The crystal structure of the porcine heart catalytic subunit of cAMP‐dependent protein kinase in a ternary complex with the MgATP analogue MnAMP‐PNP and a pseudosubstrate inhibitor peptide, PKI(5‐24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine‐rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three‐dimensional space. The structure supports an in‐line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.
The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1- Isoquinolinesulfonamide protein kinase inhibitors of the H series are among the most widely used inhibitors of Ser/Thr kinases and are indispensable in cellular and signal transduction research. Protein phosphorylation (central to cellular regulation) is mediated by the individual action of several hundred different protein kinases. Recent progress in the understanding of the highly complex cellular signaling networks depends largely on the availability and quality of specific agents that interfere with the pathways under investigation. The usefulness of a protein kinase inhibitor is defined by its ability to permeate cell membranes, its solubility, and its relative degree of specificity. Each year hundreds of publications describe work using isoquinolinesulfonamide inhibitors, despite the fact that the mode of specific inhibition by these compounds is not completely understood.(We have used the cAMP-dependent protein kinase (cAPK), 1 which binds many H series inhibitors, as a model system to investigate the factors governing inhibitor binding and specificity. Three of the most frequently used members of this class are H7, H8 (1), and H89 (2) (Fig. 1). They all act in competition to ATP but not to substrate. The highest selectivity and affinity is found with H89, which has a K i of 48 nM for cAPK, whereas H8 has a moderate affinity for both cGPK and cAPK, and H7 inhibits protein kinase C in addition to cGPK and cAPK (Table I). The inhibitors have enabled assignment of specific roles of these protein kinases in numerous regulatory interrelations (3-8). They are, however, not only valuable for the cell biologist. Pharmacologists are increasingly interested in the possibility of interfering with cellular signaling, especially in the area of anticancer drug discovery. There is evidence that isoquinolinesulfonyl inhibitors may be promising in this field (9 -14). Recently, crystal structures from several different protein kinases have been solved (for review see . The structures confirmed not only the high degree of structural conservation of the highly homologous catalytic kinase core (20) but simultaneously showed how subtle differences are responsible for individual properties of protein kinases. Most of the residues that are highly conserved or invariant in the protein kinase family line the active site, and many of them interact with ATP (21,22). Despite high similarity of K m values for ATP binding among protein kinases, the H inhibitors have remarkably different K i values. The crystal structures of kinase bound inhibitor molecules described here now show the mode of inhibitory action and the factors governing selectivity and will provide a firm basis for the design of new protein kinase inhibitors. EXPERIMENTAL PROCEDURESProtein Expression and Purification-Exp...
The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.
Loss of phosphoric acid is the most effective fragmentation reaction of pSer- and pThr-containing phosphopeptides of small size (up to 10-15 residues) in low-energy collision-induced dissociation. Therefore, tandem mass spectrometry with neutral loss scanning was evaluated for its utility to analyze protein phosphorylation using protein kinase A (PKA) catalytic subunit, which is phosphorylated at Thr197 and Ser338, as an example. Analysis of tryptic digests of phosphoproteins by tandem mass spectrometry with scanning for neutral loss of phosphoric acid resulted in spectra with poor signal-to-noise ratio, mainly because of the large size of the phosphopeptides formed (>2 kDa). This unfavorable size was caused by the distribution of tryptic cleavage sites in PKA and by interference of phosphorylation with tryptic cleavage. To generate a set of smaller peptide fragments, digestion was performed using the low-specificity protease elastase. Analysis of the total elastase digest with neutral loss scanning resulted in observation of a set of partially overlapping phosphopeptides with high abundance, providing a complete coverage of PKA phosphorylation sites. The peptide size generated by elastase (0.5-1.5 kDa) is ideally suited for this scan mode, which was found to provide the highest specificity for detection of singly charged phosphopeptides (neutral loss of 98). Identification of the PKA phosphorylation sites was performed by mass spectrometric sequencing of the elastase-derived phosphopeptides, which provided highly informative product ion spectra.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.