Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase.

Fabian Baumann,Diana Angela Pippig,Martin Rees,Alexander Alexandrovich,Magnus Sebastian Bauer,Hermann Eduard Gaub,Mathias Gautel

doi:10.7554/elife.26473

Abstract

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region.

Highlights

All cells need to withstand as well as actively generate forces during division, differentiation or for their differentiated function
We investigated the effects of the presence of different ligands such as ATP, Ca2+/CaM or regulatory light chain (RLC) peptide substrate or a combination of these on Smooth muscle myosin light chain kinase (smMLCK)’s pathway through its different conformational states until fully unfolded
For force spectroscopy experiments, the smMLCK is tethered by an N-terminal Strep-tag II via an atomic force microscope (AFM) cantilever tip that is functionalized with a monovalent variant of Strep-Tactin (Baumann et al, 2016)

Summary

Introduction

All cells need to withstand as well as actively generate forces during division, differentiation or for their differentiated function. These mechanically governed adaptive processes require the translation of mechanical signals into physicochemical signals inside the cell that trigger an appropriate biological response (Shivashankar et al, 2015). A number of mechanosensors have evolved for these purposes, including mechanosensitive ion channels. The regulation of actomyosin contraction differs significantly between the calcium-activated striated muscles and myosin-light chain phosphorylation in smooth and non-muscle cells. Four myosin light chain kinases (MLCK) exist, transcribed from the MYLK1 to 4 genes (reviewed in Chang et al, 2016)

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Conclusion