Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/ serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRMcontaining SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPKspecific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro. Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM-and two RRMcontaining SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

SRPKs (serine/arginine protein kinases) are highly specific kinases that recognize and phosphorylate RS (Arg-Ser) dipeptide repeats. It has been shown previously that SRPK1 phosphorylates the RS domain of SRSF1 (serine/arginine splicing factor 1) at multiple sites using a directional and processive mechanism. Such ability to processively phosphorylate substrates is proposed to be an inherent characteristic of SRPKs. SRPK2 is highly related to SRPK1 in sequence and in vitro properties, yet it has been shown to have distinct substrate specificity and physiological function in vivo. To study the molecular basis for substrate specificity of SRPK2, we investigated the roles of the non-kinase regions and a conserved docking groove of SRPK2 in the recognition and phosphorylation of different substrates: SRSF1 and acinusS. Our results reveal that a conserved electronegative docking groove in SRPK2, but not its non-kinase regions, is responsible for substrate binding regardless of their identities. Although SRPK2 phosphorylates SRSF1 in a processive manner as predicted, an electronegative region on acinusS restricts SRPK2 phosphorylation to a single specific site despite the presence of multiple RS dipeptides. These results suggest that primary structural elements on the substrates serve as key regulatory roles in determining the phosphorylation mechanism of SRPK2.