RESUMEN
Proteins with a bric-à-brac, tramtrack, broad-complex/Poxvirus zinc fingers (BTB/POZ) domain are implicated in a broad variety of biological processes, including DNA binding, regulation of gene transcription and organization of macromolecular structures. Kelch domain containing BTB/POZ proteins like Mayven and Keap1 display limited sequence similarity with the actin-fragmin kinase from Physarum, a protein kinase with a kelch domain. We show that mouse Keap1, a Caenorhabditis elegans protein that we named CKR, and human Mayven bind 5'-p-fluorosulfonyl-benzoyl-adenosine (FSBA), a covalently modifying ATP analogue. Binding with 2-azido-ATP or ATP-Sepharose is also demonstrated. In contrast to Mayven, FSBA binding by CKR and Keap1 was specifically inhibited by excess ATP. The ATP binding pocket is located in the N-terminal half of Keap1. Our findings indicate that several, but not all, BTB/POZ-kelch domain proteins possess an inconspicuous ATP binding cassette.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfato/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso , Proteínas Represoras/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , ADN Complementario/genética , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Physarum polycephalum/enzimología , Physarum polycephalum/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Homología de Secuencia de AminoácidoRESUMEN
We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.