Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments

ABSTRACT

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.

ACKNOWLEDGMENTS

This research was supported by National Cancer Institute/NIH grant R29-CA63366 (to E.A.G.) and core funds CA-06927 (to Fox Chase Cancer Center) and by American Cancer Society grant CB-74749 to E.A.G. Over the course of this study, S.F.L. was supported by NIH postdoctoral training grant T32 CA09035, American Cancer Society fellowship PF-4383, and NIH fellowship F32 GM18223 and Y.-Z.Z. was supported by NIH training grant T32 CA09035.

Ying Tong Wang and Joanne Estojak provided outstanding technical help on this project. We are grateful to Serge Manie and Arnie Freedman for cell lines and much helpful discussion; to Maggie Kasten, Chuck Clevenger, and Mary Ann Sells for cell lines; and to T. Gustafson for LexA fusion constructs used in specificity tests. We are very grateful to Jonathan Boyd for help with confocal microscopy. We thank Jonathan Chernoff, David Wiest, Sarah Fashena, and Tim Yen for incisive comments on the manuscript.