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bioRxiv · 10.1101/2020.10.25.354415

Distinct autoinhibitory mechanisms regulate vinculin binding by alpha-T-catenin and alpha-E-catenin

Heier, J. A.·Pokutta, S.·Dale, I. W.·Kim, S. K.·Hinck, A. P.·Weis, W. I.·Kwiatkowski, A. V.
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Abstract

-catenin binds directly to {beta}-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates -catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of T(testes)-catenin, an -catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that T-catenin binds the {beta}-catenin/N-cadherin complex with a similar low nanomolar affinity to that of E-catenin. Limited proteolysis revealed that the T-catenin M-region adopts a more open conformation than E-catenin. The T-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated T-catenin M-region is not autoinhibited and thereby distinct from E-catenin. However, the T-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, T-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results indicate that the T-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of T-catenin allow it to function as a scaffold for building specific adhesion complexes.

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Heier, J. A., Pokutta, S., Dale, I. W., Kim, S. K., Hinck, A. P., Weis, W. I., Kwiatkowski, A. V.. 2020-10-26. Distinct autoinhibitory mechanisms regulate vinculin binding by alpha-T-catenin and alpha-E-catenin. https://doi.org/10.1101/2020.10.25.354415

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