Autophosphorylation of serine/threonine-protein kinase PAK1 (PAK1) is required for complete activation. PAK1 is autophosphorylated at several sites, but serine 144 (S144) in the GTPase binding domain and threonine 423 (T423) in the activation loop are the two conserved sites that regulate catalytic activity. As a result, active PAK1 phosphorylates substrates like LIMK (LIM domain kinase) that are involved in remodelling of the actin cytoskeleton.

PAK1 needs autophosphorylation for complete activation. PAK1 is autophosphorylated at several sites, but S144 flanking the kinase inhibitor region and T423 within the catalytic domain are the two conserved sites that regulate the catalytic activity (Chong et al. 2001, Parrini et al. 2001).

G-Protein Coupled Receptors (GPCR) sense extracellular signals and activate different Guanine nucleotide binding proteins (G-proteins) that have alpha, beta and gamma subunits. Upon activation, the alpha subunit of G-proteins dissociates from beta-gamma and the both are then free to regulate downstream effectors. Serine/threonine-protein kinase PAK 1 can directly bind with Rho guanine nucleotide exchange factor 6 (ARHGEF6, PIX-Alpha) in the cytosol. This complex is then translocated to the plasma membrane where Cell division control protein 42 homolog (CDC42) is activated. CDC42 is known to be involved in epithelial cell polarization processes.

LIM kinases are serine protein kinases with a unique combination of two N-terminal LIM motifs, a central PDZ domain, and a C-terminal protein kinase domain. LIMK1 is one of the downstream targets of PAK1 and is activated through phosphorylation by PAK1 on T508 within its activation loop (Edwards et al. 1999, Aizawa et al. 2001). LIM-kinase is responsible for the tight regulation of the activity of cofilin (a protein that depolymerizes actin filaments) and thus maintains the balance between actin assembly and disassembly. Phosphorylated cofilin is inactive, resulting in stabilization of the actin cytoskeleton.

NF2 (Merlin) is a product of the tumor suppressor gene neurofibromatosis type 2 (NF2). NF2 binds to the PBD of PAK1 and prevents its activation (Kissil et al. 2003). In primary schwannoma tumor samples derived from patients with germline mutations in the NF2 gene, PAK1 activity is highly elevated (Yi et al. 2008) and essential for the malignant growth of NF2-deficient cells (Hirokawa et al. 2004). In complex with ERBIN (ERBB2IP), NF2 may also be involved in the inhibition of PAK2 activation (Wilkes et al. 2009).

The localization and function of NF2 may be modulated by PAK2-mediated phosphorylation and subsequent sumoylation (Rong et al. 2004, Qi et al. 2014).

G-Protein Coupled Receptors (GPCR) sense extracellular signals and activate different Guanine nucleotide binding proteins (G-proteins) that have alpha, beta and gamma subunits. Upon activation, the alpha subunit of G-proteins dissociates from beta-gamma and the both are then free to regulate downstream effectors. Serine/threonine-protein kinase PAK 1 binds with Rho guanine nucleotide exchange factor 6 (ARHGEF6, PIX-Alpha) in the cytosol and is subsequently translocated by the G-protein beta-gamma complex to the plasma membrane. Here, ARHGEF6 activates Cell division control protein 42 homolog (CDC42) by acting as a GEF. Once active, CDC42 can facilitate the activation of PAK1. CDC42 is known to be involved in epithelial cell polarization processes.

The PAKs (p21-activated kinases) are a family of serine/threonine kinases mainly implicated in cytoskeletal rearrangements. All PAKs share a conserved catalytic domain located at the carboxyl terminus and a highly conserved motif in the amino terminus known as p21-binding domain (PBD) or Cdc42/Rac interactive binding (CRIB) domain. There are six mammalian PAKs that can be divided into two classes: class I (or conventional) PAKs (PAK1-3) and class II PAKs (PAK4-6). Conventional PAKs are important regulators of cytoskeletal dynamics and cell motility and are additionally implicated in transcription through MAPK (mitogen-activated protein kinase) cascades, death and survival signaling and cell cycle progression (Chan and Manser 2012).

