CaMKK2 Regulates Mechanosensitive Assembly of Contractile Actin Stress Fibers

Stress fibers are contractile actomyosin bundles that guide cell adhesion, migration, and morphogenesis. Their assembly and alignment are under precise mechanosensitive control. Thus, stress fiber networks undergo rapid modification in response to changes in biophysical properties of the cell's surroundings. Stress fiber maturation requires mechanosensitive activation of 5'AMP-activated protein kinase (AMPK), which phosphorylates vasodilator-stimulated phosphoprotein (VASP) to inhibit actin polymerization at focal adhesions. Here, we identify Ca²⁺-calmodulin-dependent kinase kinase 2 (CaMKK2) as a critical upstream factor controlling mechanosensitive AMPK activation. CaMKK2 and Ca²⁺ influxes were enriched around focal adhesions at the ends of contractile stress fibers. Inhibition of either CaMKK2 or mechanosensitive Ca²⁺ channels led to defects in phosphorylation of AMPK and VASP, resulting in a loss of contractile bundles and a decrease in cell-exerted forces. These data provide evidence that Ca²⁺, CaMKK2, AMPK, and VASP form a mechanosensitive signaling cascade at focal adhesions that is critical for stress fiber assembly.