A common feature o f many o f the Rac and Cdc42 effector proteins the conserved p21 binding domain that was first identified by Manser et al, as a conserved sequence present in rat p65PAK, ACK and Ste20, the yeast PAK homologue (Manser et al., 1994). The binding sequence was described to span over 40 amino acid residues (Manser et al., 1994). Later a report by Burbelo et al redefined the motif to a minimal consensus binding sequence encompassing 16 amino acids. This motif has been called the Cdc42/Rac interactive binding (CRIB) motif (Burbelo et al., 1995) and has been identified in over 25 proteins. Many other Cdc42 and Rac binding partners have also been identified that do contain the conserved p21 binding domain including p67***‘°’‘, POR-1, pl40Sra-l, POSH, p35/Cdk5 and CIP4.
1.8.1.1: PAK-family kinases
Mammalian tissues contain at least four PAK isoforms (see table below). PAK homologues also exist in other organisms. For example the yeast protein Ste20 is involved in G-protein mediated pheromone MAPK signalling (Ramer, 1993) and the Drosophila homologue DPAK is involved in dorsal closure (Harden et al., 1996). PAK was first identified in a p21 overlay assay (Manser et al., 1994). It was shown to bind specifically to Cdc42 and Rac in a GTP-dependent maimer.
Binding to Cdc42 or Rac was reported to lead to PAK activation and subsequent PAK autophosphorylation (Manser et ah, 1994).
Rat Isoform Human isoform Size in kDa Tissue distribution
PAKa PAKl 68 Brain, muscle and
spleen
Manser et a/., 1994
PAKP PAK3 65 Brain-specific Manser et a/., 1995 PAKy PAK2 62 Ubiquitous Too et ah, 1995
PAK4 Prostrate and testis enriched
A b o e ta l, 1998
The study o f PAKs role in Rac and Cdc42-mediated pathways, has been approached using two different experimental angles. Either, by the direct expression o f PAK constructs (Manser et ah, 1997; Sells et ah, 1997: Zhao et al 1998) or by using Rac effector domain mutants that no longer bind PAK (Westwick et ah, 1996; Joneson et ah,
1996; Lamarche et ah, 1996).
Various PAK mutants have been generated that result in constitutively active PAK, kinase-deficient PAK, p21-binding deficient PAK and variations thereof. Expression o f these constructs have supported a role for PAK in the reorganisation of the actin cytoskeleton specifically in the formation o f lamellipodia and filopodia (Sells et ah, 1997). PAK also appears to antagonise Rho activity as PAK expression has been shown to induce the loss of stress fibres and cause focal complex turnover (Manser et ah, 1997, Zhao et ah, 1998). Similar effects have also been reported in PC12 cells where PAK has been shown to induce neurite extension (Daniels et ah, 1998). pPAK expression in PCI2 cells has been shown to induce a Rac phenotype, including membrane ruffling and lamellipodia formation at growth cones and long neurite shafts (Obermeier et ah, 1998). PAK is reported however to instead act upstream o f Rac through its interaction with PAK interacting nucleotide exchange factor (PIX), a Rac exchange factor also named Cool (cloned out o f hbrary) (Manser et ah, 1998; Bagrodia et ah, 1998). Another study investigating the localisation o f PAK, showed that upon stimulation with PDGF, PAKl is redistributed to dorsal and membrane ruffles and lamellipodia, colocalising with polymerised actin. Further supporting a role for PAK in the regulation o f Cdc42/Rac induced actin remodelling (Dharmawardhane et ah, 1997).
