CELL-CYCLE-REGULATED PROCESSING OF HEF1 TO MULTIPLE PROTEIN FORMS DIFFERENTIALLY TARGETED TO MULTIPLE SUBCELLULAR COMPARTMENTS

HEF1, p130(Cas), and Efs/Sin constitute a family of multidomain dockin g proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferr ing association with focal adhesion kinase; a domain rich in SH2-bindi ng sites, phosphorylated by or associating with a number of oncoprotei ns, including Abl, Crk, Fyn, and others; and a highly conserved carbox y-terminal domain. In this report, we show that the HEF1 protein is pr ocessed in a complex manner, with transfection of a single cDNA result ing in the generation of at least four protein species, p115(HEF1), p1 05(HEF1), p65(HEF1), and p55(HEF1), We show that p115(HEF1) and p105(H EF1) are different phosphorylation states of the full-length HEF1. p55 (HEF1), however, encompasses only the amino-terminal end of the HEF1 c oding sequence and arises via cleavage of full-length HEF1 at a caspas e consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to d ate. In MCF-7 cells, we find that expression of the endogenous HEF1 pr oteins is cell cycle regulated, with p105(HEF1) and p115(HEF1) being r apidly upregulated upon induction of cell growth, whereas p55(HEF1) is produced specifically at mitosis. While p105(HEF1)and p115(HEF1) are predominantly cytoplasmic and localize to focal adhesions, p55(HEF1) u nexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 iden tifies the human homolog of the G(2)/M spindle-regulatory protein Dim1 p as a specific interactor with a region of HEF1 encompassed in p55(HE F1). In sum, these data suggest that HEF1 may directly connect morphol ogical control-related signals with cell cycle regulation and thus pla y a role in pathways leading to the progression of cancer.