UC San Diego

Serine/arginines-rich (SR) proteins play important roles in constitutive and alternative pre-mRNA splicing, mRNA export and translation. The C-terminal RS domains of these proteins contain repeats of consecutive arginine-serine (RS) dipeptides, which are extensively phosphorylated and mediate protein-protein and protein-RNA interactions. One of the kinase families that phosphorylate SR proteins is the SR protein kinase family. SRPKs are unusual members of the kinase superfamily in that their kinase domains are bifurcated by spacer regions of 250 to 300 residues. Furthermore, they are constitutively active and do not require any post-translational modification or interaction with regulatory factors. In this study, I employed a combination of X-ray crystallography and biochemical techniques to investigate the molecular basis of constitutive activity, catalytic mechanism and substrate recognition of human SRPK1. An appropriate fragment of SRPK1 was designed for crystallization. The determination of the SRPK1 X-ray structure at 1.73 Å revealed that SRPK1 contains unique secondary structural elements that mimic in-trans activation mechanisms observed in different kinases. Moreover, the global network of interactions allows the activation loop to adopt a catalytically competent conformation without relying upon any direct stabilization mechanism. SRPK1 binds its SR protein substrate, ASF/SF2, stably and processively phosphorylates only half of the serines of the RS domain. The crystal structure of SRPK1 bound to a peptide and ADP has led to the identification of a docking groove in SRPK1 and a docking motif in ASF/SF2. Detailed biochemical studies of the docking interactions suggested mechanisms for the restriction in the number of phosphorylated sites and the mode of regulation of phosphorylation. Finally, the current model of the X-ray crystal structure of SRPK1 bound to AMP-PNP and a natural substrate, ASF/SF2, has revealed the structural basis for substrate recognition. Unexpectedly, ASF/SF2 engages SRPK1 at both small and large lobes upon binding. Detailed analysis of the complex structure is underway and will provide insights into the substrate specificity and the mechanism of processive phosphorylation