The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing

Abstract

The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper-and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under-and overphosphorylation of SR proteins can modulate splicing.SR proteins constitute a family of essential pre-mRNA splicing factors that are highly conserved throughout metazoa (reviewed in references 14, 26, and 42). They have been shown to function in vitro as essential factors required for all splices, as concentration-dependent regulators of alternative splice site selection, and as activators-inhibitors of splicing as components of complexes assembled on splicing enhancer-silencer elements in the pre-mRNA. Although most studies have employed in vitro assays, individual SR proteins have been shown to be capable of modulating alternative splice site selection when overexpressed in transient transfection assays (2,36,45). One SR protein, B52/SRp55, is required for proper development in Drosophila (33, 34), and another, ASF/SF2, is essential for the viability of a cultured cell line (46), and in vivo depletion of the protein can affect alternative splicing (47). SR proteins contain one or two N-terminal RNP-type RNA binding domains (RBDs) and C-terminal regions enriched in repeated arginine-serine dipeptides (RS domains). The RBDs function in binding the pre-mRNA (1, 39, 51), while RS domains have been implicated in protein-protein interactions (24,48). SR proteins are extensively phosphorylated (35).Reversible protein phosphorylation plays an important role in pre-mRNA splicing, and SR proteins are likely to be key targets. Initial in vitro studies, employing phosphatases and their inhibitors, provided evidence that protein phosphorylation, possibly of SR proteins, is required for spliceosome assembly (28); that dephosphorylation is necessary at a subsequent step (27, 40); and that phosphatase treatment of nuclear extract can influence alternative splice site selection (5). More recent experiments have begun to suggest possible molecular mechanisms for these effects. Phosphorylation of serines within RS domains can influence RNA binding by SR...

Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

“…Another study suggests that splicing factors regulate apoptosis, perhaps by regulating caspase activity (Jiang et al, 1998). It is speculated (Prasad et al, 1999) that the phosphorylation of splicing factors at unique residues facilitates the inactivation of these proteins either by rendering them inactive or by irreversibly complexing them with other proteins of the splicing machinery and thus titrating them out of the splicing reaction.…”

“…Further dephosphorylation reduces their activity. Similarly, in mammalian cells, both hyper-and hypophosphorylation inhibit the splicing activity of SR proteins (Prasad et al, 1999) and disassemble nuclear speckles (Misteli and Spector, 1996), thus suggesting that intermediate levels of phosphorylation are needed for the functional and structural integrity of these proteins. An alternative interpretation of the differential phosphorylation of PSF, during mitosis and particularly in apoptosis, would be that this protein has functions that are unrelated to splicing.…”

“…SRPK-1/2, Clk-1/2/3/4, cdc2-kinase, cyclin Ecdk2, topoisomerase I, U1 70-kDa associated kinase, cGMPdependent kinase, and lamin B-receptor kinase were all shown to phosphorylate SR proteins and other splicing factors in vitro (Woppmann et al, 1993;Gui et al, 1994;Colwill et al, 1996;Nikolakaki et al, 1996;Rossi et al, 1996;Nayler et al, 1997;Duncan et al, 1998;Kuroyanagi et al, 1998;Okamoto et al, 1998;Seghezzi et al, 1998;Wang et al, 1998;Koizumi et al, 1999;Wang et al, 1999). In addition, some of these kinases were shown to affect the splicing activity of such factors (Mermoud et al, 1994;Cao et al, 1997;Xiao and Manley, 1998;Prasad et al, 1999). How all of these enzymes work in concert in vivo, if at all, is unknown.…”

“…Another study suggests that splicing factors regulate apoptosis, perhaps by regulating caspase activity (Jiang et al, 1998). It is speculated (Prasad et al, 1999) that the phosphorylation of splicing factors at unique residues facilitates the inactivation of these proteins either by rendering them inactive or by irreversibly complexing them with other proteins of the splicing machinery and thus titrating them out of the splicing reaction.Our data substantiate the concept of the dynamic nucleus that reorganizes its components through a mechanism of phosphorylation, depending on the functional stage of the cell. Our data together with the data accumulated on SR proteins, point to the involvement of several different kinases and phosphatases in the phosphorylation status of nuclear factors.…”

Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages

“…In a yeast two-hybrid screen, Colwill and co-workers identified several RNA-binding proteins, all members of the SR family of splicing factors [17]. It was also shown that clk/STY phosphorylates SR proteins and that the phosphorylation status influences their subnuclear localization [17,18]. Clk/STY has been shown to interact and phosphorylate other protein kinases [19] as well as protein phosphatases [20].…”