Nucleotide Release Sequences in the Protein Kinase SRPK1 Accelerate Substrate Phosphorylation

Abstract

Protein kinases are essential signaling enzymes that transfer phosphates from bound ATP to select amino acids in protein targets. For most kinases, the phosphoryl transfer step is highly efficient while the rate-limiting step for substrate processing involves slow release of the product ADP. It is generally thought that structural factors intrinsic to the kinase domain and the nucleotide-binding pocket control this step and consequently the efficiency of protein phosphorylation for these cases. However, the kinase domains of protein kinases are commonly flanked by sequences that regulate catalytic function. To address whether such sequences could alter nucleotide exchange and, thus, regulate protein phosphorylation, the presence of activating residues external to the kinase domain was probed in the serine protein kinase SRPK1. Deletion analyses indicate that a small segment of a large spacer insert domain and a portion of an N-terminal extension function cooperatively to increase nucleotide exchange. The data point to a new mode of protein kinase regulation in which select sequences outside the kinase domain constitute a nucleotide release factor that likely interacts with the small lobe of the kinase domain and enhances protein substrate phosphorylation through increases in ADP dissociation rate.

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

“…kdSRPK1, inactive SRPK1 containing K109M, was expressed and purified from a pET19b vector. Deletion constructs SRPK1(ΔN), SRPK1(ΔS) and SRPK1(S) (residues 222-492) were expressed and purified as previously described (Aubol et al, 2014; Aubol et al, 2012). CLK1 virus was transfected and expressed in Hi5 insect cells and CLK1 was purified with a Nickel resin and a published procedure (Keshwani et al, 2015b).…”

Regulation of splicing by SR proteins and SR protein-specific kinases

“…SRPK cellular partition is regulated, at least in part, by the unique spacer domain in each SRPK family member, as deletion of the spacer shifts the kinase to the nucleus (Ding et al, 2006; Ngo et al, 2008; Nolen et al, 2001). Initial evidence suggests that the spacer has little effect on the kinase activity, but a more recent study indicates that it can enhance the catalysis by increasing the rate-liming ADP release step in the kinase reaction (Aubol et al, 2012). In contrast, all Clk family members are constitutively localized in the nucleus and show extensive colocalization with SR proteins in nuclear speckles (Colwill et al, 1996b; Duncan et al, 1998).…”

Recruiting a Silent Partner for Activation of the Protein Kinase SRPK1

“…21 To evaluate whether the RS2 segment functions as a molecular trigger for activation through the NRF or possibly through other sequences in the SID, we investigated SR(RS2)-dependent changes in the phosphorylation of SR(ΔRS2) using several truncated forms of SRPK1 (Figure 4A). We initially showed that SR(RS2) did not increase the maximal velocity of SRPK1(ΔN) or SRPK1(ΔS) consistent with the idea that RS2 enhances catalysis through sequences in the N-terminus and SID (Figure 4D and Table 2).…”