Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2ß1.

Transformer 2ß1 (Tra2ß1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2ß1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2ß1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2ß3), RS1 blocks phosphorylation of these serines in the full-length Tra2ß1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2ß1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2ß1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.

Splicing kinase SRPK1 conforms to the landscape of its SR protein substrate.

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 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although 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 Tra2ß that contains a C-terminal RS domain with short arginine-serine repeats [Tra2ß(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2ß(ΔN) where the rate-limiting step is the dissociation of the protein substrate rather than nucleotide exchange as in the case of SRSF1. Although a functioning docking groove is required for efficient SRSF1 phosphorylation, this conserved structural element is dispensable for Tra2ß(ΔN) phosphorylation. These large shifts in mechanism are likely to account for the slower net turnover rate of Tra2ß(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.

Substrate-specific reorganization of the conformational ensemble of CSK implicates novel modes of kinase function.

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.

Proteins at work: a combined small angle X-RAY scattering and theoretical determination of the multiple structures involved on the protein kinase functional landscape.

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.