RHO GTPase Effectors

One of the best studied RHO GTPase effectors are protein kinases ROCK1 and ROCK2, which are activated by binding RHOA, RHOB or RHOC. ROCK1 and ROCK2 phosphorylate many proteins involved in the stabilization of actin filaments and generation of actin-myosin contractile force, such as LIM kinases and myosin regulatory light chains (MRLC).

PAK1, PAK2 and PAK3, members of the p21-activated kinase family, are activated by binding to RHO GTPases RAC1 and CDC42 and subsequent autophosphorylation and are involved in cytoskeleton regulation (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Edwards et al. 1999, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009).

RHOA, RHOB, RHOC and RAC1 activate protein kinase C related kinases (PKNs) PKN1, PKN2 and PKN3, bringing them in proximity to the PIP3-activated PDPK1 (PDK1) and thus enabling PDPK1-mediated phosphorylation of PKN1, PKN2 and PKN3. PKNs play important roles in cytoskeleton organization, regulation of cell cycle, receptor trafficking and apoptosis. PKN1 is also involved in the ligand-dependent transcriptional activation by the androgen receptor.

Citron kinase (CIT) binds RHO GTPases RHOA, RHOB, RHOC and RAC1, but the mechanism of CIT activation by GTP-bound RHO GTPases has not been elucidated. CIT and RHOA are implicated to act together in Golgi apparatus organization through regulation of the actin cytoskeleton. CIT is also involved in the regulation of cytokinesis through its interaction with KIF14.

RHOA, RHOG, RAC1 and CDC42 bind kinectin (KTN1), a kinesin anchor protein involved in kinesin-mediated vesicle motility. The effect of RHOG activity on cellular morphology, exhibited in the formation of microtubule-dependent cellular protrusions, depends both on RHOG interaction with KTN1, as well as on the kinesin activity. RHOG and KTN1 also cooperate in microtubule-dependent lysosomal transport.

IQGAP proteins IQGAP1, IQGAP2 and IQGAP3, bind RAC1 and CDC42 and stabilize them in their GTP-bound state. IQGAPs bind F-actin filaments and modulate cell shape and motility through regulation of G-actin/F-actin equilibrium. Binding of IQGAPs to F-actin is inhibited by calmodulin. IQGAP1 is involved in the regulation of adherens junctions through its interaction with E-cadherin (CDH1) and catenins (CTTNB1 and CTTNA1). IQGAP1 contributes to cell polarity and lamellipodia formation through its interaction with microtubules.

RHOQ (TC10) regulates the trafficking of CFTR (cystic fibrosis transmembrane conductance regulator) by binding to the Golgi-associated protein GOPC (also known as PIST, FIG and CAL). In the absence of RHOQ, GOPC bound to CFTR directs CFTR for lysosomal degradation, while GTP-bound RHOQ directs GOPC:CFTR complex to the plasma membrane, thereby rescuing CFTR.

RAC1 and CDC42 activate WASP and WAVE proteins, members of the Wiskott-Aldrich Syndrome protein family. WASPs and WAVEs simultaneously interact with G-actin and the actin-related ARP2/3 complex, acting as nucleation promoting factors in actin polymerization (reviewed by Lane et al. 2014).

RHOA, RHOB, RHOC, RAC1 and CDC42 activate a subset of formin family members. Once activated, formins bind G-actin and the actin-bound profilins and accelerate actin polymerization, while some formins also interact with microtubules. Formin-mediated cytoskeletal reorganization plays important roles in cell motility, organelle trafficking and mitosis (reviewed by Kuhn and Geyer 2014).

Rhotekin (RTKN) and rhophilins (RHPN1 and RHPN2) are effectors of RHOA, RHOB and RHOC and have not been studied in detail. They regulate the organization of the actin cytoskeleton and are implicated in the establishment of cell polarity, cell motility and possibly endosome trafficking. Similar to formins, cytoskeletal changes triggered by RTKN activation may lead to stimulation of SRF-mediated transcription.

RHO GTPases RAC1 and RAC2 are needed for activation of NADPH oxidase complexes 1, 2 and 3 (NOX1, NOX2 and NOX3), membrane associated enzymatic complexes that use NADPH as an electron donor to reduce oxygen and produce superoxide (O2-). Superoxide serves as a secondary messenger and also directly contributes to the microbicidal activity of neutrophils.