PAK is a multi-domain protein consisting o f a C-terminal kinase domain, four N- terminal pro line-rich sequences and a kinase inhibitory domain as well as a p21-binding domain. PAK has been shown to interact with SH3 domain containing proteins such as Nek adaptor protein and PIX (Bagrodia and Cerione, 1999). PAK is reported to mediate its effects on Cdc42/Rac-induced morphologies by its recruitment to Cdc42 and Rac driven focal complexes via PIX (Manser et n/.1998). More recently PAK has been reported to be associated with paxillin, a focal adhesion adaptor protein. Paxillin is reported to bind the PAK-PIX complex via a 95kDa ARF-GAP protein that contains ankyrin repeats (Turner et a l, 1998; Bagrodia et al., 1999).
1.6.1.2: n-Chimaerin
n-Chimaerin was first identified to display preferential GAP activity for Rac in vitro (Manser et al., 1992; Ahmed et al., 1993) which was also shown in vivo using a GAP domain construct o f n-chimaerin (Kozma et al., 1996). In contrast, microinjection o f full-length n-chimaerin induced lamellipodia and filopodia formation and a reduction in stress fibres in Swiss 3X3 cells suggesting an effector function for n-chimaerin for both Rac and Cdc42-induced F-actin reorganisation (Kozma et al., 1996). Rac and Cdc42 morphological phenotypes were also induced at the leading edge o f growth cones o f NlE-115 cells following n-chimaerin microinjection. The effects o f full-length n chimaerin in Swiss 3T3 cells were inhibited by RacN17, Cdc42N17, RhoGDI and phorbol ester treatment. Inhibition by dominant-negative Rac and Cdc42 suggests a requirement for active Rac and Cdc42 for n-Chimaerin induced effects. RhoGDI prevents the interaction o f GAPs with the Rho-p21 proteins, hence the inhibition by RhoGDI suggests that n-chimaerin interaction with Rac and Cdc42 is required, which is supported by the observation using a p21 binding mutant o f n-chimaerin that was defective in inducing the morphological effects o f full length n-Chimaerin. The inhibition by PMA is a result o f stimulation o f GAP activity and therefore downregulation o f Rac, but also downregulation o f Cdc42 as filopodia were also blocked following PMA treatment. n-Chimaerin was also shown to co-localise with F- actin, via its N-terminal and its expression resulted in the redistribution o f vincullin to the newly formed microspikes. These data suggest an effector role for n-Chimaerin in Rac and Cdc42 morphological pathways (Kozma et al., 1996).
1.6.1.3: pe?’’"*”'
p67”'°’‘ is a component o f the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The NADPH oxidase is a multi-component plasma membrane-associated enzyme found in phagocytes that is used to generate superoxide against microbial infections (Chanock et ah, 1994; Morel et al., 1991). Chronic granulomatous disease (CGD) is characterised by a failure o f phagocytes to produce superoxide (Cumutte. 1993). The other components include the transmembrane flavocytochrome b558, consisting o f the subunits, gp91^*‘°* and p22^^°^, and three regulatory cytosolic components P47****®*, p67^^°* and Racl or Rac2. Using a yeast two-hybrid approach p^yphox p^jphox shown to interact (Dorseuil et al., 1996).
p67?hox is a Rac specific target and binds Rac in a GTP dependent manner (Diekmann et al., 1994; Prigmore et al., 1995), but exhibits a preference for the Rac2 isoform (Dorseuil et al., 1996). The interaction between p67^'’°* and Racl is thought to be essential for translocation o f the cytosolic proteins, p47‘’*^°’^ and p67*’*’°* to the plasma membrane and subsequent activation of the NADPH oxidase (Leusen et al., 1996). p67Phox does not contain the consensus p21-binding domain nor does its Rac binding domain exhibit similarity to other non-CRIB Rac binding domains found in PORI or pMOSral. p67P*'°* contains two SH3 domains and a poly-proline rich sequence adjacent to its Rac binding domain (Ahmed et al., 1998).
p6 7?^ox is reported to be phosphorylated in vivo (Dusi et al., 1993). p67P*“”‘
phosphorylation was shown in vitro to be mediated by PAK (Ahmed et al., 1998). p^yPhox uji(ie2-goes phosphorylation at serine and threonine residues situated close to the
Rac binding domain. The interaction between PAK and p67^^°* is proposed to be a possible means o f regulation o f the NADPH oxidase (Ahmed et al., 1998).
