Human Enhancer of Filamentation 1, a Novel p130^cas-Like Docking Protein, Associates with Focal Adhesion Kinase and Induces Pseudohyphal Growth in Saccharomyces cerevisiae

Abstract

Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domaincontaining protein that is similar in structure to p130 cas , a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130 cas , the expression of HEF1 appears to be tissue specific. Further, whereas p130 cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130 cas , as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130 cas , transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.

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

“…The C-terminal parts of BCAR1 and HEF1 exhibit extensive sequence homology (54% identity). This region of HEF1 contains a four helix bundle domain [47] and was shown to be important for filamentation of fungi [48], and both BCAR1 and HEF1 C-termini bind to BCAR3, another gene recovered from our functional screen for anti-estrogen resistance and predictive for tamoxifen resistance in breast cancer patients [38,42,47,49]. Furthermore, p85 PI3 K was shown to bind to the C-terminus of p130Cas and to contribute to the growth of v-crk transformed cells [45].…”

Role of the adapter protein CRKL in signal transduction of normal hematopoietic and BCR/ABL-transformed cells

“…29,30 CAS (for Crk-associated substrate) was cloned as a major 130 kDa tyrosine phosphorylated protein in response to v-crk transformation. 27 Currently, there are three CAS-related molecules (the 130 kDa protein CAS, the 110 kDa protein HEF1/CAS-L, 67,68 and the 95 kDa protein SIN/EFS 69,70 ). The structure for CAS-family members includes an amino-terminal SH3 domain, multiple YXXP motifs (substrate domain), and potential SRC-binding sites at the Cterminus.…”

“…Expression of the HEF1 C-terminus induces pseudohyphae in S. cerevisiae. 67 Interestingly, in the human megakaryoblastic cell line MO7e, CRKL was induced to bind to only CBL, and in the T cell line H9 CRKL was found to bind to only HEF1 after ␤1 integrin cross-linking, even though both cell lines express HEF1 and CBL. 30 This provides for the possibility of an intracellular signaling 'switch' that could couple integrin signaling to different biological effects as related to CRKL.…”

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

“…HEF1 was first identified by complementation assay for its ability to induce pseudohyphal growth in S. Cerevisiae [2]. Later on, its role in intracellular signalling and regulation of cytoskeleton has been further characterized.…”