| SR proteins are critical players in regulating both constitutive and alternative pre-mRNA splicing, during which the phosphorylation by SR P&barbelow;rotein K&barbelow;inases (SRPKs) is required. Classical SR proteins contain one or two RNA Recognition Motifs (RRM) in their N terminus and a stretch of Arginine-Serine (RS) dipeptides in C terminus. Phosphorylation status of SR proteins regulates their subcellular localization as well as physiological function. In addition, complex structure of SRPK1 with ASF/SF2, a prototype of SR protein, shows that substrate binding requires non-canonical RRM2 domain and RS domain, which can be extensively phosphorylated. However, the canonical RRM1 domain is dispensable for such interaction.;Here we show that SRPK2 binds and phosphorylates SRp20, a classical single RRM domain-containing SR protein, at its RS domain. Similarly with ASF/SF2, the canonical RRM domain of SRp20 is dispensable for interacting with SRPK2. Meanwhile, we also find that a docking groove that is critical for substrate binding in SRPK1 is also conserved in SRPK2, since mutations on four key residues in docking groove impair its binding affinity with SRp20.;In addition, SRPK2 is now known to function more then regulating mRNA splicing, such as cell proliferation and cell apoptosis. Recently, SRPK2 is also shown to be a kinase phosphorylating Tau and mediate the cognitive defects in Alzheimer's disease (AD). Besides, an intrinsic character of SRPKs lies in that they are constitutively active, but the regulation mechanism is not well understood. Therefore, in order to obtain a better recognition about SRPK2, we applied yeast two-hybrid assay and eventually a new interaction partner called ZNF187 was identified.;ZNF187 is a transcriptional factor that binds with Serum Response Element (SRE). Our studies showed that it is a positive regulator of SRE activity. However, SRPK2 showed inhibiting effect on SRE activation with the treatment of EGF, which could induce its nucleus entry, when co-transfected, it reversed the stimulating effect on SRE by ZNF187 to basal level. Furthermore, knockdown of SRPK2 by RNAi would enhance ZNF187-stimuated SRE activation. Studies on truncation and mutations that have the similar effect with EGF-induced subcellular localization of SRPK2 also generated the same inhibiting phenomenon. In contrast, mutant that has distinct localization with SRPK2 wild type failed to exert suppression. Therefore, we conclude that with the treatment of EGF, SRPK2 moves into nucleus and negatively regulates ZNF187-stimulated transactivation of SRE. Surprisingly, when cells were treated with FBS, SRPK2 showed stimulation on SRE activity and it synergized ZNF187-stimulated effect on SRE, indicating that transcriptional regulation of SRPK2 on ZNF187-stimulated SRE activity is stimuli-dependent. |
