Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Li, Shawn

Abstract

Protein tyrosine kinases (PTKs, or TKs) have emerged as one of the most intensively pursued targets in the development of anti-cancer therapeutics, due to their critical roles in the phosphotyrosine (pTyr)-mediated signaling network that regulates many cancer-related cellular activities. The TKs, tyrosine phosphorylation phosphatases (PTPs) and pTyr recognition SH2 proteins are intensively tyrosine phosphorylated, which play a pivotal role in determining the signaling outcome of this network. More than 50% of all human proteins are tyrosine phosphorylated and many of these TK substrates have been proven functional in TK regulated cellular activities. Therefore, proteomics studies of tyrosine phosphorylation are of great value for expanding our understanding of this network and its role in tumorigenesis.

Taking advantage an engineered SH2 domain with superb pTyr binding affinity, an SH2-affinity purification mass spectrometry (SH2-AP-MS) assay was developed and optimized for global TK status profiling by determining the abundance of functionally significant TK pTyr peptides including the activation loops. Multiple known TK biomarkers were identified by this assay from a minute amount of protein extracts from cultured cells or clinical leukemia or solid tumor samples. In the quantitative AP-MS study of cancer cells treated by targeted therapeutics, differential TK responses were monitored. The receptor c-Kit was found to be dramatically upregulated with long-term trastuzumab treatment which contributed to acquired trastuzumab resistance in the SK-BR-3 breast cancer cells. These results revealed the potential applications of SH2-AP-MS in the discovery of novel TK biomarkers and examination of TK positive cancers in the clinical setting. In addition, a SH2 superbinder modified yeast two hybrid (Y2H) system was developed that is potentially capable of identifying TK substrate in a high-precision and high-throughput manner. This system consisted of an SH2-superbinder-fused bait that greatly promoted the reporter gene readout, a TK substrate prey or cDNA library preys, and a co-expressed TK under the control of a conditional promoter that allowed for reverse screen to eliminate false positives. In a medium-scale cDNA library screening for Src kinase substrates, 94 positive colonies were isolated representing 48 unique in-frame proteins or protein fragments, of which at least 9 proteins are known Src substrates or direct Src interactors.

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