Kinetic mechanisms of the forward and reverse pp60c-src tyrosine kinase reactions

Tyrosine-specific protein kinases are known to utilize short synthetic tyrosine-containing peptides as substrates and, as a consequence, a number of inhibitory peptides have been prepared by replacing the tyrosine moiety in these peptides with a nonphosphorylatable phenylalanine residue. Unfortunately, the inhibitory efficacy of these phenylalanine-based peptides is often disappointing. These results demonstrate the need for nonphosphorylatable tyrosine surrogates that enhance enzyme affinity. As a consequence, we prepared nearly two dozen different phenethylamine derivatives, attached them to the C terminus of an active site-directed peptide (Glu-Glu-Leu-Leu), and examined their effectiveness as inhibitors of pp60 c-src . Three derivatives exhibit enhanced inhibitory activity (relative to phenethylamine), including para-substituted sulfonamide and guanidino analogs as well as a pentafluoro-containing species. The para-sulfonamide derivative was selected for further study and was found to function as a competitive inhibitor versus variable peptide substrate and as a noncompetitive inhibitor versus variable ATP. In short, the enhanced inhibitory activity of the sulfonamide derivative is not due to the association of this moiety with the ATP binding site. Furthermore, peptides containing the para-guanidino and pentafluoro derivatives of phenylalanine were prepared. These species also display enhanced inhibitory activity toward pp60 c-src relative to the corresponding phenylalaninebased peptide.More than 2 decades ago, the cAMP-dependent protein kinase was shown to phosphorylate short synthetic peptides containing sequences that correspond to phosphorylated sites in intact proteins (1-4). This key observation, which has been demonstrated innumerable times for other protein kinases in the intervening years, has had a profound impact on the ability to examine the chemistry and biochemistry of this important family of enzymes. Synthetic peptides are readily available and, unlike common protein kinase substrates such as histones, can be prepared containing only a single site of phosphorylation. As a consequence, peptidic substrates have proven to be an indispensable tool in the many detailed enzymological studies that have been described for protein kinases.In addition to their clear enzymological utility, peptide-based substrates immediately suggest the likelihood that structurally analogous inhibitors can be prepared. Indeed, the incorporation of an alanine residue, in place of the phosphorylatable serine moiety in a cAMP-dependent protein kinase-directed peptide, generates a species that serves as a competitive inhibitor versus both peptide and protein substrates (4). Feramisco and Krebs (5) subsequently introduced other residues in place of serine, including glycine, valine, aspartic acid, and asparagine. However, none of these substitutions provides an inhibitor as powerful as the alanine-based peptide. Interestingly, a naturally occurring inhibitor of the cAMP-dependent protein kinase also contains an alanine res...

Section: Resultssupporting

confidence: 84%

Nonphosphorylatable Tyrosine Surrogates

Niu

Lawrence

1997

“…The catalytic mechanism of the full-length ligand-stimulated activated PDGF ␤-receptor kinase is also found to be a sequential rather than ping-pong mechanism (37), and the epidermal growth factor receptor (38) and insulin receptor kinase (39) reactions are reported to occur by a sequential Bi Bi rapid equilibrium random mechanism. In addition, sequential mechanisms are reported for other tyrosine kinases, such as csk (40) and pp60 src (41), and for the serine/threonine kinases, cAMP-dependent protein kinase (42), and p38 kinase (43), the latter utilizing an ordered sequential mechanism in which the peptide substrate binds first (43).…”

Section: Identification Of the Sequence Of Enzymatically Active Kdrmentioning

confidence: 99%

Vascular Endothelial Growth Factor Receptor KDR Tyrosine Kinase Activity Is Increased by Autophosphorylation of Two Activation Loop Tyrosine Residues

Kendall

Rutledge

Mao

et al. 1999

116

Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on k cat , the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme.

“…A continuous spectrophotometric assay (22) was used to measure initial rates of phosphorylation and to determine kinetic constants for some peptides. Reactions were carried out in volumes of 100 l using 5 g of purified enzyme.…”

mentioning

confidence: 99%

Engineering the Substrate Specificity of the Abl Tyrosine Kinase

Till

Chan

Miller

1999

c-Abl is a non-receptor tyrosine kinase that is involved in a variety of signaling pathways. Activated forms of c-Abl are associated with some forms of human leukemia. Presently, no high resolution structure of the tyrosine kinase domain of Abl is available. We have developed a structural homology model of the catalytic domain of Abl based on the crystal structure of the insulin receptor tyrosine kinase. Using this model as a guide, we selected residues near the active site predicted to play a role in peptide/protein substrate recognition. We expressed and purified 15 mutant forms of Abl with single amino acid substitutions at these positions and tested their peptide substrate specificity. We report here the identification of seven residues involved in recognition of the P؊1, P؉1, and P؉3 positions of bound peptide substrate. Mutations in these residues cause distinct changes in substrate specificity. The results suggest features of Abl substrate recognition that may be relevant to related tyrosine kinases.The c-abl proto-oncogene encodes a multidomain non-receptor tyrosine kinase that is expressed ubiquitously in human tissues (reviewed in Refs. 1-4). Mutant forms of c-abl are found in patients with Philadelphia chromosome-positive chronic myelogenous leukemia and acute lymphocytic leukemia (1-4). In these diseases, a chromosomal translocation event produces a chimeric oncogene consisting of 5Ј-sequences of bcr fused to abl. The BCR-Abl fusion protein has elevated tyrosine kinase activity relative to c-Abl, and the tyrosine kinase activity of the BCR-Abl fusion protein is necessary for disease progression. Similarly, tyrosine kinase activity is necessary for transformation of fibroblasts or hematopoietic cells by BCR-Abl (5, 6).In addition to its tyrosine kinase catalytic domain, c-Abl has a short amino-terminal unique domain followed by SH3 and SH2 domains (1-4). This domain organization is found in many non-receptor tyrosine kinases. Abl also possesses a large carboxyl-terminal region that includes a DNA-binding domain, an F-actin-binding domain, a nuclear localization signal, and a proline-rich region implicated in mediating protein-protein interactions. Studies aimed at understanding the normal physiological role of c-Abl have shown the enzyme to be involved in signal transduction, cytoskeletal rearrangement, RNA polymerase II activation, DNA repair, and cell cycle control (1-4). c-Abl has been shown to physically associate with at least seven unique proteins, including p53 and the nuclear Rb protein (4). Mice with targeted disruptions in the c-abl gene have high neonatal mortality rates and are more susceptible to infection, suggesting a role for c-abl in B-lymphocyte development (7).At least eight in vivo substrates for Abl have been identified (4). The amino acid sequences surrounding the phosphorylation sites for two of these proteins, RNA polymerase II (8) and c-Crk (9), have been described. These sequences do not share a common primary sequence motif, suggesting that Abl may have a broad range of subs...