All ETDs from UAB

Advisory Committee Chair

Stuart J Frank

Advisory Committee Members

Joseph L Messina

Robert W Hardy

Chenbei Chang

Andra R Frost

Document Type

Dissertation

Date of Award

2008

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

EGF receptor (EGFR) is a receptor tyrosine kinase, mediating cell growth from ectodermal and mesodermal origin. The overexpression and aberrant function of EGFR are involved in a wide range of human carcinomas. Growth hormone receptor (GHR) coexists with EGFR in various cell types and implicated in malignant tumor behavior. Previously in our lab, we found that via ERK activation GH can threonine phosphorylate EGFR and reduce EGF-induced EGFR downregulation. GH also synergizes with EGF in activating ERK. In this project, using reconstitution, we studied the mutation of the ERK phosphorylation consensus residue on EGFR, 669T. CHO-GHR cells, which lack EGFR and express GHR, were stably transfected to express human wild-type or T669A EGFR. GH or EGF caused phosphorylation of WT, but not T669A EGFR indicating that 669T is required for this phosphorylation. Notably, EGF induced more rapid downregulation of EGFR in cells expressing EGFR T669A or WT EGFR with inhibited ERK activation, suggesting 669T phosphorylation serves as a brake in EGF-induced receptor downregulation. In signaling experiments, EGFR T669A displayed enhanced acute EGFR tyrosine phosphorylation, suggesting that 669T phosphorylation negatively modulates EGF-induced EGFR kinase activity. Similar findings were observed in a human fibrosarcoma cell line that harbors an activating Ras mutation that results in constitutive ERK activation. Collectively, these data indicate that ERK activation mediates threonine phosphorylation in the EGFR and modulates EGFR signaling. Then ii we study the mechanism of signaling synergy between GH and EGF. We found that the signaling synergy existed specifically in MEK/ERK pathway, and at the level between Raf-1 and MEK. Previous reports suggest that KSR (Kinase Suppressor of Ras) is a major molecule modulating growth factor-induced ERK activation at Raf-1/MEK level; hence we examined KSR activation by detecting phosphorylation on serine 392. Notably, GH and EGF synergize in KSR activation, suggesting that KSR might be involved in the signaling synergy between GH and EGF. This study help us better understand GH’s effect on EGF signaling and EGF receptor behavior.

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