Jennifer Shaffer

Processive phosphorylation of alternative splicing factor/splicing factor 2.

SR proteins, named for their multiple arginine/serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF/SF2. By using the start–trap strategy, we monitored the progress curve for ASF/SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF/SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme–substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF/SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains “locked” onto SRPK1 during RS domain modification.

Phosphorylation Driven Motions in the COOH-terminal Src Kinase, Csk, Revealed Through Enhanced Hydrogen–Deuterium Exchange and Mass Spectrometry (DXMS)

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.

Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding HYDROGEN-DEUTERIUM EXCHANGE STUDIES USING MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY DETECTION

Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999)Biochemistry 38, 12072–12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.