1.6.1.4: ACK and ACK2
ACK (Activated Cdc42Hs associated kinase) is a non-receptor tyrosine kinase that binds specifically to Cdc42Hs-GTP (Manser et al., 1993). ACK2 is a structural variant o f ACK that lacks 344 residues within the carboxy-terminal tail (Yang and Cerione, 1997). Both ACK and ACK2 are brain-enriched proteins that exhibit similarity with the kinase domain o f tyrosine kinases such as FAK and PYK2. The exact fimction o f these proteins has not been clarified. Data suggests however a role for these kinases in
modulating cell adhesion. ACK2 phosphorylation has been reported to be increased by cell attachment and stimulation by EGF and bradykinin (Yang and Cerione, 1997). More recently ACK has been associated with the cell surface antigen Melanoma chondroitin sulphate proteoglycan (MCSP) that has been implicated in tumour growth and invasion. A signalling pathway involving ACK, Cdc42Hs and pl30^^, an adaptor protein, is proposed to enhance integrin-mediated melanoma cell spreading (Eisenmann et al., 1999)
1.6.1.5: MRCK
The Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) family consists o f two members MRCKa and MRCKp. The MRCKs were identified from a human brain cDNA library using expression screening with radio labelled GTP-bound Cdc42. They also exhibit weak binding with Racl-GTP but not to Rho-GTP. The MRCK proteins encode the conserved p21-binding domain. The protein domain structure o f MRCKs domain shows considerably homology to both myotonic dystrophy kinase and the Rho effector, ROK. They all contain a kinase domain, coiled-coil a-helix region, a cysteine rich region and PH domain (Leung et al., 1998).
MRCK is reported to be an effector for Cdc42Hs-induced cytoskeletal changes. Co-transfection o f MRCK and Cdc42 V12 has been shown to result in a Cdc42 type morphology in HeLa cells where MRCK is reported to colocahse with Cdc42 at cell cell junctions and Cdc42-induced surface protrusions. The localisation o f MRCK and the resulting morphological changes are dependent on the PH domain. Cdc42V12- mediated microspike and focal complex formation were blocked when kinase dead MRCK was injected prior to Cdc42Hs injection. MRCK co-injection with limited concentrations o f Cdc42, that alone is unable to induce a visible phenotype, resulted in enhanced formation o f cellular extensions and localisation o f MRCK to cortical regions. These effects were not observed with either a kinase dead or p21-binding deficient construct suggesting a requirement o f these domains (Leung et al., 1998). MRCKa has also been implicated down stream o f Cdc42Hs/ Rac in NGF-induced neurite outgrowth in PC 12 cells. The kinase activity and the C-terminal o f MRCKa that encodes the cysteine-rich/PH domain and a citron homology region in MRCKa were shown to be required for neurite outgrowth (Chen et al., 1999).
1.6.1.6: WASP-related proteins
WASP, N-WASP and WAVE are related sequences that share considerable protein domain homology. Two domains have been identified, WH 1 and WH2 (WASP homology) that share homology with vasodilator-stimulated phosphoprotein (VASP) and verprolin respectively (Symons et al., 1996). VASP and verprolin have been reported to be involved in the organisation o f the actin cytoskeleton (Haffiier et al., 1995). The conserved domains include a PH domain, CRIB motif, verprolin (WH2) and a cofilin domain. WAVE shares structural similarity to WASP and N-WASP however WAVE does not encode a p21-binding domain CRIB motif.
1.6.1.6.1: WASP
The WASP (Wiskott-Aldrich syndrome protein) gene is mutated in WAS patients and is linked to a recessive disorder characterised by thrombocytopenia (Derry et al., 1994, 1995). Interestingly, the cellular defects o f WAS patients include, cytoskeletal abnormalities o f T cells and platelets (Molina et al., 1992), failure o f B cells to respond to polysaccharide antigens and defective chemotaxis in neutrophils (Ochs et al., 1980).
