Regulation of protein interaction domains is required for cellular signaling dynamics. Here, we show that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function as mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation.

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Cdc42 recruits Par-6–aPKC to establish cell polarity from worms to mammals. Although Cdc42 is reported to have no function in Drosophila neuroblasts, a model for cell polarity and asymmetric cell division, we show that Cdc42 colocalizes with Par-6–aPKC at the apical cortex in a Bazooka-dependent manner, and is required for Par-6–aPKC localization. Loss of Cdc42 disrupts neuroblast polarity: cdc42 mutant neuroblasts have cytoplasmic Par-6–aPKC, and this phenotype is mimicked by neuroblast-specific expression of a dominant-negative Cdc42 protein or a Par-6 protein that lacks Cdc42-binding ability. Conversely, expression of constitutively active Cdc42 leads to ectopic Par-6–aPKC localization and corresponding cell polarity defects. Bazooka remains apically enriched in cdc42 mutants. Robust Cdc42 localization requires Par-6, indicating the presence of feedback in this pathway. In addition to regulating Par-6–aPKC localization, Cdc42 increases aPKC activity by relieving Par-6 inhibition. We conclude that Cdc42 regulates aPKC localization and activity downstream of Bazooka, thereby directing neuroblast cell polarity and asymmetric cell division.

Summary Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing—Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds—occurs in single cells (1,2) and in epithelia (3-10). Here we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions as well as around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single cell wound response (2,11), also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.

SummaryPDZ protein interaction domains are typically selective for C-terminal ligands, however, non-Cterminal, "internal" ligands have also been identified. The PDZ domain from the cell polarity protein Par-6 binds C-terminal ligands and an internal sequence from the protein Pals1/Stardust. The structure of the Pals1-Par-6 PDZ complex reveals that the PDZ ligand-binding site is deformed to allow for internal binding. While binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ regulates binding of C-terminal ligands, the conformational change that occurs upon binding of Pals1 renders its binding independent of Cdc42. These results suggest a mechanism by which the requirement for a C-terminus can be readily bypassed by PDZ ligands and reveal a complex set of cooperative and competitive interactions in Par-6 that are likely to be important for cell polarity regulation.Protein interaction domains form the backbone of cellular information flow 1 . The PDZ protein interaction domain participates in a wide variety of signaling pathways and is one of the most common in metazoan genomes 2 . Because they often occur in multiple instances in the same polypeptide, PDZ domains are thought to serve an organizational role in signal transduction pathways 3 . Given the large number of PDZ domains, several modes of ligand recognition exist whose mechanisms are still being elucidated.PDZ domains bind to short sequences of five to seven residues in their target proteins 4,5 . Although these recognition sequences have a low information content, specificity is typically enhanced by the requirement that the sequence occurs at the C-terminus. C-terminal recognition is found to fall into several different classes, depending on the identity of critical binding residues 3 . For example, class I PDZ ligands have a consensus sequence of -S-X-V-COOH.The requirement for a C-terminus results from a steric rather than electrostatic mechanism. The peptide-binding pocket is constructed such that residues that extend past the C-terminus clash with a conserved PDZ segment known as the carboxylate-binding loop 3,4 . Although Cterminal ligands possess a negatively charged carboxylate, studies of salt effects on the binding reaction suggest that electrostatic contributions are negligible 6 . Because of the additional specificity provided by the C-terminus, enforcement of C-terminal binding is an important component of PDZ -ligand recognition.While recognition of C-terminal motifs appears to be the dominant mode of PDZ-ligand interaction, non-C-terminal (also known as "internal"), PDZ ligands also exist. Compared to our understanding of C-terminal PDZ ligands, however, the mechanism of internal ligand recognition is much less clear. The best-characterized internal PDZ interactions involve ligands that adopt a specific conformation that adheres to the steric requirements of the PDZ binding Correspondence should be addressed to K.E.P. (prehoda@molbio.uoregon.edu) . pocket. For example, in the hetero-oligomerization of the nNOS and sy...