Characterization of the biochemical and transforming properties of the neuroepithelial transforming protein 1

Rho family small G proteins are key regulators of cytoskeletal organization and oncogenic transformation whose activation is controlled by a family of proteins known as guanine nucleotide exchange factors (GEFs). In this work we have characterized the structural and biological determinants for cytoskeletal regulation and cell transformation by the neuroepithelioma transforming gene 1 (NET1), which is a GEF specific for RhoA, but not Cdc42 or Rac1. Previously it was shown that the biological activity and nuclear localization of NET1 is controlled by its amino terminus. Here we demonstrate that the amino terminus of NET1 does not function as cis-acting autoinhibitory domain, nor does it affect the ability of full-length NET1 to stimulate actin stress fiber formation. We also show that the nuclear localization of NET1 is controlled by two separate domains within its amino terminus, only one of which contains the previously identified NLS sequences. Importantly, we find that the ability of NET1 to stimulate actin stress fiber formation does not correlate with its transforming activity, because NET1 proteins that potently stimulate stress fiber formation do not transform cells. Furthermore, the presence of a potential PDZ binding site in the C terminus of NET1 is critical to its ability to transform cells, but is not required for enzymatic activity or for effects on the actin cytoskeleton. Thus, these data highlight a divergence between the ability of NET1 to stimulate cytoskeletal reorganization and to transform cells, and implicate the interaction with PDZ domain-containing proteins as critical to NET1-dependent transformation.