Characterization of the human cytoplasmic protein tyrosine phosphatases in breast cancer and the crystallographic analysis of an ENTH domain from yeast epsin Ent2

Gregory Thomas Costakes, Purdue University

Abstract

EphA2 is a receptor tyrosine kinase that is overexpressed in a number of highly metastatic cancers including breast and prostate. While non-transformed epithelial cells express low levels of EphA2 that quickly becomes hyper-phosphorylated and degraded at sites of cell-cell contact, EphA2 in transformed epithelial cells is overexpressed, hypo-phosphorylated, and localized in membrane ruffles where it cannot bind to its membrane bound ligand. The phosphorylation of EphA2 is closely regulated by the human cytoplasmic protein tyrosine phosphatase (HCPTP), which is also overexpressed in cancers overexpressing EphA2. Knockdown of overexpressed HCPTP in transformed cells that also overexpress EphA2 has been shown to increase EphA2 phosphorylation, reduce overall EphA2 levels, and reduce metastatic potential. However, studies to differentiate the roles of each HCPTP variant, HCPTP-A and HCPTP-B, have not been done. As both EphA2 and HCPTP are involved in cancer, a complete understanding of their interaction is required for the development of anti-cancer therapeutics against them. Our research aims to elucidate the structural details of their interaction using X-ray crystallography and identify the specific roles of each HCPTP variant in cell transformation through the use of novel variant specific HCPTP antibodies. We have shown that HCPTP-A is overexpressed in transformed MDA-MB-232 breast cancer cells compared to immortalized non-transformed MCF-10A cells, while HCPTP-B remains minimally expressed in each. HCPTP-A also appears to be dephosphorylated when cells become transformed. These results indicate that an overexpression of dephosphorylated HCPTP-A plays a key role in cell transformation and the future development of anti-cancer therapeutics should be targets towards HCPTP-A. Epsins are eukaryotic, endocytic adaptor proteins primarily involved in the early steps of clathrin mediated endocytosis. Two epsins exist in Saccharomyces cerevisiae, Ent1 and Ent2; with single epsin knockouts being viable, while the double knockout is not. These proteins contain a highly conserved Epsin N-terminal homology (ENTH) domain that is essential for cell viability. In addition, overexpression of the ENTH domain of Ent2 (ENTH2) was shown to play a role in cell division by interacting with the septin organizing, Cdc42 GTPase activating protein, Bem3, leading to increased cytokinesis failure. In contrast, overexpression of the ENTH domain of Ent1 (ENTH1) does not affect cytokinesis, despite being 75% identical to ENTH2. An ENTH2N112D, S114E, E118Q mutant that switches residues in loop 7 to those found correspondingly in ENTH1 was incapable of inducing the cytokinesis phenotype. In order to better understand the role of loop 7 in the ENTH2-induced phenotype at a molecular level, X-ray crystallography was used to elucidate the structures of yeast ENTH2WT and ENTH2DEQ. Our results indicate that mutations did not affect the conformation of loop 7, but rather introduce an increased negative charge on a potential interaction interface. Morphological analysis of cells overexpressing ENTH2 loop 7 mutants showed that the cytokinesis failure phenotype was abolished by the single mutants N112D, E118Q, and to a lesser extent by S114E. Taken together, our results indicate that the interaction surface that contains loop 7 and the specific nature of these residues are crucial for ENTH2 involvement in cytokinesis.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Molecular biology|Biophysics

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