Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

Design, Synthesis, and Evaluation of Dasatinib–Amino Acid and Dasatinib–Fatty Acid Conjugates as Protein Tyrosine Kinase Inhibitors

“…Binding assays . To determine the binding of compound 26 (Das‐G‐Flu) toward the kinase‐deficient Src catalytic domain (kdSrcCata), fluorescence polarization (FP) binding assays were carried out as described previously . The enzyme is a kinase‐deficient form of Src catalytic domain.…”

“…Regression analysis was carried out in software (http://www.labfit.net). To determine the binding of unlabeled dasatinib, its ability to compete against the fluorogenic compound for binding to kdSrcCata was determined by an FP competition binding assay . This assay setup differs from the above in that each tube had a 50 n m concentration of compound 26 (Das‐G‐Flu), 125 n m kdSrcCata (held constant), and concentrations of the unlabeled dasatinib from 200–1600 n m .…”

Conformational Basis for SH2-Tyr(P)527 Binding in Src Inactivation

“…Fluorescence Polarization Assay and K d Determination-The binding of a fluorescent phosphopeptide, Flu-GpYEEI, to kdSrc and kdSrc mutants was determined by the fluorescence polarization assay as described previously (23). To determine the binding of an unlabeled phosphopeptide to the SH2 domain, fluorescence polarization of 80 nM Flu-GpYEEI as the probe in the presence of 80 nM SH2 domain and variable concentrations of the unlabeled phosphopeptide was determined.…”

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase

“…In the active state the SH2 domain makes contacts with the loop preceding -helix C, while in the inactive conformation the SH2 domain is rotated 60 upwards with respect to the kinase domain, breaking these contacts. The binding of SH2 ligands has been shown to activate Csk and CHK (Zrihan-Licht et al, 1997, 1998Takeuchi et al, 2000;Ayrapetov et al, 2005), presumably by stabilizing the SH2-kinase domain contacts, but how the effect of SH2 ligand binding is propagated to the kinase domain is still unclear.…”

“…C-terminal tail phosphorylation of SFKs favours their 'closed' inactive state, thereby attenuating SFK signalling (Sicheri et al, 1997;Xu et al, 1997). The Csk-family kinases themselves are activated by phosphotyrosyl ligand binding by their Src-homology 2 (SH2) domains, thereby enhancing their ability to inhibit SFK signalling (Zrihan-Licht et al, 1997, 1998Takeuchi et al, 2000;Ayrapetov et al, 2005). As the SFKs are known proto-oncogenes that mediate proliferative responses downstream of numerous cell-surface receptors (Hunter, 1995;Pawson, 2004), it is important to understand the mechanistic basis of Csk and CHK activation and whether this could be exploited in the design of therapeutic inhibitors of SFK signalling.…”