NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability

The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na+/H+ exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. alpha-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the alpha-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/alpha-actinin-4 interaction increased alpha-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified alpha-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1.