The scaffolding protein, hematopoietic PBX-interacting protein (HPIP/PBXIP1), regulates cell migration necessary for cancer cell dissemination. However, the mechanism that governs this process remains unknown. We show here that HPIP expression is associated with stages of breast cancer where cell dissemination results in poor patient outcome. Our investigation finds a novel association of HPIP with focal adhesion kinase (FAK) regulating FA dynamics. Interestingly, this interaction that led to activation of FAK protein was mediated by the C-terminal domain of HPIP and not the typical integrin-binding motif. Further, short hairpin RNA-mediated knockdown of FAK expression significantly reduced HPIP-induced cell migration indicating participation of FAK pathway. Live-cell time-lapse imaging and biochemical analysis further established the role of HPIP in microtubule-induced FA disassembly. We also found that HPIP-mediated MAPK activation led to phosphorylation and subsequent activation of calpain2, and the activated calpain2 in turn proteolyses FA protein, talin. Interestingly, HPIP is also proteolysed by calpain2 in breast cancer cells. The proteolysis of HPIP and talin by calpain2, and the activation of calapin2 by HPIP-mediated MAPK phosphorylation, is a novel regulatory axis to modulate the cell migration signal. Together, we have determined HPIP as a novel activator of FAK and a new substrate of calpain2. These molecular interactions between HPIP and FAK, and HPIP and calpain2 regulate cell adhesion and migration through modulation of FA dynamics.

Edited by Xiao-Fan WangProper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G 2 /M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20 -mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G 2 /M transition and thereby controls mitosis and cell division.