Subcellular Localization of Multiple PREP2 Isoforms Is Regulated by Actin, Tubulin, and Nuclear Export

Abstract

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.Patterning of the embryonic antero-posterior axis comes under the control of HOX and TALE 1 class homeoproteins (1).Members of the TALE class are characterized by sequence relatedness and by a three amino acid loop extension between helices one and two of the homeodomain (2). Families within the TALE group include PBC, MEIS, and PREP. Among the PBC family members, the fly extradenticle and vertebrate PBX proteins have been most intensively studied. PBC proteins bind DNA cooperatively with many but not all HOX proteins. Additionally, they form stable DNA-binding heterodimers with MEIS and PREP family members (2). The ability of PBC proteins to interact with both HOX and MEIS/PREP partners results in DNA-bound heterotrimeric complexes that have been implicated in gene control in vitro and in vivo (3-8). However, the instances of HOX-independent gene control by TALE heterodimers have also been documented (9, 10), consistent with the presence of these proteins in tissues devoid of Hox gene expression. In addition to the homeodomain, two conserved N-terminal domains, PBC-A and PBC-B, have been defined in PBC family proteins (11). These regions are implicated in numerous processes including nuclear export, transcriptional repression, MEIS/PREP interaction, and binding to non-muscle myosin heavy chain II B (2). Verte...

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