PAK1, PAK2 and PAK3 are direct effectors of RAC1 and CDC42 GTPases. RAC1 and CDC42 bind to the CRIB domain. This binding induces a conformational change that disrupts inactive PAK homodimers and relieves autoinhibition of the catalytic carboxyl terminal domain (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009). Autophosphorylation of a conserved threonine residue in the catalytic domain of PAKs (T423 in PAK1, T402 in PAK2 and T436 in PAK3) is necessary for the kinase activity of PAK1, PAK2 and PAK3. Autophosphorylation of PAK1 serine residue S144, PAK2 serine residue S141, and PAK3 serine residue S154 disrupts association of PAKs with RAC1 or CDC42 and enhances kinase activity (Lei et al. 2000, Chong et al. 2001, Parrini et al. 2002, Jung and Traugh 2005, Wang et al. 2011). LIMK1 is one of the downstream targets of PAK1 and is activated through PAK1-mediated phosphorylation of the threonine residue T508 within its activation loop (Edwards et al. 1999). Further targets are the myosin regulatory light chain (MRLC), myosin light chain kinase (MLCK), filamin, cortactin, p41Arc (a subunit of the Arp2/3 complex), caldesmon, paxillin and RhoGDI, to mention a few (Szczepanowska 2009).

Class II PAKs also have a CRIB domain, but lack a defined autoinhibitory domain and proline-rich regions. They do not require GTPases for their kinase activity, but their interaction with RAC or CDC42 affects their subcellular localization. Only conventional PAKs will be annotated here.

Besides adhesive roles in cell cell interaction, L1 functions as a signal transducing receptor providing neurons with cues from their environment for axonal growth and guidance. L1 associates with beta1 integrins on the cell surface to induce a signaling pathway involving sequential activation of pp60csrc, Vav2 -GEF, Rac1, PAK1, MEK and ERK1/2. L1 stimulates cell migration and neurite outgrowth through the MAP kinases ERK1/2. CHL1 also associates with integrins and activates a MAPK signaling pathway via pp60c-src, MEK and ERK1/2.
L1 also binds the Sema3A receptor neuropilin1 and acts as an obligate coreceptor to mediate Sema3A induced growth cone collapse and axon repulsion. This repulsion can be converted to attraction by homophilic binding of L1 on an apposing cell in trans with L1 complexed with Neuropilin1 (NP1) in the responding neuron.
L1 also interacts with FGF receptor and activates PLC gamma and DAG, resulting in the production of arachidonic acid and subsequent opening of voltage-gated channels.

RHO GTPases regulate cell behaviour by activating a number of downstream effectors that regulate cytoskeletal organization, intracellular trafficking and transcription (reviewed by Sahai and Marshall 2002).

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) (Amano et al. 1996, Ishizaki et al. 1996, Leung et al. 1996, Ohashi et al. 2000, Sumi et al. 2001, Riento and Ridley 2003, Watanabe et al. 2007).

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 (Maesaki et al. 1999, Zong et al. 1999, Owen et al. 2003, Modha et al. 2008, Hutchinson et al. 2011, Hutchinson et al. 2013), bringing them in proximity to the PIP3-activated PDPK1 (PDK1) and thus enabling PDPK1-mediated phosphorylation of PKN1, PKN2 and PKN3 (Flynn et al. 2000, Torbett et al. 2003). PKNs play important roles in cytoskeleton organization (Hamaguchi et al. 2000), regulation of cell cycle (Misaki et al. 2001), receptor trafficking (Metzger et al. 2003) and apoptosis (Takahashi et al. 1998). PKN1 is also involved in the ligand-dependent transcriptional activation by the androgen receptor (Metzger et al. 2003, Metzger et al. 2005, Metzger et al. 2008).

Citron kinase (CIT) binds RHO GTPases RHOA, RHOB, RHOC and RAC1 (Madaule et al. 1995), 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 (Camera et al. 2003). CIT is also involved in the regulation of cytokinesis through its interaction with KIF14 (Gruneberg et al. 2006, Bassi et al. 2013, Watanabe et al. 2013).

RHOA, RHOG, RAC1 and CDC42 bind kinectin (KTN1), a kinesin anchor protein involved in kinesin-mediated vesicle motility (Vignal et al. 2001, Hotta et al. 1996). 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 (Vignal et al. 2001). RHOG and KTN1 also cooperate in microtubule-dependent lysosomal transport (Vignal et al. 2001).

