The insert region of Rac1 is essential for membrane ruffling but not cellular transformation

The Rho family of Ras-related proteins, which includes Rac1, RhoA, and Cdc4 2, is distinguished from other members of the Ras superfamily of small GTPa ses in that its members possess additional sequences positioned between bet a -strand 5 and alpha -helix 4, designated the insert region. Previous stud ies have established the importance of an intact insert region for the tran sforming, but not actin cytoskeletal reorganization, activities of Cdc42 an d RhoA, Similarly, the insert region was determined to be essential for Rad -mediated mitogenesis. Additionally, an intact insert region was also deter mined to be required for the antiapoptotic activity of Rad as well as for R ad activation of reactive oxygen species and the NF-kappaB transcription fa ctor. However, it has not been determined whether the insert region is impo rtant for Rad-mediated growth transformation. In this study, we assessed th e requirement for the insert region in Rad transformation and signaling in NIH 3T3 cells. Unexpectedly, we found that a mutant of constitutively activ ated 1Rac1 that lacked the insert region retained potent transforming activ ity. The insert region of 1Rac1 was dispensable for Rad stimulation of tran scription from the cyclin D1 promoter and for activation of the cJun, NF-ka ppaB, and E2F-1 transcription factors but was essential for Rad induction o f serum response factor activity. While an intact insert region was dispens able for inducing reactive oxygen species production in vivo, it was requir ed for Rad induction of lamellipodia, When taken together, these results sh ow that the insert region of Rad serves roles in regulating actin organizat ion and cell growth that are distinct from those of the analogous regions o f Cdc42 and RhoA and support its involvement in regulating specific downstr eam effector interactions.