Signal Transduction by the Epidermal Growth Factor Receptor: a Dissertation

Abstract

The experimental data included in this thesis examines two events involved in signal transduction by the Epidermal Growth Factor Receptor. The first event (receptor oligomerization) occurs at the cell membrane and is proposed to be involved in activating the tyrosine protein kinase activity of the EGF receptor. Activation of the tyrosine protein kinase is an initial step in signal transduction by the EGF receptor. The second event examined (activation of an EGF stimulated serine/threonine protein kinase activity) occurs in the cytosol and may potentially be involved in final transmission of the EGF signal to the cell nucleus. The role of oligomerization in regulating the EGF receptor tyrosine protein kinase was examined by testing two hypotheses: 1) that PMA controls EGF receptor function by regulating the oligomeric state of the receptor and 2) that oligomerization is required to activate the EGF receptor tyrosine protein kinase. The oligomeric state of the EGF receptor was examined by chemical cross-linking and sucrose density gradient centrifugation analysis. It was determined that protein kinase C inhibition of the EGF receptor tyrosine protein kinase activity is independent of the oligomeric state of the receptor. It was also determined that the tyrosine protein kinase of the EGF receptor can be activated in the absence of receptor oligomerization. Threonine 669 is the major site of phosphorylation of the EGF receptor after treatment of cells with EGF. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. The kinetics of activation of this T669 kinase activity is rapid. Furthermore, it was demonstrated that EGF could activate the T669 kinase in the absence of detectable tyrosine kinase activity. Together, these data suggest that the T669 kinase has a role in intracellular signal transduction. Therefore, the T669 kinase was purified and characterized in order to help understand how EGF binding to its receptor at the cell membrane ultimately leads to signal transduction to the cell nucleus.