IQGAP proteins IQGAP1, IQGAP2 and IQGAP3, bind RAC1 and CDC42 and stabilize them in their GTP-bound state (Kuroda et al. 1996, Swart-Mataraza et al. 2002, Wang et al. 2007). IQGAPs bind F-actin filaments and modulate cell shape and motility through regulation of G-actin/F-actin equilibrium (Brill et al. 1996, Fukata et al. 1997, Bashour et al. 1997, Wang et al. 2007, Pelikan-Conchaudron et al. 2011). Binding of IQGAPs to F-actin is inhibited by calmodulin (Bashour et al. 1997, Pelikan-Conchaudron et al. 2011). IQGAP1 is involved in the regulation of adherens junctions through its interaction with E-cadherin (CDH1) and catenins (CTTNB1 and CTTNA1) (Kuroda et al. 1998, Hage et al. 2009). IQGAP1 contributes to cell polarity and lamellipodia formation through its interaction with microtubules (Fukata et al. 2002, Suzuki and Takahashi 2008).

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 (Neudauer et al. 2001, Cheng et al. 2005).

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 (Sudo et al. 2006, Watanabe et al. 1996, Fujita et al. 2000, Peck et al. 2002, Mircescu et al. 2002). Similar to formins (Miralles et al. 2003), cytoskeletal changes triggered by RTKN activation may lead to stimulation of SRF-mediated transcription (Reynaud et al. 2000).

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 (Knaus et al. 1991, Roberts et al. 1999, Kim and Dinauer 2001, Jyoti et al. 2014, Cheng et al. 2006, Miyano et al. 2006, Ueyama et al. 2006).

The Rho family of small guanine nucleotide binding proteins is one of seven phylogenetic branches of the Ras superfamily (Bernards 2005), which, besides Rho, Miro and RHOBTB3 also includes Ran, Arf, Rab and Ras families (Boureux et al. 2007). Miro GTPases and RHOBTB3 ATPase are sometimes described as Rho family members, but they are phylogenetically distinct (Boureux et al. 2007). Phylogenetically, RHO GTPases can be grouped into four clusters. The first cluster consists of three subfamilies: Rho, RhoD/RhoF and Rnd. The second cluster consists of three subfamilies: Rac, Cdc42 and RhoU/RhoV. The third cluster consists of the RhoH subfamily. The fourth cluster consists of the RhoBTB subfamily. Based on their activation type, RHO GTPases can be divided into classical (typical) and atypical (reviewed by Haga and Ridley 2016, and Kalpachidou et al. 2019). Classical RHO GTPases cycle between active GTP-bound states and inactive GDP-bound states through steps that are tightly controlled by members of three classes of proteins: (1) guanine nucleotide dissociation inhibitors or GDIs, which maintain Rho proteins in an inactive state in the cytoplasm, (2) guanine nucleotide exchange factors or GEFs, which destabilize the interaction between Rho proteins and their bound nucleotide, the net result of which is the exchange of bound GDP for the more abundant GTP, and (3) GTPase activating proteins or GAPs, which stimulate the low intrinsic GTP hydrolysis activity of Rho family members, thus promoting their inactivation. GDIs, GEFs, and GAPs are themselves subject to tight regulation, and the overall level of Rho activity reflects the balance of their activities. Many of the Rho-specific GEFs, GAPs, and GDIs act on multiple Rho GTPases, so that regulation of these control proteins can have complex effects on the functions of multiple Rho GTPases (reviewed by Van Aelst and D'Souza-Schorey 1997, Schmidt and Hall 2002, Jaffe and Hall 2005, Bernards 2005, and Hodge and Ridley 2016). Classical RHO GTPases include four subfamilies: Rho (includes RHOA, RHOB and RHOC), Rac (includes RAC1, RAC2, RAC3 and RHOG), Cdc42 (includes CDC42, RHOJ and RHOQ) and RhoD/RhoF (includes RHOD and RHOF) (reviewed in Haga and Ridley 2016). Atypical RHO GTPases do not possess GTPase activity. They therefore constitutively exist in the active GTP-bound state. Atypical RHO GTPases include three subfamilies: Rnd (includes RND1, RND2 and RND3), RhoBTB (includes RHOBTB1 and RHOBTB2), RhoH (RHOH is the only member) and RhoU/RhoV (includes RHOU and RHOV). Members of the Rho family have been identified in all eukaryotes. Among Rho GTPases, RHOA, RAC1 and CDC42 have been most extensively studied.