WASP interaction with Cdc42Hs was reported by three groups (Aspenstrom et al., 1996); Symons et al., 1996; Kolluri et al., 1996). The interaction was shown to be mediated by an N-terminal p21 binding domain (Symons et al., 1996; Kolluri et al., 1996). A weak interaction with Racl is also observed. WASP also contains a polyproline rich domain that binds the third SH3 domain o f the adaptor molecule. Nek (Rivero-Lezcano et al., 1995). Expression o f WASP results in the reorganisation o f the actin cytoskeleton causing stress fibre reduction and the formation o f F-actin clusters. Cluster formation was also dependent on Cdc42 activity, as clustering could be inhibited with Cdc42Hs N17 but not with dominant-negative Rac or Rho. The WH2 domain was also able to block clustering activity. WASP-induced cluster formation was shown to be an active actin polymerisation process as it was inhibited with the actin polymerisation inhibitor, cytochalasin D (Symons et al., 1996). WASP effects may be mediated by the proline-rich WASP interacting protein WIP, that contains actin and profilin binding sites (Ramesh et al., 1997). WIP also binds to the adaptor protein Nek via its second SH3 domain that is distinct fi*om the SH3 binding interaction with WASP (Anton et a l, 1998).
1.6.1.6.2: N-WASP
N-WASP was first identified through its binding to the SH3 domains o f Ash/GRB2 (Miki et al., 1996). Overexpression o f N-WASP is reported to cause surface protrusions in COS-7 cells where there is a co-localisation o f N-WASP and F-actin. N-WASP is also reported to induce microspike formation in EGF treated cells (Miki et al., 1998a). Miki et al also show that N-WASP can induce extremely long actin microspikes when co-expressed with Cdc42V12 implicating a role for N-WASP in Cdc42- induced filopodia formation (Miki et al., 1998a). The actin monomer binding protein, profilin was shown to associate with N-WASP in vivo and in vitro at proline-rich sites. The association between N-WASP and profilin was shown to be required for efficient N- WASP-induced microspike elongation. Microspike formation was shown to require profilin, N-WASP and activated Cdc42, in an actin polymerisation reconstitution assay (Suetsugu aA, 1999).
N-WASP has also been reported to co-operate with the Arp2/3 complex in actin polymerisation. Actin polymerisation activity was shown to be enhanced with Cdc42 and phophatidylinositol 4,5 bisphosphate (PIP2) (Rohatgi et al., 1999).
1.6.1.6.3: WAVE
WAVE (WASP family Verprolin-homologous protein) was identified following a database search with the conserved VPH domain (Miki et al., 1998b). WAVE is reported to induce actin reorganisation downstream o f Rac. However a direct binding interaction between Rac and WAVE is not reported. Like WASP, WAVE has been reported to be involved in the polymerisation o f actin into clusters. Profilin is shown to be required for WAVE-induced cluster formation. WAVE is most abundant in cell lines o f neuronal origin. Endogenous WAVE in NlE-115 neuroblastoma cells is shown to localise to areas o f RacV 12-induced membrane ruffling, where WAVE is described to re localise fi*om the cytoplasm to the plasma membrane. In addition WAVE is proposed to be specifically involved in RacV 12-induced membrane ruffle formation. Co expression o f a WAVE VPH deletion mutant reported to specifically block Rac-induced membrane ruffle formation in Cos 7 cells and partially in N lE -115 neuroblastoma cells. PDGF-induced ruffling in Swiss 3T3 cells could also be blocked by the WAVE VPH deletion mutant. Neurite formation induced by serum starvation is also blocked by the WAVE VPH deletion mutant resulting increased cell flattening (Miki et al., 1998b).