Publication Date

2014-05-09

Availability

Embargoed

Embargo Period

2016-05-08

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2014-02-18

First Committee Member

Ralf Landgraf

Second Committee Member

Feng Gong

Third Committee Member

Xiangxi Xu

Fourth Committee Member

Thomas K Harris

Abstract

The complex of ligand-binding deficient ERBB2 with kinase-dead ERBB3 represents the most potent signaling partner among the ERBB receptor family. The canonical model of receptor activation only provides an explanation for ERBB3 phosphorylation, leaving the ERBB2 part unresolved.Traditional therapeutic agents including monoclonal antibodies or kinase inhibitors against ERBB2 are effective in treatment, but their mechanism of action remains unknown. Meanwhile, patients often develop high levels of drug resistance over time. Since the process of ERBB2 signaling activation and transduction is elusive, we are interested in looking into the underlying molecular mechanisms, and thereby developing new medications in an effort to treat ERBB2-overexpressed breast cancer. Higher order complexes of ERBB receptors have been observed biophysically and offer a theoretical route for ERBB2 phosphorylation, but it is not clear whether such complexes provide functionality beyond the constituent dimers. In my first project, we show that a previously selected inhibitory RNA aptamer which targets the extracellular domain (ECD) of ERBB3 acts by sterically disrupting these higher order interactions. Ligand binding, heterodimerization, phosphorylation of ERBB3, and AKT signaling are only minimally affected, whereas ERBB2 phosphorylation and MAPK signaling are selectively inhibited. The mapping of the binding sites and the creation of aptamer-resistant point mutants are consistent with a model of side-by-side oriented heterodimers to facilitate proxy phosphorylation. Additional modes of signaling with relevance to pathological ERBB expression states emerge at high receptor levels. Hence, higher order complexes of nonoverexpressed ERBB receptors are an integral and qualitatively distinct part of normal ERBB2/ERBB3 signaling. Earlier literature has indicated Serine/Threonine phosphorylation at the carboxyl terminus of EGFR could promote receptor desensitization and reduce tyrosine kinase activity, which associates with maintaining the efficacy of chemotherapy-induced cell death. However, the details of serine/threonine phosphorylation profile at ERBB2 C-tail remain unknown. In the second project, I identified multiple serine/threonine phosphorylation sites by mass spectrometry at ERBB2 carboxyl end and evaluated their role in inhibition of receptor function. Our preliminary results suggest the possibility of p38 as a candidate responsible for phosphorylating these serine residues. Since phospho-serine is capable of retaining ERBB2 at relatively low activation status, either by changing receptor allosteric activation, or increasing its internalization, it is feasible to design drugs aimed at inducing the serine phosphorylation signaling. Besides receptor activation control from its phosphorylated status, the receptor can also interact with intracellular proteins under quiescent or unphosphorylated state. Grb7 is coamplified with ERBB2 on chromosome 17q12 in ERBB2 overexpression breast cancer. It can exist in equilibrium between monomer and dimer and the dimer can be disrupted by single point mutation. It is possible that the shift of this equilibrium can result in differences in adaptor recruitment to receptors. We ask the question how Grb7 is recruited to ERBB2/3 complex mechanistically and how the potential differences in recruitment impact the tumorigenesis of ERBB2. Our CoIP studies identified a novel second mode of Grb7 interaction with ERBB2, but not with ERBB3. Combined with the receptor higher structure activation model and the dimerization properties of Grb7, we predict the dual binding mode may add to the signaling diversity of ERBB2/ERBB3 complex.

Keywords

ERBB2; serine phosphoprylation; Grb7

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