Research on HER2 and HER3 signaling pathway and the enzymatic properties of HER2 dimers using single-molecule co-immunoprecipitation단분자 면역침강법을 이용한 HER2와 HER3의 신호전달 체계와 HER2 이합체의 효소적 특성에 관한 연구

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A successful targeted therapy, trastuzumab is treated to breast cancer patients who are diagnosed as HER2 positive which means HER2 gene-amplification, HER2 overexpression or both. Here, we applied single-molecule pull-down, immunolabeling, and co-IP to HER2 and HER3 signaling within breast cancer cell lines. We observed highly heterogeneous signaling phenotypes of them while their drug responses were also heterogeneous. Finally, we found that HER3-$p85 \alpha$ PPI was highly correlated to trastuzumab efficacy while HER2-related signaling phenotypes showed low correlation. Ligand induced heterodimer between the orphan ligand human epidermal growth factor receptor (HER2/ErbB2) and its catalytically impaired homolog HER3 is known to the most proliferative among all the dimers formed within HER family. Moreover, the heterodimer is well known for rescuing many cancer from respective targeted therapy. The superiority of HER2-HER3 heterodimer have not been explained through in vitro kinase assay even using vesicle anchored kinase assay. Using single-molecule pull-down and co-immunoprecipitation (co-IP), we specifically pulled down intact HER2 homo- or HER2-HER3 heterodimer which are formed on cell membrane on glass surface and observed their properties. By virtue of the mild detergent digitonin, the dimers maintain their kinase activity after they are extracted from cell. When ATP and magnesium are introduced, the dimers phosphorylates their tyrosine sites. By introducing eGFP labeled downstream proteins (grb2, $PLC \gamma$, and $p85 \alpha$), we found that known grb2 binding site of phosphorylated HER2 (pY1139) did not directly recruit grb2. Instead, grb2 recruitment was increased when a famous scaffold of HER2, SHC1, and its known tyrosine kinase, Src were introduced during phosphorylation step. We also measured enzymatic properties of intact HER2 dimers extracted from SKBR3. Although both dimer equally use HER2 kinase as an effective enzyme, HER2-HER3 heterodimer shows about 150 times faster maximum catalytic rate for single phosphorylation site than HER2 homodimer while their Michaelis constants for ATP are similar. HER2-HER3 heterodimer also endures HER2 Tyrosine Kinase Inhibitor (TKI), Lapatinib with ATP and magnesium while HER2 homodimer is effectively inhibited. ed. However, when HER2-HER3 heterodimer treated with Lapatinib without ATP and magnesium, it loses the resistance. Moreover, when we added PTPN1 during Lapatinib inhibition, we observed that the endurance of the heterodimer against lapatinib severally impaired. From these results, we suggest that the resistance is based on the overwhelming catalytic rate of HER2-HER3 heterodimer.
Advisors
Park, Yong Keunresearcher박용근researcher
Description
한국과학기술원 :물리학과,
Publisher
한국과학기술원
Issue Date
2018
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 물리학과, 2018.8,[vii, 63 p. :]

Keywords

Single-molecule co-immunoprecipitation▼acell signaling▼amembrane protein▼aHER2-HER3 heterodimer▼aHER2 homodimer▼alapatinib resistance; 단분자 면역침강법▼a세포 신호 전달▼a막 단백질▼aHER2-HER3 이종이합체▼aHER2 동종이합체▼a라파티닙 저항성

URI
http://hdl.handle.net/10203/264645
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=828267&flag=dissertation
Appears in Collection
PH-Theses_Ph.D.(박사논문)
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