HEF1, p130^Cas, and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115^HEF1, p105^HEF1, p65^HEF1, and p55^HEF1. We show that p115^HEF1 and p105^HEF1 are different phosphorylation states of the full-length HEF1. p55^HEF1, however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase 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 date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105^HEF1 and p115^HEF1 being rapidly 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 unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G₂/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55^HEF1. In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following β1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130^Cas and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130^Cas and HEF1, phosphorylation of p130^Cas, but not of HEF1, was dependent on an intact autophosphorylation site (Tyr⁴⁰²) on RAFTK. To determine if RAFTK phosphorylated p130^Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr⁴⁰² mutant to phosphorylate purified HEF1 and p130^Cas domains. RAFTK was able to phosphorylate the substrate domains of both p130^Cas and HEF1, but only the C-terminal domain of p130^Cas. Furthermore, Tyr⁴⁰², which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130^Cas. These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130^Cas.