Prenylation-independent activation of RhoA and Rac1: Effects on localization and neurite outgrowth
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Rho family guanine nucleotide triphosphatases (GTPases) are molecular switches that play a fundamental role in axon growth and guidance by directing growth cone cytoskeletal changes through interaction with downstream effectors. Small GTPases are considered active when bound to guanosine triphosphate (GTP), a process promoted by plasma membrane-associated guanine exchange factors (GEFs), and inactive when bound to guanosine diphosphate (GDP). RhoA, a member of the Rho family GTPases, participates in the formation of focal adhesions, retraction of neuron processes in response to injury or insult and growth cone collapse. Rac1, another Rho GTPase, is implicated in regulating cell proliferation, cell cycle entry, and extension of neuronal processes. Both RhoA and Rac1 require prenylation for membrane targeting, which is believed to be required for activation of these GTPases via interaction with GEFs. Meta-analyses in the early 2000's showed that people who took statins, global inhibitors of protein prenylation, have reduced incidence of neurodegenerative disorders. We performed neurite outgrowth studies on cells treated with lovastatin and found statin administration increased neurite branching. Global inhibitors do not give us a good indication of what is causing the observed effect as they do not implicate how inhibiting the prenylation of a specific protein leads to the observed affect. We constructed vectors expressing GFP-fused non-prenylatable RhoA and Rac1 as a protein-specific way of determining how prenylation affects neurite morphology and Rho GTPase activation. An active, but non-prenylated form of GTP-bound Rac1 and RhoA were found in cellular compartments other than the membrane after construct transfection. The non-prenylatable constructs affected cell morphology and neurite outgrowth in B35 neuroblastoma cells, which indicated that RhoA and Rac1 can be activated independent of prenylation, possibly leading to differential signal transduction cascades based on the localization of active GTPases. Similar results were obtained after transfection in primary rat cortical neurons. Active RhoA was found in primary rat cortical neurons after treatment with lovastatin, while Rac1 activation was suppressed. These studies suggest that Rac1 and RhoA can be differentially activated, localized, and regulated. Understanding how prenylation of RhoA and Rac1, and how their competition impacts function in neurite outgrowth may identify novel targets to facilitate axon regeneration in traumatic or degenerative neurological conditions.