Functional characterization of multiple domains involved in the subcellular localization of the hematopoietic Pbx interacting protein (HPIP).
We have previously reported the cloning of the Hematopoietic Pbx Interacting Protein (HPIP), a novel protein discovered through its interaction with Pbx1. HPIP is expressed in early hematopoietic precursors, can bind all members of the Pbx family and can inhibit the transcriptional activation of the oncogene E2A-Pbx. To further understand the function of HPIP, we have analysed its cellular localization and characterized its functional localization domains. Using fluorescence microscopy to follow the distribution of different HPIP sequences fused to GFP, we found that HPIP localizes predominantly to cytoskeletal fibers but has the potential ability to shuttle between the nucleus and the cytosol. The cytoskeletal localization of HPIP is mediated by an N-terminal leucine rich region (between aa 190-218) and can be disrupted by the microtubule destabilizing drug vincristine. The HPIP C-terminal domain (aa 443-731) bears a nuclear export activity that is blocked by the CRM1 inhibitor Leptomycin B. In addition, we found two basic amino acid regions located between aa 485-505 and aa 695-720 that contain nuclear import activities attenuated by nuclear export. These observations support a model in which the constitutive attachment of HPIP to the cytoskeleton could be modified by changes in functional domains implicated in nuclear export, import and cytoskeleton binding sequences, allowing the molecule to shuttle between the nucleus and the cytosol.
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D056970:Co-Repressor Proteins; D003599:Cytoskeleton / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D056970:Co-Repressor Proteins; D019913:Nuclear Localization Signals; C413449:PBXIP1 protein, human; D014157:Transcription Factors