Non-prenylatable, cytosolic Rac1 alters neurite outgrowth while retaining the ability to be activated

Rac1 is an important regulator of axon extension, cell migration and actin reorganization. Like all Rho guanine triphosphatases (GTPases), Rac1 is targeted to the membrane by the addition of a geranylgeranyl moiety, an action thought to result in Rac1 guanosine triphosphate (GTP) binding. However, the role that Rac1 localization plays in its activation (GTP loading) and subsequent activation of effectors is not completely clear. To address this, we developed a non-prenylatable emerald green fluorescent protein (EmGFP)-Rac1 fusion protein (EmGFP-Rac1C189A) and assessed how expressing this construct affected neurite outgrowth, Rac1 localization and activation in neuroblastoma cells. Expression of EmGFP-Rac1C189A increased localization to the cytosol and induced cell clustering while increasing neurite initiation. EmGFP-Rac1C189A expression also increased Rac1 activation in the cytosol, compared to cells expressing wild-type Rac1 (EmGFP-Rac1). These results suggest that activation of Rac1 may not require plasma membrane localization, potentially leading to differential activation of cytosolic signaling pathways that alter cell morphology. Understanding the consequences of differential localization and activation of Rho GTPases, including Rac1, could lead to new therapeutic targets for treating neurological disorders.