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

Abstract

HEF1, p130Cas , 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 G 2 /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.

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation

“…However, in the HCT-116 cell line, none of these proteins interacts with HEF1, which suggests that HEF1 degradation is not mediated through AIP-4, Smad3 or CDH1 (data not shown). In addition, besides its degradation through the proteasome, Law et al showed that upon TNFα stimulation in MCF-7 cells, HEF1 can be processed in a caspase-dependent manner [20,40]. Although the ser-369 is very close to the D 363 LVD 367 consensus caspase cleavage site, we did not observe a stabilization of this HEF1 isoform with addition of caspase inhibitor (data not shown), indicating that HEF1 clearance does not occur in a caspase dependent manner.…”

“…1). This two bands profile (p105 & p115) is a product of 2 different ser/thr phosphorylation states of HEF1, previously described in the litterature [20,21]. We then decided to examine the significance of this difference.…”

“…However, HEF1 is also phosphorylated on serine and threonine residues; indeed, HEF1 appears as a doublet: p105 and p115 (105 and 115 kDa) which mirrors two different phosphorylation states of the protein [20,21]. In this study we investigated the ser/thr phosphorylation of HEF1 in cells.…”

“…Phosphorylation of HEF1 on serine/threonine residues and appearance of the p115 isoform have been described as a result of cell adhesion [20,21]. However, the serine/threonine kinase(s) responsible for this HEF1 phosphorylation have not been identified in most cases.…”

The substrate domain of BCAR1 is essential for anti-estrogen-resistant proliferation of human breast cancer cells

“…HEF1 was barely detectable in the parental cells (not shown), while transfectants carrying the HEF1 expression construct carried two proteins (Fig. 2b) most likely related to differential phosphorylation [44].…”