C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle x-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology.The Src family of tyrosine kinases (SFK) 2 are modular signaling enzymes involved in the control of cellular growth and differentiation (1). The members of this family contain three important structural domains: a C-terminal tyrosine kinase domain, comprised of a small and large lobe, preceded by the noncatalytic regulatory SH2 and SH3 domains (2). SFKs also contain a unique region and an N-terminal myristic acid for membrane association. The activity of c-Src, the prototype for the SFKs, is up-regulated by phosphorylation of Tyr-416 (in the activation loop of the kinase domain) and dephosphorylation of Tyr-527 (in the C-terminal tail) (Fig. 1A) (3, 4). While phosphorylation of the activation loop is autocatalytic, phosphorylation of the C-terminal tail is inhibitory and requires Csk (5). Like the SFKs, Csk contains a C-terminal tyrosine kinase domain and N-terminal SH2 and SH3 domains (Fig. 1A) (6). Unlike SFKs, Csk lacks an inhibitory C-terminal tail, is not regulated through phosphorylation, and does not possess an N-terminal sequence for membrane localization. Instead, Csk is constitutively active and increased activity is coupled to its association with membrane adaptor proteins, for example Csk-binding protein (Cbp) (7). Cbp localizes Csk to the membr...

Background: Serine-arginine-rich (SR)-like proteins regulate the alternative splicing of human genes. Results: The serine-arginine protein kinase 1 (SRPK1) phosphorylates transformer 2␤1 (Tra2␤1) at numerous sites regulating RNA binding, splicing of the survival motor neuron 2 gene, and catalytic function of the kinase domain. Conclusion:The two RS domains interact and regulate Tra2␤1 activity in a phosphorylation-dependent manner. Significance: SRPK1 is a regulator of Tra2␤1.

The splicing function of SR proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine-serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR protein SRSF1 using a directional mechanism in which eleven serines flanked by arginines are sequentially feed from a docking groove in the large lobe of the kinase domain to the active site. While this process is expected to operate on lengthy arginine-serine repeats (≥8), many SR proteins contain smaller repeats of only 1–4 dipeptides raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2β that contains a C-terminal RS domain with short arginine-serine repeats [Tra2β(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2β(ΔN) where the rate-limiting step is dissociation of the protein substrate rather than nucleotide exchange as in the case of SRSF1. While a functioning docking groove is required for efficient SRSF1 phosphorylation, this conserved structural element is dispensable for Tra2β(ΔN) phosphorylation. These large shifts in mechanism are likely to account for the slower, net turnover rate of Tra2β(ΔN) compared to SRSF1 and may signal fundamental differences in phosphorylation among SR proteins with distinctive arginine-serine profiles. Overall, these data indicate that SRPK1 conforms to changes in RS domain architecture using a flexible kinetic mechanism and selective usage of a conserved docking groove.

Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.