利用序列標靶導向定量蛋白體方法測量激酶下游訊息傳導之磷酸化計量

Measuring kinase-specific protein phosphorylation stoichiometry by motif-targeting quantitative proteomic approach

江品璉(Pin-Lian Jiang)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801828

摘要

磷酸化後轉譯修飾在細胞訊息調控中扮演著重要角色，而蛋白質磷酸化變化可
能源自蛋白質表現量差異或特定的胺基酸位點磷酸化程度變化兩個層次。磷酸化計量定義為磷酸化程度之百分比，特定胺基酸位點之磷酸化程度在整個蛋白質此位點總含量之比率，唯有精準測量磷酸化計量來描述位點磷酸化量變化，才能區分磷酸化量的轉變是由上游激酶/脫磷酸酶造成化亦或是蛋白質本身量的變化。
我們利用非小細胞肺癌和其主要的致癌蛋白質－表皮生長因子受體激酶（EGFR）之訊息傳遞路徑作為本研究的實驗模型，發展以定量蛋白體學為基礎的方法，測量加入表皮生長因子(EGF)活化後和加入酪胺酸激酶抑制劑後(TKI)的表皮生長因子受體以及其下游蛋白質的磷酸化劑量的變化。這方法結合了穩定同位素標定細胞培養(SILAC)、激酶反應(kinase reaction)和靜相金屬螯合層析法(IMAC)。
細胞培養使用碳-13/氮-15 的精胺酸(R)與離胺酸(K)將細胞內的蛋白質都標定上重的穩定同位素，蛋白質水解後，利用在氧-18 同位素三磷酸腺苷(18O-ATP)反應下之EGFR 激酶反應，將所有未磷酸化的EGFR 受質胜肽上都標定上重同位素的磷酸根。最後，結合IMAC 純化磷酸化胜肽，再以液相層析串聯質譜儀進行分析。同時帶有SILAC 的重同位素和氧-18 同位素的磷酸化胜肽代表在細胞中原本未被磷酸化的潛在EGFR 的受質，而在細胞中原本已經被磷酸化的受質磷酸化胜肽則帶有SILAC 的重同位素和氧-16 同位素胜肽。因此，我們可以利用氧16 胜肽/(氧16 胜肽+氧18 胜肽)的比例，計算特定磷酸化位點之磷酸化計量。
我們成功鑑定及定量來自4,425 蛋白質之13,104 條磷酸化胜肽，其中5,426 個磷酸化胜肽 可測到其氧-18 激酶反應的訊號。更重要的是，在95 個已報導過的EGFR 之下游蛋白質中測得49 個磷酸化位點之計量。另外，我們也辨識到在EGFR下游路徑中的多個蛋白質以及其磷酸化位點如Src pS225, pY419 , Shc1 pY427,iiiRASA1 pY460, STAT1 pY170, MAPK pY204 等。我們進一步進行活化表皮生長因子以及酪胺酸激酶抑制劑的實驗，測量到數個已報導過或者被預測會EGFR 激酶作用的磷酸化位點，如EGFR pY1197, pY1172, Shc1 pY427, Src pY419, ERK1 pY204和ERK pY187，其磷酸化計量受到表皮生長因子被活化或抑制之影響。這些位點上磷酸化的變化趨勢則另外使用西方墨點法(western blotting)進行驗證。除了EGFR之外，我們還利用MAPK 以及CK2 激酶測量在非小細胞肺癌的傳訊路徑中其他受質的磷酸化計量。利用多重激酶實驗的結果顯示，除了EGFR 激酶的受體之外，亦偵測到在非小細胞肺癌的訊息傳遞路徑中之其他受質蛋白，如STAT1/3、 AKT1A1、RAF1 等，也表現出對表皮生長因子活化或抑制劑的變化。
綜上所述，本研究的新策略可探究在一般狀態下以及不同刺激下的訊息傳遞路徑中蛋白質磷酸化的計量變化。透過此精準量測，探討酪胺酸激酶抑制劑如何影響下游磷酸化之動態變化，此資訊有助於了解評估抑制劑成效與活體抗藥性程度。這個方法可以被應用在更多的樣品種類例如細胞株、組織或者體液之分析。

