Regulating SR Protein Phosphorylation through Regions Outside the Kinase Domain of SRPK1

Abstract

SR proteins are essential splicing factors whose phosphorylation by the SRPK family of protein kinases regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, non-homologous spacer insert domain that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, hydrogen-deuterium exchange and steady-state kinetic experiments were performed on SRPK1. Unlike the kinase core, the large spacer insert domain lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and spacer insert domain equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to stable structure to facilitate high affinity SR protein interactions and phosphorylation rates.

N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

“…This is not a substrate-specific phenomenon but rather appears to reflect a general mechanism exploited with other known physiological substrates including SRSF2, SRSF5 and Tra2β1. Interestingly, although the related SR-directed kinase SRPK1 also contains an N-terminal extension, this segment is not an RS domain, does not affect the phosphorylation level or specificity in SRSF1 and has very little effect on binding affinity [29]. Thus, CLKs appear to possess unique structural features compared to other SR-specific kinases that are vital for attracting and hyper-phosphorylating SR proteins.…”

SRPK2 Phosphorylates Tau and Mediates the Cognitive Defects in Alzheimer's Disease

“…Removal of the unique spacer between the two kinase domains in SRPK has little effect on the kinase activity but triggers its nuclear translocation to induce aggregation of splicing factors in the nucleus (Ding et al, 2006). Interestingly, this region is an intrinsically disordered motif that shares little homology between SRPK2 and SRPK1 (Wang et al, 1998; Plocinik et al, 2011). Thus, it remains unclear why tau associates with both SRPKs on a region that is structurally different between the two isoforms (Fig 3B).…”

Nucleotide Release Sequences in the Protein Kinase SRPK1 Accelerate Substrate Phosphorylation

“…The SID is highly charged and predicted to lack regular secondary structure. Recent hydrogen-deuterium [H-D] exchange data indicate that most of the SID with the exception of two short segments near the SID/kinase boundaries is unstable and likely to be unfolded (30). Such a large, unstructured region in the SID could provide attachment points for the chaperones, thus, pinning the kinase in the cytoplasm.…”

“…A small segment of the N- and C-terminal ends of the SID, included in the expression construct to aid in crystallization, adopts helical conformations possibly explaining the observed stability of these regions in the H-D exchange experiments. While not included in the X-ray structure, the N-terminus appears to possess stable regions based on H-D exchange studies suggesting that it may adopt some secondary structure and/or pack onto the kinase domain (30). …”