Structure and functional domains of Cdc42p

Cdc42p belongs to the Rho subfamily of the Ras superfamily of small GTPases. Together with the other Rho-type GTPases belonging to the same class (Rho1-4p), Cdc42p plays an essential role in regulating the signal transduction pathways that control the generation and maintenance of cell polarity. The major functions of Cdc42p seem to be in regulating the rearrangements of the actin cytoskeleton in response to extracellular and intracellular

signals as well as in modulating protein kinase cascades that result in the transcriptional activation of genes required for growth control and other processes. The Cdc42p GTPase was initially identified from a S. cerevisiae mutant strain carrying a temperature-sensitive (ts) mutation, cdc42-1^ts, that affected bud formation at the restrictive temperature and resulted in formation of greatly enlarged, unbudded cells (Adams et al., 1990). Staining of those mutants with rhodamine phalloidin showed that the cells had severe defects in polarized organization of the actin cytoskeleton, whereas isotropic growth was not affected. Due to its significant role in cell polarization, S. cerevisiae cdc42 null mutants are inviable (Johnson and Pringle, 1990). Structural and functional Cdc42p homologs have been identified in a large number of eukaryotes (Johnson, 1999), and the high degree of identity (80 to 95%) in the predicted amino acid sequences highlights the conservation and significance of Cdc42p proteins among different eukaryotic organisms (Fig. 7). The similarity of Cdc42p to other GTPases of the Ras superfamily is only ~40%, but it is clustered in several important domains. Mutational analysis of Cdc42p and determination of the solution structure of Cdc42 from Homo sapiens (Cdc42Hs) by nuclear magnetic resonance (NMR) spectroscopy (Feltham et al., 1997), along with the determination of the X-ray crystal structure of Cdc42Hs in complex with various regulatory or effector proteins (Garrard et al., 2003; Hoffman et al., 2000; Nassar et al., 1998; Rossman et al., 2002) have greatly aided in defining functional domains within Cdc42p (Fig. 7). Four domains are implicated in binding and hydrolysis of GTP, and their structure is highly conserved among GTPases of the different subfamilies. Two cdc42 point mutations analyzed early in S. cerevisiae (Ziman et al., 1991) were mapped to these sites, cdc42^G12V and cdc42^Q61L. They are both lethal to yeast because they reduce the intrinsic GTPase activity, thereby shifting the mutant protein to an activated, GTP-bound form which constitutively interacts with downstream effectors. According to current knowledge, GTPases bind to GEFs when in the nucleotide-free or GDP-bound state and bind to GAPs and downstream effectors when in the GTP-bound state. Two domains of Cdc42p called switch I (also referred to as effector domain) and switch II undergo the most significant conformational changes upon binding the different guanine nucleotides, suggesting that these regions are important specificity determinants required for differential interaction with downstream effectors and regulatory proteins. This hypothesis has been corroborated by the analysis of interaction of many different point-mutated Cdc42p variants with downstream effectors (Davis et al., 1998; Mösch et al., 2001; Richman and Johnson, 2000; Richman et al., 1999). However,

mutations in the switch I domain not only affect binding to downstream effectors of Cdc42p, but also interaction with the GEF of Cdc42p, Cdc24p (Davis et al., 1998).

Another important domain of Cdc42p is the Rho insert domain, which is unique to members of the Rho subfamily of Ras GTPases. This domain mediates interaction with one of the Cdc42p effectors, Iqg1 (Osman and Cerione, 1998). Moreover, the Rho-insert domain is, at least in the human homologue, important for interaction with its GDI (Wu et al., 1997b).

Cdc42Sc MQTLKCVVVGDGAVGKTCLLISYTTNQFPADYVPTVFDNYAVTVMIGDEPYTLGLFDTAGQEDY ||| |||||||||||||||||||||| || |||||||||||||||| ||||||||||||||||

Cdc42Hs MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDY

Cdc42Sc DRLRPLSYPSTDVFLVCFSVISPPSFENVKEKWFPEVHHHCPGVPCLVVGTQIDLRDDKVIIEK ||||||||| |||||||||| || ||||||||| || |||| | | |||||||||| |||

Cdc42Hs DRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHHCPKTPFLLVGTQIDLRDDPSTIEK

Cdc42Sc LQRQRLRPITSEQGSRLARELKAVKYVECSALTQRGLKNVFDEAIVAALEPPVIKKSKKCTIL | |||| | ||| ||||||||||||||||||||||||| |||||| || | Cdc42Hs LAKNKQKPITPETAEKLARDLKAVKYVECSALTQRGLKNVFDEAILAALEPPETQPKRKCCIF

GTP/GDP Switch I GTP/GDP

GTP/GDP

Switch II Rho

GTP/GDP Insert

Fig. 7. Sequence alignment of Cdc42p from S. cerevisiae (Cdc42Sc) and from human (Cdc42Hs). Vertical

lines indicate identical residues. Known GTP-binding/hydrolysis domains (GTP/GDP), switch I and switch II domains, and the Rho-insert domain are underlined.

With two exceptions, all Cdc42p proteins identified so far contain the C-terminal Cys-Xaa-Xaa-Leu sequence. This conserved domain is necessary for proper membrane anchorage.

Therefore, Cdc42p is prenylated with a C₂₀ geranylgeranyl isoprene group at its C-terminal Cys residue (Cys¹⁸⁸). This modification is dependent on geranylgeranyltransferase β-subunit encoded by CDC43 (Finegold et al., 1991). Membrane anchorage of Cdc42p after geranylgeranylation on Cys¹⁸⁸ is thought to be followed by proteolytic cleavage of the last three amino acids and carboxyl methylation of the now C-terminal Cys residue. A second localization determinant is a polylysine region found next to the Cys¹⁸⁸ residue. This

positively charged stretch might interact with negatively charged components of the membrane. Cdc42p is present in two cellular pools in yeast (Miller and Johnson, 1994;

Ziman et al., 1993). The majority of Cdc42p was found in the particulate fraction, but up to

~20% were found in a soluble pool. This fraction is supposed to be either nonprenylated or complexed with the Rho-GDI Rdi1p (Koch et al., 1997; Masuda et al., 1994).

Immunofluorescence and immunoelectron microscopy studies revealed that Cdc42p localized to the plasma membrane at sites of polarized growth, i.e. to the tips of growing buds and of mating projections in pheromone-treated cells (Ziman et al., 1991; Ziman et al., 1993). Moreover, a GFP-Cdc42p fusion also localized to internal (vacuolar, nuclear) membranes and to the mother-bud neck region, suggesting that Cdc42p also plays a role in cytokinesis and/or septation (Richman et al., 2002).