PORI, POSH and pl40Sral have all been implicated in Rac-mediated pathways. PORI (Partner o f Rac) was identified fi’om a Yeast two-hybrid screen o f a Jurkat library (Van- Aelst et al., 1996). It was found to interact specifically to Racl in a GTP-dependent manner. It shares no homology to known Rac-interacting proteins, such as RhoGAP, bcrGAP, Tiaml PAK, Ost or PI3K. POR-1 expression alone does not induce membrane ruffling but is thought to act synergistically with RasV12 in inducing membrane ruffles. Co-expression o f full-length POR-1 and Racl does not exhibit synergistic tendencies but truncated constructs o f POR-1 have been reported to Rac 1V 12-induced membrane ruffles. PORI has been reported to interact with ARF6 GTPase, a member o f the ADP ribosylation factor (ARF) family that is involved endocytic trafficking. Overexpression of a GTPase negative ARF6 mutant is reported to induce cytoskeletal rearrangements that were inhibited with deletion mutants o f PORI but were unaffected by RacN17 co-expression (D’Souza-Schorey et al., 1997).
Another protein associated with the Rac regulation o f the actin cytoskeleton is pl40Sra-l (Specifically Racl associated protein). pl40sral specifically binds Racl- GTP and is found to co-localise with Rac V I2 at cortical actin o f membrane ruffles induced by RacV 12 in KB cells and is therefore suggested to be specifically required for membrane ruffle formation (Kobayashi et al., 1998).
POSH (Plethora Of SH3s) was also identified fi*om a yeast two-hybrid screen o f a Ras transformed mouse cDNA library. POSH too shows little homology with other proteins. POSH however does contain four SH3 domains. The exact role o f POSH is unclear as overexpression o f POSH was reported to induce cell death (Tapon et al.,
1998).
1.6.1.8: p35
p35 is a neuronal-specific Cdk5 regulator that activates Cdk5 activity upon binding. p35 is described as an effector for Rac involved in neurite outgrowth. p35 colocahses with Rac in neuronal growth cones. p35 binds Rac in a GTP-dependent manner. PAK is also localised with the p35-Rac complex. PAK is hyperphosphorylated by active p35 in a Rac-dependent manner, which results in the downregulation o f PAKl kinase activity. It
is proposed that this modification o f PAK by p35 is required for p35 induced neurite outgrowth (Nikolic et al., 1998).
1.6.1.9: CIP4
CIP4 (Cdc42 Interacting Protein) was isolated in a two-hybrid screen o f a B-cell library. CIP4 specifically binds Cdc42-GTP. CIP4 expression in Swiss 3T3 cells is described to induce the loss o f stress fibres and the formation o f dorsal surface foci. CIP4 encodes a domain that exhibit sequence similarity with the ERM (Erzin, Radixin and Moesin) proteins involved in signalling and cytoskeletal control, and is suggested to be a likely candidate for Cdc42 mediated signalling and regulation o f the actin cytoskeleton (Aspenstrom, 1997).
1.6.1.10: IQGAP
IQGAP was initially identified as a protein containing a potential calmodulin binding (IQ) motif. It sequence also revealed homology to the GAP domain o f RasGAP (Weissbach et al., 1994) however IQGAP failed to exhibit GAP activity toward Ras, Racl or Cdc42 (Brill et al., 1996; Kuroda et al., 1996). In addition IQGAP also contains a CHD (calponin homology domain) present in several actin binding proteins, a WW domain and a putative SH3-binding domain but does not contain a CRIB-motif (Brill et al., 1996; Kuroda et al., 1996; McCallum et al., 1996). IQGAP was also identified using affinity chromatography to isolate GTP-Cdc42 dependent binding proteins. IQGAP also exhibits Racl- GTP binding activity but does not bind RhoA-GTP (Kuroda et al., 1996; Brillera/., 1996).
