RESUMEN
The PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Here we report that murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. We further show that cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. We suggest that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems.
Asunto(s)
Actinas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/química , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Tubulina (Proteína)/metabolismo , Actinas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Antibióticos Antineoplásicos/farmacología , Secuencia de Bases , Northern Blotting , Western Blotting , Células COS , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Citoesqueleto/metabolismo , ADN/química , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos/química , Ácidos Grasos Insaturados/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Células 3T3 NIH , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , Transfección , Tubulina (Proteína)/químicaRESUMEN
We describe Prep2, a new murine homeobox-containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain-containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes.
