Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

A simple mechanochemical model for calcium signalling in embryonic epithelial cells

Calcium signalling is one of the most important mechanisms of information propagation in the body. In embryogenesis the interplay between calcium signalling and mechanical forces is critical to the healthy development of an embryo but poorly understood. Several types of embryonic cells exhibit calcium-induced contractions and many experiments indicate that calcium signals and contractions are coupled via a two-way mechanochemical feedback mechanism. We present a new analysis of experimental data that supports the existence of this coupling during Apical Constriction. We then propose a simple mechanochemical model, building on early models that couple calcium dynamics to the cell mechanics and we replace the hypothetical bistable calcium release with modern, experimentally validated calcium dynamics. We assume that the cell is a linear, viscoelastic material and we model the calcium-induced contraction stress with a Hill function, i.e. saturating at high calcium levels. We also express, for the first time, the “stretch-activation” calcium flux in the early mechanochemical models as a bottom-up contribution from stretch-sensitive calcium channels on the cell membrane. We reduce the model to three ordinary differential equations and analyse its bifurcation structure semi-analytically as two bifurcation parameters vary - the IP3 concentration, and the “strength” of stretch activation, λ. The calcium system (λ = 0, no mechanics) exhibits relaxation oscillations for a certain range of IP3 values. As λ is increased the range of IP3 values decreases and oscillations eventually vanish at a sufficiently high value of λ. This result agrees with experimental evidence in embryonic cells which also links the loss of calcium oscillations to embryo abnormalities. Furthermore, as λ is increased the oscillation amplitude decreases but the frequency increases. Finally, we also identify the parameter range for oscillations as the mechanical responsiveness factor of the cytosol increases. This work addresses a very important and understudied question regarding the coupling between chemical and mechanical signalling in embryogenesis

A simple mechanochemical model for calcium signalling in embryonic epithelial cells

Calcium signalling is one of the most important mechanisms of information propagation in the body. In embryogenesis the interplay between calcium signalling and mechanical forces is critical to the healthy development of an embryo but poorly understood. Several types of embryonic cells exhibit calcium-induced contractions and many experiments indicate that calcium signals and contractions are coupled via a two-way mechanochemical feedback mechanism. We present a new analysis of experimental data that supports the existence of this coupling during Apical Constriction. We then propose a simple mechanochemical model, building on early models that couple calcium dynamics to the cell mechanics and we replace the hypothetical bistable calcium release with modern, experimentally validated calcium dynamics. We assume that the cell is a linear, viscoelastic material and we model the calcium-induced contraction stress with a Hill function, i.e. saturating at high calcium levels. We also express, for the first time, the “stretch-activation” calcium flux in the early mechanochemical models as a bottom-up contribution from stretch-sensitive calcium channels on the cell membrane. We reduce the model to three ordinary differential equations and analyse its bifurcation structure semi-analytically as two bifurcation parameters vary - the IP3 concentration, and the “strength” of stretch activation, λ. The calcium system (λ = 0, no mechanics) exhibits relaxation oscillations for a certain range of IP3 values. As λ is increased the range of IP3 values decreases and oscillations eventually vanish at a sufficiently high value of λ. This result agrees with experimental evidence in embryonic cells which also links the loss of calcium oscillations to embryo abnormalities. Furthermore, as λ is increased the oscillation amplitude decreases but the frequency increases. Finally, we also identify the parameter range for oscillations as the mechanical responsiveness factor of the cytosol increases. This work addresses a very important and understudied question regarding the coupling between chemical and mechanical signalling in embryogenesis