This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The shape of a cell is determined primarily by its internal actin cytoskeletal architecture and influenced by focal adhesions, which constitute points of linkage between the extracellular matrix (ECM), the integrin receptor system and actin scaffolds. In response to external adhesive signals or internally generated cues, cells rearrange their actin cytoskeletal networks, thereby altering assembly of integrin-associated signal-transducing networks. Critical biological processes affected by the combined changes in cell shape and signal transduction include cell migration, proliferation, transformation and apoptosis, making the elucidation of the mechanisms involved of considerable interest. HEF1, and the related proteins p130Cas and Efs/Sin, define the Cas (Crk-associated substrate) family of focal adhesion-associated signaling proteins. Mechanistic studies of these proteins has defined their roles in the processes of cell adhesion and cell migration, and outlined a set of upstream inducers and downstream mediators of their function. However, our work has also demonstrated that HEF1 possesses additional unique activities relevant to apoptosis and cell division. These discrete activities derive from the function of processed forms of HEF1. A 28 kD carboxyterminal form induces cellular rounding and initiates p53-dependent death signaling under some growth circumstances, opposing a pro-survival activity of p130Cas. Intriguingly, a 55 kD aminoterminal HEF1 form arising in mitosis translocates to the mitotic spindle; and we have recently determined that altering the expression level and ratios between different forms of HEF1 so as to increase abundance of full length HEF1 versus the 55 kD form results in a failure of mitosis that involves development of multipolar spindles, failure of abscission at cytokinesis, and accumulation of cells at G1. This suite of defects is compatible with defects reported for proteins involved in functions at the centrosome, based on activities that include regulation of cyclin B stability, and licensing of abscission through centrosomal migration to the site of cytokinesis and delivery of proteins such as p160ROCK that control actomyosin ring contractile function. We have also determined that endogenous HEF1 localizes to the centrosome, and further shown that mis-expressed HEF1 affects cyclin B accumulation and activation of RhoA, a p160ROCK activator. Based on these and other results, we propose that HEF1 has a unique function as a regulator of cell cycle progression through mitosis and cytokinesis, and that this effect is separable from its roles in attachment-related survival signaling. Our goal is to determine whether HEF1 uses common or unique effectors for its functions in interphase versus mitotic cells.
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