Functional Cloning and Characterization of a Novel Nonhomeodomain Protein That Inhibits the Binding of PBX1-HOX Complexes to DNA

Abstract

PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes. A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomaincontaining protein that interacts with PBX1 as well as PBX2 and PBX3. RNA analysis revealed it to be expressed in CD34؉ hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues. The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins. This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus. The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences. Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences. Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX. Together these findings suggest that HPIP is a new regulator of PBX function.The PBC protein family (mammalian PBX, C. elegans CEH-20 and Drosophila extradenticle) make critical contributions to cell fate and segmental patterning during embryogenic development (1-3). PBX1, a member of the PBX family along with PBX2 and PBX3 (4), was initially identified as the chromosome 1 participant of the t(1;19) translocation, which occurs in 25% of pediatric pre-B cell acute lymphocytic leukemia and that creates a chimeric gene designated E2A-PBX1 (5, 6). The mechanism by which E2A-PBX1 causes leukemia is still unclear. However, the structure of the protein, in which the majority of PBX1, including the homeodomain, is fused to the transcriptional activation domain of E2A (6, 7), suggests that the oncogenic properties of E2A-PBX1 result from inappropriate regulation of target genes whose expression during hematopoiesis is normally regulated by wild type PBX proteins (8 -11).In vitro and in vivo data strongly suggest that PBX functions in combination with heterologous homeodomain proteins, including class I HOX proteins. As HOX cofactors, PBC proteins improve HOX specificity due to the increased size of the cooperative binding site and the strength of DNA binding, as well as by modulating recognition of cooperative binding sites by different groups of HOX proteins (12-14). In addition, cooperative DNA binding with PBC proteins may act to change the regulatory signal of HOX proteins, from repressors to activators (15). HOX-PBC interaction optimally requires the PBC homeodomain and a short carboxyl-terminal region, called the HOX cooperativity motif (16,17).Although PBC proteins contribute to HOX DNA binding specificity, PBC-HOX complexes exhibit little transcriptional activity and thus alone do not account ...

MDM2 restrains estrogen-mediated AKT activation by promoting TBK1-dependent HPIP degradation

“…8,9 AKT activation by estrogens requires the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). 10 Initially identified as a corepressor of pre-B-cell leukemia homeobox protein 1 (PBX1), 11 HPIP assembles a signaling complex that connects the p85 subunit of PI3K and ERa to microtubules in order to properly activate AKT. 10 Likewise, HPIP also promotes the growth and differentiation of hematopoietic cells through AKT.…”

Functional characterization of multiple domains involved in the subcellular localization of the hematopoietic Pbx interacting protein (HPIP)

“…As we have previously reported, a search for novel Pbx1 interacting proteins in the hematopoietic system resulted in the identification of the Hematopoietic Pbx Interacting Protein or HPIP (Abramovich et al, 2000). HPIP can bind to the different members of the mammalian Pbx family, inhibit the binding of Pbx1/Hox complex to DNA and block the transcriptional activity of E2A-Pbx (Abramovich et al, 2000).…”

“…Interestingly, both HPIP and Pbx1 have a restricted pattern of expression in hematopoietic cells, with highest expression in early progenitor cells, e.g. CD34 + (Abramovich et al, 2000), which suggests a role for HPIP-Pbx interaction during primitive stages of hematopoiesis. High-density oligonucleotide array analysis has further confirmed the expression of HPIP in CD34 + cells and revealed highest level of expression in CD34 + G-CSF mobilized peripheral blood cells compared with their bone marrow counterparts (Graf et al, 2001).…”

“…Our previous data suggest that HPIP activity involves, at least in part, the control of Pbx mediated gene expression (Abramovich et al, 2000). Pbx1 has been shown to constitutively localize to the cytosol by a mechanism that involves its export from the nucleus by the CRM1 receptor (Berthelsen et al, 1999;Abu-Shaar et al, 1999).…”

“…The presence of Pbx1 in the nucleus is mediated by its association with the homeobox-containing protein Meis1, and depends on the Pbx1 nuclear localization signal (NLS) (Berthelsen et al, 1999;Abu-Shaar et al, 1999). We have previously reported that HPIP localizes mainly to the cytosol (Abramovich et al, 2000). As the subcellular localization usually indicates the potential function of a protein, we set out to further study HPIP localization and to map its localization signals.…”