RhoA and Rac1 prenylation: Effects on serine/threonine signaling for actin polymerization

RhoA and Rac1 are small guanosine triphosphates (GTPases) that regulate cytoskeletal rearrangements, cell polarity and axon guidance and signaling pathways. Alteration of the subcellular localization or activation of Rho GTPases is implicated in several neurological conditions. Rho GTPases are activated by binding to guanosine triphosphate (GTP) and is thought to require translocation to the plasma membrane by the addition of an isoprenoid moiety (geranylgeranyl) to the protein. However, previous experiments indicate that RhoA or Rac1 can be activated in the cytosol, without translocation to the plasma membrane. Based on this and evidence the Rho GTPases are centrally located in signaling pathways regulating actin dynamics, it was hypothesized that overexpressing non-geranylgeranylatable RhoA or Rac1 would decrease the actin filament content in neuronal growth cones by altering the location of actin regulating molecules. In particular, it was hypothesized that non-geranylgeranylatable RhoA or Rac1 would decrease the activity of actin filament promoters (i.e association of WAVE with the ARP2/3 complex and activation of JNK and ERK), and inactivation of the actin-depolymerizing protein cofilin. Overexpressing non-geranylgeranylatable (EmGFP-RhoAC190A), but not wild-type RhoA (EmGFP-RhoA), increased activation of ERK in the cytosol and increased association of WAVE with the ARP2/3 complex at the membrane, compared to cells overexpressing only the empty vector (EmGFP). However, overexpressing neither RhoA construct affected actin dynamics. Overexpressing wild-type Rac1 (EmGFP-Rac1), but not non-geranylgeranylatable Rac1 (EmGFP-Rac1C189A), increased the actin filaments content in growth cones compared to neurons expressing only EmGFP, concomitant with an increase in JNK activation. Overexpression of EmGFP-RacC189A decreased JNK activation and increased WAVE/ARP2/3 complexing, compared to cells expressing wild-type Rac1 or EmGFP alone. Studies with signaling molecule inhibitors indicated significant cross-talk between signaling pathways, which is altered by overexpressing wild-type or non-geranylgeranylatable forms of RhoA or Rac1. The results suggest that altering the subcellular localization of RhoA or Rac1 changes the activation of signaling molecules that regulate actin dynamics in neuronal growth cones. Elucidating the signaling cascades of the active GTPases may identify the distinct functions of these GTPases in the cytosol and can be used as novel targets to facilitate axon regeneration in neurodegenerative and neurological conditions.