A conserved sequence motif bridges two protein kinases for enhanced phosphorylation and nuclear function of a splicing factor

Abstract

The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.

Cdc-Like Kinases (CLKs): Biology, Chemical Probes, and Therapeutic Potential

“…Not only nuclear import but also nuclear function has been described most thoroughly for the CLK1 isoform, namely its regulation mechanism of the SR protein SRSF1 [ 55 , 68 , 85 , 86 ]. In this process, CLK1 and SRPK1 work co-operatively as a complex [ 55 ].…”

“…The complex containing CLK1 in active form first recruits hypo-phosphorylated SRSF1 from nuclear speckles (also termed interchromatin granule clusters), which act as a pool of SR proteins in the nucleus, whereby a ternary complex CLK1-SRPK1-SRSF1 is formed ( Figure 6 ). The ternary complex executes full phosphorylation of SRSF1 and subsequently releases it ( Figure 6 ) [ 55 , 84 , 85 ]. Alternatively, CLK1 itself can form a complex with SRSF1 and catalyze full phosphorylation first.…”

“…Alternatively, CLK1 itself can form a complex with SRSF1 and catalyze full phosphorylation first. Subsequently, SRPK1 can engage to create the ternary complex CLK1-SRPK1-SRSF1 [ 85 ]. The interaction between CLK1 N-terminus and SRPK1 kinase domain holds the complex CLK1-SRPK1 together but also facilitates the release of hyper-phosphorylated SRSF1 from the ternary complex, which is a prerequisite for the assembly of spliceosome [ 55 ].…”

“…These recent findings suggest it may be desirable to evaluate the effect of CLK inhibitors on splicing in the context of the SRPK1-CLK1 complex, rather than using the isolated CLK1 kinase [ 85 ].…”

“… Schematic representation of the phosphorylation mechanism involving CLK1 [ 39 , 49 , 55 , 68 , 82 , 83 , 84 , 85 ]. …”