The Clk2 and Clk3 Dual-Specificity Protein Kinases Regulate the Intranuclear Distribution of SR Proteins and Influence Pre-mRNA Splicing Peter I. Duncan,* David F. Stojdl,* Ricardo M. Marius,* Karl H. Scheit,† and John C. Bell* ,1 *Ottawa Regional Cancer Centre, 501 Smyth Road, Ottawa, Ontario, Canada, K1H 8L6; and †Max-Planck-Institut fur biophysikalische Chemi Abteilung Molekulare Biologie, D-37077 Gottingen, Germany The three members of the Clk family of kinases (Clk1, 2, and 3) have been shown to undergo conserved alternative splicing to generate catalytically active (Clk) and inactive (Clk T ) isoforms. The prototype, mu- rine Clk1 (mClk1), is a nuclear dual-specificity kinase that can interact with, and cause the nuclear redistri- bution of, SR proteins. In this study, we demonstrate that the human Clk2 and Clk3 (hClk2 and 3) are also found within the nucleus and display dual-specificity kinase activity. The truncated isoforms, hClk2 T and hClk3 T , colocalize with SR proteins in nuclear speck- les. We also show catalytically active hClk2 and hClk3 cause the redistribution of SR proteins and can regu- late the alternative splicing of a model precursor mRNA substrate in vivo. © 1998 Academic Press INTRODUCTION Signal transduction in eukaryotes is mediated by networks of proteins, transmitting extracellular stim- uli across the plasma membrane, through the cyto- plasm, and finally into the nucleus. Signalling path- ways initiated by such diverse stimuli as mitogens, hormones, and stress make use of distinct cytoplasmic kinase cascades to transmit and amplify these signals. Several of these cascades have now been well char- acterized including the MAPK (mitogen-activated pro- tein kinase)/SAPK (stress-activated protein kinase) pathways which have been conserved from yeast to man. Traditionally protein kinases have been segre- gated into two groups based on their substrate hydroxy amino acid preference. Serine/threonine kinases (PSK) were the first to be identified and represent the largest group of protein kinases. Tyrosine kinases (PTK), dis- covered later, have revealed a complexity of signal transduction pathways previously unappreciated. More recently still, another group of protein kinases have emerged: the dual-specificity kinases (DSK). As the name implies, members of this group have been shown to phosphorylate substrates on serine and/or threonine residues as well as tyrosine residues. Many DSKs, however, have only demonstrated this ability in vitro or on nonphysiological substrates in vivo, largely because the physiological substrates for many of these kinases are still unknown. The relevance of dual-spec- ificity phosphorylation has, however, been demon- strated for several signalling pathways. For example, MEKs activate MAPKs by phosphorylation on tyrosine and threonine residues [1, 2]. Weel, a DSK, regulates cyclin-dependent kinase cdc2 by tyrosine and threo- nine phosphorylation and is itself regulated by the DSK Nim1 in Schizosaccharomyces pombe (reviewed in [3]). Work in our laboratory identified one of the first members of the DSK group of kinases named variously as Sty, Sty/Clk1, or Clk1. Murine Sty/Clk (herein called mClk1) is the proto- type of a family of protein kinases which have been conserved throughout evolution [4 –11]. We have shown previously that the mClk1 primary transcript is alternatively spliced to generate mRNAs encoding a full-length catalytically active dual-specificity kinase (mClk1) and a truncated, kinase-deficient polypeptide (mClk1 T ) [12]. The biological activities of the mClk1 gene product are largely unknown although possible roles in PC12 differ- entiation [13] and in the regulation of RNA splicing have been suggested [14]. In the latter case, we have shown that mClk1 kinase can interact with and phosphorylate members of the serine/arginine-rich (SR) family of splic- ing factors [14]. Furthermore, overexpression of mClk1 kinase in mammalian cells initiates the redistribution of nuclear SR proteins, suggesting that mClk1 directly reg- ulates the compartmentalization and activity of SR splic- ing factors [14]. Additional members of the Clk family have been cloned from human cDNA libraries [10, 11] and like mClk1 all three human Clk genes express alternatively spliced mRNAs predicted to encode full-length catalyt- ically active and truncated inactive polypeptides [11]. The identification of related but distinct Clk gene prod- 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (613) 247-3524. E-mail: jbell@med.uottawa.ca. EXPERIMENTAL CELL RESEARCH 241, 300 –308 (1998) ARTICLE NO. EX984083 300 0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.