關鍵字

蛋白體學 ； 激? ； 質譜 ； 磷酸化

並列摘要

Protein phosphorylation plays an important role to regulate signal transduction pathways. Phosphorylation alteration may occur due to changes at protein expression level or site-specific phosphorylation stoichiometry, defined as the percentage of phosphorylation on specific site of a protein. Only direct measurement of the phosphorylation stoichiometry can unambiguously reveal whether the signal-induced alteration is regulated by protein abundance or by upstream kinase/phosphatase activity.
Using EGFR signaling pathway in non-small cell lung cancer as a model study, we develop a quantitative proteomic method to measure the phosphorylation stoichiometry on EGFR and its downstream substrates in response to EGF and TKI treatment. The method integrates SILAC labeling, motif-targeting kinase reaction, immobilized metal affinity chromatography (IMAC) and LC-MS/MS analysis. The cells were cultured with heavy SILAC isotopes (13C615N4Arg/13C615N2Lys). After protein digestion, the kinase reaction was performed by recombinant EGFR under the 18O-ATP; thus, EGFR substrates can be recognized by 18O-labeling. Finally, the IMAC is utilized for phosphopeptide enrichment, followed by LC-MS/MS analysis. The phosphopeptides with heavy SILAC labeling and 18O-labeling represent the initially unphosphorylated form of potential EGFR substrate sites, while its corresponding 16O-labeled phosphopeptides represent initially phosphorylated form. The phosphorylation stoichiometry can be calculated by the ratio of 16O-phosphopeptide/(16O-phosphopeptide+ 18O-phosphopeptide).
Total of 13,104 phosphopeptides from 4590 proteins were identified. Among 4425 quantified proteins, we obtained stoichiometry of 5,426 phosphopeptides by detecting their 18O-labeled peaks. The result revealed 95 reported EGFR-related proteins and stoichiometry of 49 proteins were successfully measured. In the NSCLC pathway, many EGFR downstream proteins and its sites such as Src pS225, pY419, Shc1 pY427, RASA1 pY460, STAT1 pY170, MAPK pY204 were identified. Upon EGF activation and TKI treatment, we measured altered stoichiometry of many known or predicted EGFR substrates, such as Shc1(Y427), Erk1(Y204), Erk2(Y187), and Src(Y419). The results were verified by western-blotting experiment. Besides EGFR kinase, we also applied CK2 and MAPK kinases to analyze their substrates in the NSCLC pathways. The results also obtained stoichiometry of CK2 and MAPK substrates in NSCLC downstream pathway such as STAT1/3 pS727, AKT1A1 pS211, pS212 and RAF1 S301, which phosphorylation sequence also matched to the kinase motif.
In summary, our new method revealed the first map of phosphorylation stoichiometry of a signaling pathway at the basal level and under stimuli, which provides new insight on the phosphorylation-mediated signaling pathway for better understanding the site-specific TKI response in NSCLC. The method can be generally applied to other sample types such as cells or tissue, body fluids.

並列關鍵字

proteomic ； kinase ； mass spectrometry ； phosphorylation

參考文獻

1. Oliveira, A. P.; Sauer, U., The importance of post-translational modifications in regulating Saccharomyces cerevisiae metabolism. FEMS Yeast Res 2012, 12 (2), 104-17.

2. Peck, S. C., Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates. Plant J 2006, 45 (4), 512-22.

3. Nicosia, S. V.; Diaz, J.; Nicosia, R. F.; Saunders, B. O.; Muro-Cacho, C., Cell proliferation and apoptosis during development and aging of the rabbit corpus luteum. Ann Clin Lab Sci 1995, 25 (2), 143-57.

4. Babior, B. M., NADPH oxidase: an update. Blood 1999, 93 (5), 1464-76.

5. Graves, J. D.; Krebs, E. G., Protein phosphorylation and signal transduction. Pharmacol Ther 1999, 82 (2-3), 111-21.