Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Abstract

Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low K m values) through stabilization of the enzymesubstrate complex, these contacts could reduce catalytic turnover (decrease k cat ) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a K m that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low K m through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.The phosphorylation of hydroxyl-containing amino acids (i.e. serine, threonine, and tyrosine) in eukaryotic proteins controls numerous physiological responses essential for cell function. The enzymes that catalyze this modification, the protein kinases, have been studied for their critical role in complex signaling cascades and for their potential as valuable chemotherapeutic targets in proliferative diseases (1, 2). Although much is known about the protein kinase family on the structural and functional levels (3-6), less is known about the interactions of these enzymes with their physiological protein substrates. Protein kinases recognize and phosphorylate short peptide segments (consensus sequences) of 4 -10 residues based on the known phosphorylation sequences of their targets (7-9). Although these short segments (consensus sequences) are minimal substrates, they do not provide all the binding energy offered by the full-length protein substrate. For instance, the catalytic efficiency of phosphorylating the protein substrate MAPKAP2 using p38 MAPK 1 is ϳ2 orders of magnitude higher than that for a 14-residue peptide based on the consensus sequence (10). In another example, Csk phosphorylates the protein substrate Lck Ͼ3 orders of magnitude more efficiently than a 9-residue peptide based on the phosphorylation site (11). Studies of this type support a model in which regions beyond the limited consensus sequence interact with the protein kinase scaffold and govern high efficiency protein phosphorylation.Although an x-ray structure for a protein kinase with a full-length protein substrate bound is currently unavailable, structural and functional studies are now beginning to reveal the nature of the interactin...

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

“…The linker region in the CDC6 peptide contains the KEN motif, which is a site for recognition by the anaphase promoting complex/CDH1 complex for ubiquitination of CDC6 prior to its degradation. Phosphorylation of CDC6 at Ser 54 and Ser 74 are important events that protect CDC6 from degradation, thus allowing CDC6 to play its role in the assembly of the prereplication complex during G 1 (50).…”

Kinetic Mechanism of Fully Activated S6K1 Protein Kinase

“…Adams and coworkers first noted and coined "catalytic clamping" in the reactions of (i) yeast Sky1p protein kinase with the RNA carrier protein Np13 (19) and (ii) the human C-terminal Src kinase with Src (20); we agree that natural selection of the fast chemistry coupled to weak peptide binding mechanism has been advantageous, because it facilitates high peptide specificity but with quick production and release of the phosphorylated downstream protein target (21). …”

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

“…Control experiments were performed to determine the background phosphorylation (i.e. phosphorylation of substrate in the presence of quench) using previously published protocols (22). The time-dependent concentration of phosphoproduct was then determined by considering the total cpm of the flow-though, the specific activity of the reaction mixture, and the background phosphorylation.…”

“…This poor binding affinity for both substrate and phosphoproduct is not without precedence. Recently, we showed that the protein kinase Sky1p has very poor affinity for its natural substrate, Npl3, and releases the phosphorylated product, phospho-Npl3, at a rate constant in excess of maximum turnover (22). Furthermore, it is likely that the active sites of protein kinases do not generally provide interactions that preferentially stabilize phosphorylated versus unphosphorylated ligands.…”

“…In a previous study we showed that Csk does not bind with high affinity to Src based on equilibrium sedimentation and single turnover experiments (21). Rather, the low K m for Src stems from a fast phosphoryl transfer step that clamps the substrate into the active site, a mechanism also shown to be operative in another kinase-protein substrate system (22). Given these findings, it is likely that residues out-side the active site of Csk play a large role in enhancing the phosphoryl transfer step, thereby promoting efficient recognition of poorly interacting substrates.…”