ErbB4 signal transduction and subcellular localization during prostate tumor suppression

Richard Michael Gallo, Purdue University

Abstract

Whereas three members of the ErbB family of receptor tyrosine kinases (EGFR, ErbB2, and ErbB3) have been well characterized as oncogenes, the role of ErbB4 in tumorigenesis has not yet been well defined. We hypothesize that ErbB4 functions as a tumor suppressor. We have generated constitutively active ErbB4 mutants to study the effects of ErbB4 signaling in human prostate tumor cell lines. We found that the ErbB4 Q646C mutant inhibits the colony formation on plastic of several prostate tumor cell lines. We showed that kinase activity is required for this tumor suppressor activity. The ErbB4 Q646C CT-b isoform fails to inhibit colony formation, indicating that tumor suppressor activity is isoform dependent. The putative ErbB4 phosphorylation site at Tyr1056 lies within the 16 amino acid region missing in the CT-b isoform, suggesting that this phosphorylation site might be critical for the tumor suppressor activity of ErbB4 Q646C. Indeed, substitution of a phenylalanine residue for Tyr1056 abolishes the tumor suppressor activity of ErbB4 Q646C, whereas substitution of a phosphomimic glutamate residue for Tyr1056 rescues the tumor suppressor activity of the ErbB4 Q646C mutant lacking tyrosine kinase activity. We have also developed and validated a modified EGFP tagged ErbB4 Q646C mutant that we subsequently used to evaluate the subcellular localization of various mutants in prostate cancer cells. The V673I mutation, which is predicted to disrupt ErbB4 cleavage and affects trafficking of the ErbB4 cytoplasmic domain, disrupts tumor suppressor activity of the ErbB4 Q646C mutant. Likewise, mutations that abrogate ErbB4 kinase activity and phosphorylation of Tyr1056 also affect trafficking of the ErbB4 cytoplasmic domain. In contrast, some mutations that disrupt an ErbB4 LXXLL motif or an ErbB4 BH3 motif affect ErbB4 Q646C tumor suppressor activity, but not trafficking of the ErbB4 cytoplasmic domain. Thus, ErbB4 tumor suppressor activity appears to be dependent on proper trafficking of the ErbB4 cytoplasmic domain and on interactions of the ErbB4 LXXLL and BH3 motifs with signaling effector proteins.

Degree

Ph.D.

Advisors

Riese, Purdue University.

Subject Area

Molecular biology|Cellular biology|Medicine

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