Primary structural features of SR-like protein acinusS govern the phosphorylation mechanism by SRPK2

Abstract

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.

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors

“…We first determined whether PP1␣ is capable of dephosphorylating phosphorylated RS dipeptide by monitoring the phosphorylation of acinusS in the presence of PP1␣. AcinusS was chosen because it is an SRlike protein that contains only one SRPK2-mediated phosphorylation site (20). Our result showed that PP1␣ could dephosphorylate acinusS despite the absence of its physiological regulators (Fig.…”

“…3A). This is comparable with the binding affinity of SRSF1 with SRPK2, suggesting that SRPKs may bind both subclasses of SR proteins with high affinity (20). We next investigated how SRPK2 recognizes SRSF3 by performing pulldown assays with different truncation constructs of SRPK2, where the nonconserved N-terminal and spacer regions were removed individually or in combination.…”

“…1B), suggesting that SRPK2, like SRPK1, preferentially phosphorylates SRSF1 at its N-terminal RS domain. We have previously demonstrated that SRPK2 is able to phosphorylate SRSF1 in a processive manner (20). To examine whether the phosphorylation mechanism of SRPK2 is similar to that of SRPK1, we performed start-trap assays as described previously to compare the reactions carried out by the two kinases (39).…”

“…3B). Instead, a conserved docking groove, which has been shown to be important for the interaction of SRSF1 and the SR-like protein aci-nusS, was critical for SRSF3 binding (20). To further study how the different truncations and mutation might have affected the binding affinity of SRPKs and SRSF3, we determined the K d of the different constructs with SRSF3 using MST ( Fig.…”

“…In the past decade, significant progress has been made in understanding the phosphorylation mechanism of SRSF1 by SRPK1; however, few mechanistic insights into the interaction and phosphorylation of different SR proteins by other SRPKs have been achieved (19). Previously, we investigated the roles of the nonkinase regions and a conserved docking groove of SRPK2, which is highly related to SRPK1, in the recognition and phosphorylation of two different classes of substrate, namely SR protein SRSF1 and SR-like protein acinusS (20). Our findings suggest that the electronegative docking groove in SRPK2, but not its nonkinase regions, is responsible for substrate binding regardless of their identities.…”