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Focal Adhesion Kinase as a Cellular Mechano-Sensor

Zhou, Jing

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Abstract

Mechanical signaling plays a crucial role in cell communication. Translating mechanical force into biochemical reactions is vital for virtually all cellular processes such as cell proliferation and differentiation. Mechanosensors, which can respond to an external force in form of controllable conformational changes, are key regulators in the mechanical signal transduction processes. In this work, we present results from Molecular Dynamics (MD) and biochemical network simulations that suggest Focal Adhesion Kinase (FAK) as a mechanosensing enzyme in the signaling pathway. FAK is a non-receptor tyrosine kinase, located at the cytoplasmic site of the cell membrane. During the last decade, a growing body of evidence shows that tensile stress acting on cells leads to increased FAK activity. However, the mechanism of FAK mechanical activation has not been resolved. The auto-inhibitory conformation of the FAK FERMkinase fragment suggests that the major mechanism of the regulation of FAK activity is the release of Tyr576/577 on the activation loop from the N-terminal FERM domain. We first addressed the allosteric regulation of the FAK by PIP2 binding (Chapter 3), a recently recognized stimulus for FAK activation, by performing all-atom MD simulations of FAK FERM-kinase fragment and comparing the dynamics in absence and presence of ATP and PIP2. A closing-opening motion between the kinase and FERM domain upon ATP and additional PIP2 binding was observed in close agreement with corresponding changes in fluorescence resonance energy transfer experiments. As an underlying allosteric mechanism, using Force Distribution Analysis (FDA), a signal network spanning from the PIP2 and ATP binding site to the distant interface between the FERM and kinase domains could be identified. However, our results also demonstrated that the ligand induced conformational changes are insufficient for FAK catalytic turnover, which requires full exposure of Tyr576/577. This suggests that an additional biochemical or mechanical stimulus is required for FAK activation. Following the study of FAK allosteric regulation, we suggest a mechanical model of FAK activation in which tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by relieving the occlusion of the central phosphorylation site of FAK (Tyr576/577). To test the hypothesis of FAK as a force-sensor, extensive Force Probe Molecular Dynamics (FPMD) simulations, with varying loading rates, pulling directions and membrane PIP2 concentrations, were carried out. They directly supported the notion of a specific force-induced opening and activation of FAK. This is remarkable given that force-induced unfolding of the primarily α-helical FERM and kinase domains are competitive processes but were observed to be less favored over domain-domain dissociation. To assess downstream consequences of FAK mechano-sensing, force-dependent FAK kinetics based on extrapolated MD data were implemented as parameters to establish a mechano-biochemical network model, which connects force-dependent FAK activation to the downstream exchange rate of GTP/GDP in Ras. Our computational study provides direct evidence for FAK-mediated mechanical signaling through Ras to the nucleus, with predictions directly testable by cell stretching experiments and single molecule force spectroscopy.

Item Type: Dissertation
Supervisor: Spatz, Prof. Dr. Joachim
Date of thesis defense: 18 February 2015
Date Deposited: 08 Apr 2015 06:34
Date: 2015
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Subjects: 540 Chemistry and allied sciences
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