There are two forms o f IQGAP, IQGAP 1 and IQGAP2 where IQGAP2 exhibits 62% homology with IQGAP 1. The two isoforms also show differences in their mRNA expression (Brill et al., 1996). IQGAP 1 is postulated to serve as a possible effector for Cdc42 and Rac mediated cytoskeletal reorganisation, since IQGAP 1 contains a potential actin-binding domain and was found to accumulate at insulin- or RacVI2 induced membrane ruffles (Kuroda et al., 1996). In addition IQGAP 1 was found in an actin cytoskeleton complex with GTP-bound Cdc42 in vivo, which was shown also to be stimulated with EGF treatment o f COS-7 cells (Erickson et al., 1997). The IQGAP 1: Cdc42 complex formation was shown to be disrupted by calmodulin and calcium in vitro and is postulated to provide a means o f regulation (Joyal et al., 1997). IQGAP 1
was also reported to co-sediment with F-actin for which the CHD o f IQGAP 1 is required. IQGAP 1 and exhibits F-actin cross-linking activity that is enhanced by GTP- bound Cdc42 and regulated in part by calmodulin (Fukata et al., 1997). The yeast homologue o f IQGAP is reported to be involved in cytokinesis and actin recruitment in budding (Machesky, 1998). Another report by Kuroda et al shows that IQGAP also binds directly to the adhesion proteins E-cadherin and p-catenin at cell-cell contacts and appears to be required for the formation o f Rac and Cdc42 induced cell-cell contacts (Kuroda et al., 1998).
1.6.1.11: Borg family
More recently a new family o f Cdc42 and TCIO interacting proteins have been identified. The Borg (binders o f Rho GTPases) family consists o f five members, Borg 1-5. These proteins were identified from a two-hybrid screen using TCIO as bait. All members exhibit sequence conservation in three regions referred to as Borg homology domains. They also encode the conserved CRIB binding motif. It is not clear what role these proteins play. Borg3 is reported to inhibit Cdc42-mediated JNK activation. Borg3 and Borgl are also speculated to be involved cell spreading events (Joberty et al.,
1999).
Borg5, also known as MSE55 is reported to have effector fimction for Cdc42- mediated reorganisation o f the actin cytoskeleton. MSE55 is shown to be localised to membrane ruffles in transfected COS-7 cells and is shown to induce long cellular extensions when overexpressed in NIH 3T3 cells (Burbelo et al., 1999).
1.6.2: Rho specific effectors
1.6.2.1: ROK
ROK, Rho-associated kinase was identified using GTP-labelled RhoA to screen a rat brain expression library (Leung et al., 1995, 1996). It was also identified by two other groups, giving rise to alternative nomenclature p i60 ROCK (Fujisawa et al., 1996; Ishizaki et al., 1996) and Rho-kinase (Matsui et al., 1996). ROK interacts specifically with GTP-bound RhoA (Leung et al., 1995; Ishizaki et al., 1996; Fujisawa et al., 1996; Matsui et al., 1996) and also with Rho members B and C but does not interact with Cdc42 or Rac (Leung et al., 1996). ROK contains a kinase domain at its C terminal that shares considerable homology to the myotonic dystrophy kinase, that is implicated in
the etiology o f neuromuscular degeneration (Brook et aî., 1992; Fu et al., 1992). Adjacent to the kinase domain lies a coiled-coil region. The Rho-binding domain is situated at the N terminal next to a cysteine/ histidine rich (CRD) region found within a PH domain (Leung et al., 1995; Ishizaki et al., 1996; Fujisawa et al., 1996; Matsui et a l, 1996).
Immunopreciptitation studies in Cos cells identified a physical and functional association between RhoA and p i60 ROCK resulting in stimulation o f kinase activity (Ishizaki et al., 1996). A RhoA-dependent membrane association o f ROK was also reported (Leung et a l, 1995; Matsui et a l, 1996) which was shown to be specifically too peripheral actin microfilaments in COS-7 cells and notably no association to Rho- induced stress fibres (Leung et a l, 1995). Two isoforms o f ROK have been identified, ROKa and ROKp. ROKp exhibits 64% overall identity with ROKa but both differ in their tissue distribution. ROKa expression in serum starved HeLa cells results in the formation o f stress fibres and focal adhesion complexes. These effects were shown to be dependent on kinase activity and the presence o f the extreme N terminal sequence. The expression o f a C-terminal truncated ROKa in cells grown in serum with pre-existing stress fibres resulted in enhanced stress fibre formation and focal adhesion formation. The cytoskeletal effects induced by ROKa were not inhibited by C3, suggesting that ROKa lies downstream o f RhoA. The increased cytoskeleton reorganising activity o f the C-terminal truncation constructs o f ROKa were shown to also display an increase in kinase activity, suggesting that the C-terminal sequences behave as negative regulator o f the kinase domain. Furthermore, the expression o f kinase-deficient ROKa and an N- terminal truncation o f ROKa in growing cells resulted in a loss o f stress fibres and focal adhesion complexes. This was shown to be accompanied with cell spreading, that was independent o f Racl activity but was dependent on the Rho binding domain, PH domain and CRD region (Leung et al., 1996).