Comparative Analysis of Mutant Tyrosine Kinase Chemical Rescue

Abstract

Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically-deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole, however the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue is somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs. pH studies with these imidazole analogs suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells.Protein tyrosine kinases (PTKs 1 ) are key enzymes in cell signaling and play roles in a wide range of diseases including cancer (1,2). Several PTKs, including Abl, are targeted clinically with therapeutic agents, and others are the subject of pharmaceutical development (3). As members of the protein kinase superfamily, PTKs share conserved sequences and folds (2), and yet they show distinct substrate selectivity (4,5) and modes of regulation (6). Evidence *To whom correspondence should be addressed: P.A.C.: Tel, Fax, suggests that the mechanism of PTK-catalyzed phosphoryl transfer is dissociative, in which the bond to the leaving group ADP is largely broken prior to tyrosine phenol attack on the gamma phosphorus of ATP (7). The alignment, orientation, and spacing of the tyrosine and ATP substrates by key active site residues are believed to be crucial in facilitating catalysis based on structural (8), mutagenic (9), and kinetic (10) studies.The catalytic loop sequence, DLAARN, is conserved throughout the vast majority of the 90 human PTKs, but differs in the nine members of the Src family, which have the sequence DLRAAN ( Figure 1A) (2). This relocation of the Arg from the D+4 position in most PTKs to the D+2 position in the Src family suggests an unusual functional plasticity. A crystal structure of an insulin receptor tyrosine kina...

Identification of Targets of c-Src Tyrosine Kinase by Chemical Complementation and Phosphoproteomics

“…However, the chemical rescue of the R390A c-Src mutant provides a strategy that perfectly fits this purpose because it provides a rapid, specific, and reversible mechanism of c-Src activation by the small molecule imidazole, allowing the detection of the immediate effects of the restored c-Src activity in cells (3). Prior in vitro studies on the tyrosine kinases Csk, Src, and Abl have indicated that the imidazole concentration used here (Ͻ30 mM) has no effect (Ͻ10%) on wild-type kinase activity (3,12,14). Furthermore, previous experiments on chemical rescue of R/A Csk and Src with imidazole in cell culture have revealed that this chemical rescue approach recapitulates, without apparent toxic effects, expected tyrosine kinase activities involved in stress fiber formation, vascular tube formation, cell migration and transformation, and guanylyl cyclase modulation (3, 12-14, 53, 54).…”

“…It has previously been shown that mutation of a highly conserved Arg (390 in c-Src) in protein-tyrosine kinases re-sults in a dramatic reduction in catalytic activity (200 -5000-fold), presumably because of the loss of a key hydrogenbonding side chain responsible for orienting the substrate tyrosine phenol for phosphoryl transfer (12)(13)(14). A variety of di-and triamino compounds added to the enzyme reaction buffer have been shown to complement this defective kinase activity, the most efficient being imidazole (12)(13)(14).…”

“…A variety of di-and triamino compounds added to the enzyme reaction buffer have been shown to complement this defective kinase activity, the most efficient being imidazole (12)(13)(14). Structural and pH studies suggest that positively charged imidazolium occupies the unnatural cavity found in R/A mutant proteintyrosine kinases and serves to rescue the catalytic function without significantly affecting c-Src substrate selectivity (14) (see Fig.…”

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

“…The insoluble pellet was resuspended in 8 M urea, diluted 1:10 in suspension buffer, incubated with glutathione agarose (Sigma) and washed three times before use as substrate for in vitro phosphorylation (described below). Purified recombinant wildtype Abl kinase domain (residues 229-511) (Seeliger et al, 2005) and corresponding mutant R367A (Muratore et al, 2009) were gifts from Philip Cole (Johns Hopkins School of Medicine, Baltimore, MD).…”

“…We tested emerin as a potential substrate for the purified recombinant His-tagged kinase domain (residues 229-511) of human Abl (Seeliger et al, 2005). As the control, we used Abl mutant R367A, which has 5000 times lower catalytic efficiency (Muratore et al, 2009). Recombinant His-tagged emerin (residues 1-176) was incubated 30 minutes alone or with each Abl kinase domain.…”