Maria L. McGlone

Role of the Glycine Triad in the ATP-binding Site of cAMP-dependent Protein Kinase*

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed inEscherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on bothK m and k cat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5–8-fold reduction in K m  (ATP), an 8–12-fold increase in K m  (peptide), and a 3–5-fold drop in k cat. TheK m  (ATP) andK m  (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and thek cat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.

Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases.

SR proteins are essential splicing factors whose biological function is regulated through phosphorylation of their C-terminal RS domains. Prior studies have shown that cytoplasmic-nuclear translocalization of the SR protein SRSF1 is regulated by multisite phosphorylation of a long Arg-Ser repeat in the N-terminus of the RS domain while subnuclear localization is controlled by phosphorylation of a shorter Arg-Ser repeat along with several Ser-Pro dipeptides in the C-terminus of the RS domain. To better understand how these two kinases partition Arg-Ser versus Ser-Pro specificities, we monitored the phosphorylation of SRSF1 by CLK1 and SRPK1. Although SRPK1 initially binds at the center of the RS domain phosphorylating in an orderly, N-terminal direction, CLK1 makes widespread contacts in the RS domain and generates multiple enzyme-substrate complexes that induce a random addition mechanism. While SRPK1 rapidly phosphorylates N-terminal serines, SRPK1 and CLK1 display similar activities toward Arg-Ser repeats in the C-terminus, suggesting that these kinases may not separate function in a strict linear manner along the RS domain. CLK1 induces a unique gel shift in SRSF1 that is not the result of enhanced Arg-Ser phosphorylation but rather is the direct result of the phosphorylation of several Ser-Pro dipeptides. These prolines are important for binding and phosphorylation of the SR protein by CLK1 but not for the SRPK1-dependent reaction. The data establish a new view of SR protein regulation in which SRPK1 and CLK1 partition activities based on Ser-Pro versus Arg-Ser placement rather than on N- and C-terminal preferences along the RS domain.

Prokaryotic expression of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase

The prokaryotic expression of the Cα subunit of cAPK provides a system for the production of milligram quantities of wild-type and mutant protein with relative ease and little expense. The protocols described here have been optimized for the production of soluble and active protein kinase, with kinetic parameters similar to those found for the enzyme isolated from mammalian tissue. The Cα subunit expressed in E. coli is a phosphoprotein and appears to contain the sites of phosphorylation identified in the mammalian protein. The free N-terminal Gly indicates the lack of an N-terminal myristic acid; however, coexpression of the gene encoding the yeast N-myristoyltransferase allows for myristoylated Cα subunit to be produced in E. coli. Finally, the purified recombinant Cα subunit has been used in X-ray crystallographic studies which have yielded the crystals that will allow the first three-dimensional structure of a protein kinase to be solved. Therefore, although the employment of prokaryotic expression in the production of functional protein kinases has been limited, it is hoped that the methods presented here for the C subunit of cAPK will encourage the use of this simple and versatile expression system.

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

SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of protein kinases. The SRPKs (SR-specific protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine-proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1 (SR protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 (transformer 2β1) by 2-3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

Distal Loop Flexibility of a Regulatory Domain Modulates Dynamics and Activity of C-Terminal Src Kinase (Csk)

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Csk and SFKs share a modular design with the kinase domain downstream of the N-terminal SH2 and SH3 domains that regulate catalytic function and membrane localization. While the function of interfacial segments in these multidomain kinases are well-investigated, little is known about how surface sites and long-range, allosteric coupling control protein dynamics and catalytic function. The SH2 domain of Csk is an essential component for the down-regulation of all SFKs. A unique feature of the SH2 domain of Csk is the tight turn in place of the canonical CD loop in a surface site far removed from kinase domain interactions. In this study, we used a combination of experimental and computational methods to probe the importance of this difference by constructing a Csk variant with a longer SH2 CD loop to mimic the flexibility found in homologous kinase SH2 domains. Our results indicate that while the fold and function of the isolated domain and the full-length kinase are not affected by loop elongation, native protein dynamics that are essential for efficient catalysis are perturbed. We also identify key motifs and routes through which the distal SH2 site might influence catalysis at the active site. This study underscores the sensitivity of intramolecular signaling and catalysis to native protein dynamics that arise from modest changes in allosteric regions while providing a potential strategy to alter intrinsic activity and signaling modulation.

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 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 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 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.

Nucleotide Release Sequences in the Protein Kinase SRPK1 Accelerate Substrate Phosphorylation

Protein kinases are essential signaling enzymes that transfer phosphates from bound ATP to select amino acids in protein targets. For most kinases, the phosphoryl transfer step is highly efficient, while the rate-limiting step for substrate processing involves slow release of the product ADP. It is generally thought that structural factors intrinsic to the kinase domain and the nucleotide-binding pocket control this step and consequently the efficiency of protein phosphorylation for these cases. However, the kinase domains of protein kinases are commonly flanked by sequences that regulate catalytic function. To address whether such sequences could alter nucleotide exchange and, thus, regulate protein phosphorylation, the presence of activating residues external to the kinase domain was probed in the serine protein kinase SRPK1. Deletion analyses indicate that a small segment of a large spacer insert domain and a portion of an N-terminal extension function cooperatively to increase nucleotide exchange. The data point to a new mode of protein kinase regulation in which select sequences outside the kinase domain constitute a nucleotide release factor that likely interacts with the small lobe of the kinase domain and enhances protein substrate phosphorylation through increases in ADP dissociation rate.