. Since more than 45RS domain-containing proteins have been found in the human genome [31], SRPKs may exert effects on diverse cellular functions other than pre-mRNA processing through phosphorylating many of these potential substrates [6]. Furthermore, studies on pathological conditions, such as viral infection and tumor development underscore the fundamental importance of SRPKs to cellular life [32], [33].
S. pombe members of SRPKs and Clk/Sty family are Dsk1 and Kic1/Lkh1 (kinase in the Clk/Sty family1/LAMMER kinase homolog1), respectively [5], [34], [35]. Although neither is essential, deletion in both dsk1+ and kic1+ results in a severe growth defect [5], [35]. The kinases are involved in both cell cycle and pre-mRNA splicing. Dsk1 promotes metaphase/anaphase transition whereas Kic1 influences cytokinesis [35], [36]. Similar to its mammalian counterparts, Dsk1 displays high specificity in phosphorylating the bona fide SR proteins in S. pombe, Srp1 and Srp2, as well as the orthologue of human large subunit of U2 auxiliary factor (U2AF), Prp2/Mis11 (spU2AFLG) [4], [5], [37], [38], [39], [40]. Consistent with its substrate specificity for SR proteins, dsk1-gene deletion impairs pre-mRNA splicing in fission yeast [35], [41].
S. pombe, as a genetically tractable system, provides a valuable organism to investigate the regulation of cell cycle and RNA metabolism. Both cell-division cycle (cdc) and pre-mRNA processing (prp) temperature-sensitive (ts) mutants have been isolated in S. pombe. Notably, a splicing defect is coupled with a cdc phenotype at restrictive temperature in many prp mutants in fission yeast [42], [43], [44]. Moreover, about 43% of the S. pombe genes contain introns and 25% of them harbor multiple introns [45]. Genetic, proteomic, and bioinformatic analyses suggest that the splicing machinery in S. pombe closely reflects the prototype of a mammalian spliceosome [46], [47]. However, not much is understood about what and how Dsk1 contributes to splicing. It is also unknown whether Dsk1 functions in additional steps of pre-mRNA processing other than splicing in fission yeast. Addressing these questions about Dsk1 in S. pombe will shed light on the conserved mechanisms that govern the action of the SRPK family in eukaryotes.
To investigate the biological functions of Dsk1, in this study we examined the factors interacting with Dsk1, which led us to the finding of the aberrant poly(A)+ RNA distribution in dsk1-null mutant cells. The identities of the Dsk1-associated factors provide not only further evidence for Dsk1 involvement in pre-mRNA processing and cell cycle, but also clues about how the kinase contributes to these cellular processes. We also determined the cellular localization and protein oscillation patterns of Dsk1 during the cell cycle to explore the mechanisms of the kinase regulation.
Section snippets
S. pombe strains and cell culture
The strains used in this study are listed in Table 1. Routine cell culture and standard genetic methods were carried out as described previously [48], [49], [50]. Strains were grown in yeast extract plus supplements (YES) or minimal medium (MM) with appropriate supplements of 225 mg/l each. Cells were incubated at 30 °C unless otherwise specified.
Construction of in vivo tagging strains of S. pombe
Strains expressing GFP (green fluorescent protein)-and TAP (tandem-affinity purification)-tagged versions of Dsk1 were constructed by PCR
Tagged Dsk1 proteins are functional, like wild-type
To identify the interacting factors, and to determine the expression and localization patterns of the kinase produced from its own promoter during the cell cycle, we TAP or GFP tagged dsk1+ gene at its genomic locus to encode the C-terminal fusion kinase. Prior to biochemical and cytological analyses using the tagged strains, we ensured the endogenous production of the fusion kinase in S. pombe cells. TCA extracts of the tagged strains, dsk1TAP and dsk1GFP, were prepared and processed for
Discussion
In this study we have isolated more than 10 in vivo interacting factors of Dsk1. Consistent with its reported functions, pre-mRNA processing and cell cycle factors are prominent among the Dsk1-associated factors (Table 2). We have demonstrated a novel kinase-substrate relationship between Dsk1 and one of its interacting factors, Rsd1, a RS domain-containing protein (Fig. 3). Interestingly, finding Pab1 in the list of Dsk1-interacting factors has led us to discover the deficiency of poly(A)+ RNA
Acknowledgments
We thank Dr. Kathleen Gould (Howard Hughes Medical Institute and Vanderbilt University Medical School, Nashville, TN, USA) for kindly providing cwf11 tagged and dim1 strains, as well as genomic tagging vectors, Dr. Adrian Krainer (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) for GST-Rsd1 protein, Dr. David Stead (COGEME, proteomics facility, Aberdeen University, Aberdeen, UK) and Dr. Alice Copsey (Genome Damage and Stability Center, University of Sussex, Falmer, UK) for
References (78)
The age of crosstalk: phosphorylation, ubiquitination, and beyond
Mol. Cell.
(2007)- et al.
Fission yeast mitotic regulator is an SR protein-specific kinase
J. Biol. Chem.
(1998) - et al.
Molecular cloning of a novel human cdc2/CDC28-like protein kinase
J. Biol. Chem.
(1991) - et al.
A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1
Mol. Cell.
(2008) - et al.
More than a splicing code: integrating the role of RNA, chromatin and non-coding RNA in alternative splicing regulation
Curr. Opin. Genet. Dev.
(2011) - et al.
TREX, SR proteins and export of mRNA
Curr. Opin. Cell Biol.
(2005) - et al.
Negative regulation of filamentous growth and flocculation by Lkh1, a fission yeast LAMMER kinase homolog
Biochem. Biophys. Res. Commun.
(2001) - et al.
The kic1 kinase of Schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell–cell separation
Exp. Cell. Res.
(2003) - et al.
LAMMER kinase Kic1 is involved in pre-mRNA processing
Exp. Cell. Res.
(2011) - et al.
Molecular genetic analysis of fission yeast Schizosaccharomyces pombe
Methods Enzymol.
(1991)