Abstract
The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.
Highlights
The Rho family small GTP-binding proteins RhoA, Rac1, and Cdc42 have been known to regulate a variety of cell biological events involving actin cytoskeletal reorganization [1, 2]
Cdc42 is at least 50% identical in amino acid sequences to other Rho family members such as RhoA (Fig. 1) and is about 30% identical to Ras; it shares a similar overall three-dimensional folding with RhoA and Ras (Fig. 1, Ref. 39)
To examine the requirement of individual residues of the switch I and neighboring regions of Cdc42 for effector recognition in more detail, we have compared the ability of Cdc42 mutants made at five conserved residues in switch I and at seven nonconserved residues in switch I and immediate adjacent regions (Fig. 1) to bind to the p21-binding domain (PBD) of the implicated effectors, PAK1, Wiskott-Aldrich Syndrome protein (WASP), and IQGAP1
Summary
The Rho family small GTP-binding proteins RhoA, Rac1, and Cdc42 have been known to regulate a variety of cell biological events involving actin cytoskeletal reorganization [1, 2].
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