Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones

Pre-mRNA processing consists of a highly regulated cascade of events that are critical for gene expression in higher eukaryotic cells. SR proteins are an important class of splicing factors or regulators because of their involvement in both constitutive and regulated splicing (Lin and Fu 2007). More recent studies reveal an even broader role of SR proteins in gene expression from transcriptional elongation to protein synthesis (Sanford et al. 2004; Lin et al. 2008; Michlewski et al. 2008). These fundamentally important functions render SR proteins essential for viability of proliferating cells (Wang et al. 1996; Lin et al. 2005; Xiao et al. 2007). SR proteins are regulated in development, which are translocated from the cytoplasm to the nucleus during zygotic activation of gene expression (Sanford and Bruzik 1999). Individual SR proteins are also autoregulated, suggesting critical importance in maintaining their homeostasis in somatic cells (Lareau et al. 2007). Indeed, some SR proteins exhibit altered expression in human cancers (Ghigna et al. 1998; Stickeler et al. 1999; Pind and Watson 2003), and a recent study demonstrated that overexpression of a specific SR protein, SF2/ASF, is sufficient to trigger cellular transformation (Karni et al. 2007). These observations indicate that SR proteins must be tightly regulated and alteration of such regulation can have a profound impact on the physiological state of the cell.

Typical SR proteins contain one or two RNA recognition motifs (RRMs) followed by a signature serine/arginine-rich sequence known as the RS domain at the C terminus. The RS domains are extensively phosphorylated. While SR protein phosphorylation is essential for nuclear import of SR proteins as well as for their functions in mediating spliceosome assembly (Roscigno and Garcia-Blanco 1995; Xiao and Manley 1997; Yeakley et al. 1999; Yun and Fu 2000; Lai et al. 2001), partial dephosphorylation of SR proteins is also critical for progression of the assembled spliceosome to catalysis (Mermoud et al. 1994; Cao et al. 1997) and for a series of post-splicing events from the interaction with the mRNA transport machinery to SR protein-mediated translational control (Huang et al. 2004; Lai and Tarn 2004; Lin et al. 2005; Sanford et al. 2005). Consequently, it may not be surprising that experimental induction of both SR protein hypo- and hyperphosphorylation inhibits splicing (Prasad et al. 1999). While these observations clearly suggest that SR protein phosphorylation is under precise control, little is known about the mechanism of such regulation in the cell.

Multiple protein kinases have been implicated in SR protein phosphorylation. Among the growing list of SR protein kinases, the SRPK and Clk/Sty families are best characterized. Mammalian cells express two SRPKs and four members of the Clk/Sty family of kinases. Interestingly, SRPK1 and SRPK2 were shown recently to differentially associate with U1 and tri-snRNP particles, respectively, indicating that these kinases have both overlapping and unique functions in mammalian cells (Mathew et al. 2008). Enzymatic analysis reveals that SRPKs use a highly processive mechanism to phosphorylate a defined region in the RS domain in each SR protein (Aubol et al. 2003; Ngo et al. 2008), and Clk/Sty can further phosphorylate the remaining sites in the RS domain (Ngo et al. 2005; Velazquez-Dones et al. 2005), suggesting the possibility that these kinases may catalyze a cooperative phosphorylation relay to modulate SR protein function at different biochemical steps and/or in various cellular locations (Ngo et al. 2005; Hagopian et al. 2008). This idea is consistent with their cellular distributions: While members of the Clk/Sty family of kinases are predominately localized in the nucleus (Colwill et al. 1996; Nayler et al. 1998), the SRPK family of kinases are detected in both the cytoplasm and the nucleus (Wang et al. 1998; Ding et al. 2006).

Despite its importance, little is known about how SR protein phosphorylation might be regulated in the cell and how a specific signal might be transduced to control RNA processing via modulation of SR protein phosphorylation. Recent discoveries from the Manley laboratory shed a critical light on these important questions by revealing dramatic dephosphorylation of a specific SR protein (SRp38) in response to heat shock (Shin et al. 2004; Shi and Manley 2007). The regulation is achieved by an increased exposure of the protein to the activated protein phosphatase PP1 in combination with limited accessibility of the protein to SR protein kinases under heat-shock conditions, underscoring the importance of a balanced action between SR protein kinases and phosphatases in controlling the phosphorylation state of SR proteins.

In the present study, we focused on understanding how SR protein kinases are regulated. Our previous work showed that SRPK1 is a constitutively active kinase (Nolen et al. 2001; Lukasiewicz et al. 2007) and that an accessory domain (a spacer sequence that splits conserved kinase domains into two blocks) is involved in partitioning of the kinase between the cytoplasm and nucleus, suggesting that the cellular distribution of the kinase, rather than activity, is subject to regulation (Ding et al. 2006). We now show that SRPK1 directly interacts with two specific cochaperones for major heat-shock proteins and that the ATPase activity of Hsp90 plays a critical role in regulating the cellular distribution of the kinase in the cell. We further demonstrate that osmotic stress can induce SRPK1 nuclear translocation by modulating the dynamic interaction of SRPK1 with the chaperone complexes, thereby inducing differential SR protein phosphorylation and alternative splice site selection. These findings reveal a novel strategy by which to regulate SR protein phosphorylation and alternative splicing in higher eukaryotic cells.