N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

Abstract

SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing

“…Furthermore, we showed that the N-terminus also interacts with its kinase domain but is not a substrate, suggesting that it can bind different proteins in intra- and intermolecular fashions (Aubol et al, 2014; Colwill et al, 1996). To determine whether the CLK N-terminus also interacts with SRPK, we performed in vitro pull-down experiments using GST-tagged SRPK1 with either full-length His-tagged CLK1 or a form lacking its N-terminus (CLK1(ΔN)).…”

“…We showed previously that the CLK1 N-terminus binds with high affinity to the RS domain of its substrate SRSF1 (Aubol et al, 2014). Furthermore, we showed that the N-terminus also interacts with its kinase domain but is not a substrate, suggesting that it can bind different proteins in intra- and intermolecular fashions (Aubol et al, 2014; Colwill et al, 1996).…”

“…First, while phosphorylation is thought to enhance the activity of SR proteins in splice-site recognition, high levels of CLK paradoxically inhibit this function (Prasad et al, 1999). Second, as both SRPKs and CLKs are also present in the nucleus, this sets up a potential competitive rather than a cooperative relationship because we recently found that although SRPK has built-in sequences that assist in the release of SR proteins after phosphorylation (Aubol et al, 2014; Koizumi et al, 1999), CLK does not release fully phosphorylated SR proteins (Aubol et al, 2014). These findings also suggest that CLKs may require a release factor to generate functional SR proteins for splicing function.…”

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

“…One prior report identified EWS-FLI1 increasing TERT activity independently of DNA binding; however, no isoform analysis was performed (56). These four genes were recognized as putative contributors to oncogenesis based on the published literature (57)(58)(59)(60)(61)(62)(63)(64)(65). None of these genes were alternatively spliced by WT EWS reduction.…”

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1

“…Tra2␤1 constructs (1 M) were incubated with SRPK1 (200 nM) and 0.3 mM ATP in 25 mM Mops (pH 7.2) and 10 mM free Mg 2ϩ for 10 min or 2 h in a total volume of 100 l. Reaction quenching and desalting were performed according to a prior method (34). The binding of RNA to the Tra2 proteins was measured using a nitrocellulose membrane and a Bio-Dot apparatus (Bio-Rad) according to a previously published protocol (36).…”