RHO GTPases regulate cell behavior by activating a number of downstream effectors that regulate cytoskeletal organization, intracellular trafficking and transcription (reviewed by Sahai and Marshall 2002). They are best known for their ability to induce dynamic rearrangements of the plasma membrane-associated actin cytoskeleton (Aspenstrom et al. 2004; Murphy et al. 1999; Govek et al. 2005). Beyond this function, Rho GTPases also regulate actomyosin contractility and microtubule dynamics. Rho mediated effects on transcription and membrane trafficking are believed to be secondary to these functions. At the more macroscopic level, Rho GTPases have been implicated in many important cell biological processes, including cell growth control, cytokinesis, cell motility, cell-cell and cell-extracellular matrix adhesion, cell transformation and invasion, and development (Govek et al., 2005). One of the best studied RHO GTPase effectors are protein kinases ROCK1 and ROCK2, which 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) (reviewed in Riento and Ridley 2003). The p21-activated kinase family, which includes PAK1, PAK2 and PAK3, is another well characterized family of RHO GTPase effectors involved in cytoskeleton regulation (reviewed in Daniels and Bokoch 1999, Szczepanowska 2009). Protein kinase C related kinases (PKNs), PKN1, PKN2 and PKN3 play important roles in cytoskeleton organization (Hamaguchi et al. 2000), regulation of cell cycle (Misaki et al. 2001), receptor trafficking (Metzger et al. 2003), apoptosis (Takahashi et al. 1998), and transcription (Metzger et al. 2003, Metzger et al. 2005, Metzger et al. 2008). Citron kinase (CIT) is involved in Golgi apparatus organization through regulation of the actin cytoskeleton (Camera et al. 2003) and in the regulation of cytokinesis (Gruneberg et al. 2006, Bassi et al. 2013, Watanabe et al. 2013). Kinectin (KTN1), a kinesin anchor protein, is a RHO GTPase effector involved in kinesin-mediated vesicle motility (Vignal et al. 2001, Hotta et al. 1996), including microtubule-dependent lysosomal transport (Vignal et al. 2001). IQGAP proteins, IQGAP1, IQGAP2 and IQGAP3, are RHO GTPase effectors that modulate cell shape and motility through regulation of G-actin/F-actin equilibrium (Brill et al. 1996, Fukata et al. 1997, Bashour et al. 1997, Wang et al. 2007, Pelikan-Conchaudron et al. 2011), regulate adherens junctions (Kuroda et al. 1998, Hage et al. 2009), and contribute to cell polarity and lamellipodia formation (Fukata et al. 2002, Suzuki and Takahashi 2008). WASP and WAVE proteins (reviewed by Lane et al. 2014), as well as formins (reviewed by Kuhn and Geyer 2014), are RHO GTPase effectors that regulate actin polymerization and play important roles in cell motility, organelle trafficking and mitosis. Rhotekin (RTKN) and rhophilins (RHPN1 and RHPN2) are RHO GTPase effectors that regulate the organization of the actin cytoskeleton and are implicated in the establishment of cell polarity, cell motility and possibly endosome trafficking (Sudo et al. 2006, Watanabe et al. 1996, Fujita et al. 2000, Peck et al. 2002, Mircescu et al. 2002). Cytoskeletal changes triggered by the activation of formins (Miralles et al. 2003) and RTKN (Reynaud et al. 2000) may lead to stimulation of SRF-mediated transcription. 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-), are also regulated by RHO GTPases (Knaus et al. 1991, Roberts et al. 1999, Kim and Dinauer 2001, Jyoti et al. 2014, Cheng et al. 2006, Miyano et al. 2006, Ueyama et al. 2006). Every RHO GTPase activates multiple downstream effectors and most effectors are regulated by multiple RHO GTPases, resulting in an elaborate cross-talk.

Serine/threonine-protein kinase PAK 1