Regulatory mechanisms controlling the subcellular localization and activity of the RhoA GEF Net1 in breast cancer

Abstract

Net1 is a RhoA/B‐specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for efficient cancer cell motility and invasion in vitro. Net1 knockout mice are viable but exhibit defects in tumor initiation and lung metastasis in a genetic model of breast cancer. Net1 is unusual among RhoGEFs in that it localizes to the nucleus in quiescent cells, but relocalizes to the cytoplasm in response to motility stimuli. However, regulatory mechanisms controlling its cytoplasmic accumulation are not well understood. In the present work we show that accumulation of the Net1A isoform in the cytosol requires CRM1‐dependent nuclear export and also requires phosphorylation by stress activated MAP kinases. Overexpression of constitutively active MKK7 or MKK3, which stimulate the Jnk and p38 MAPKs respectively, stimulates Net1A cytosolic localization in a ligand independent manner. Mutation of the main Jnk phosphorylation site, Ser52, to alanine prevents ligand stimulated cytosolic localization. Mechanistically, Net1A phosphorylation by Jnk prevents nuclear re‐import of cytosolic Net1A. Glutamic acid substitution of Ser52 promotes constitutive cytosolic localization and results in elevated RhoA activation and signaling. Expression of Net1A S52E promotes increased motility, extracellular matrix invasion, and invadopodia formation in breast cancer cells that are normally not invasive. Taken together, these data identify a novel regulatory mechanism controlling the subcellular localization and proinvasive functions of Net1A, and suggest novel therapeutic avenues to target Net1‐dependent cancers.Support or Funding InformationThis work was supported by NCI grant to CA172129 J.A.F. and a CPRIT fellowship RP160015 to A.U.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.