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Visualizing and manipulating focal adhesion kinase regulation in live cells.

Ritt M, Guan JL, Sivaramakrishnan S.

J Biol Chem. 2013 Mar 29;288(13):8875-86.

Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.




Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of FRET sensors that retain all of the individual FAK domains but modulate a key intramolecular regulatory interaction between the band 4.1/ezrin/radixin/moesin (FERM) and kinase domains of FAK. We demonstrate systematic control and quantitative measurement of the FERM-kinase interaction at focaladhesions, which in turn allows us to control cell migration. Using these sensors, we find that Tyr-397 phosphorylation, rather than kinase activity of FAK, is the key determinant of cell migration. Our sensors directly demonstrate, for the first time, a pH-dependent change in a protein-protein interaction at a macromolecular structure in live cells. The FERM-kinase interaction at focal adhesions is enhanced at acidic pH, with a concomitant decrease in Tyr-397 phosphorylation, providing a potential mechanism for enhanced migration of cancer cells.


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Additional Information

Focal Adhesion Kinase (FAK) has long been accepted as being essential for cell migration and motility and has an integral role in tumor metastasis. Here, we utilize a toolbox of FAK FRET sensors to dissect the interactions between the kinase domain and the autoinhibitory band 4.1/ezrin/radixin/moesin (FERM) domain of FAK. Through the use of a series of genetically encoded alpha helices, we are able to modulate the intramolecular interactions within FAK through distance rather than truncation or mutation and thereby gain unique insight into the mechanisms of FAK regulation. In this paper, we leverage our sensors to demonstrate that phosphorylation of tyrosine 397 and not the activity of the kinase domain is the key event in FAK signaling. In addition, we provide new insights on the influence of pH on cell motility and we are able to directly observe differences in FAK signaling at the focal adhesion due to changes pH.


Visualizing and Manipulating Focal Adhesion Kinase Regulation in Live Cells