RESUMO
Rho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid-binding regions, they also participate in protein-protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is fourfold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in engaging Rho GTPases directly for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Alinhamento de SequênciaRESUMO
Kazrin binds to periplakin and ARVCF catenin, and regulates adhesion and differentiation of cultured human keratinocytes. To explore kazrin function in vivo, we generated a kazrin gene-trap mouse in which only exons 1-4 were expressed, fused to ß-galactosidase. On transient transfection, the protein encoded by exons 1-4 did not enter the nucleus, but did cause keratinocyte shape changes. The mice had no obvious defects in skin development or homeostasis, and periplakin and desmoplakin localization was normal. Expression of the kazrin-ß-galactosidase fusion protein faithfully reported endogenous kazrin expression. Kazrin was not expressed in embryonic epidermis and was first detected at postnatal day 1. In adult mice, epidermal kazrin expression was less widespread than in humans and Xenopus, being confined to the bulb of anagen hair follicles, the infundibulum, and parakeratotic tail epidermis. In anagen bulbs, kazrin was expressed by a band of cells with elongated morphology and low desmoplakin levels, suggesting a role in morphogenetic cell movements. We conclude that exons 5-15 of kazrin, encoding the nuclear localization signal and C-terminal domain, are not required for epidermal development and function. The previously reported role of kazrin in regulating cell shape appears to reside within the N-terminal coiled-coil domain encoded by exons 1-4.