晚期癌癥通常會(huì)繞過抗癌藥物設(shè)置的路障而繼續(xù)迅速生長(zhǎng),，近日,，國(guó)際著名雜志《國(guó)家科學(xué)院院刊》PNAS在線刊登了來自美國(guó)普渡大學(xué)研究者的最新研究成果“Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates，”,，文章中,，研究者繪制了激酶與它們的靶蛋白的互作圖譜，以幫助制藥者找到捷徑,，開發(fā)出更有效的抗癌藥物,。
蛋白激酶將磷酸基團(tuán)連接到目的蛋白的氨基酸殘基上，使蛋白的特定位點(diǎn)發(fā)生磷酸化的過程就稱之為磷酸化作用,。通過這一機(jī)制可開啟或關(guān)閉細(xì)胞的某種功能,。磷酸化作用失常可導(dǎo)致細(xì)胞生長(zhǎng)失控,，是癌癥的一種標(biāo)志,。
許多有效的癌癥藥物都是激酶抑制劑，它們通過阻斷激酶與細(xì)胞內(nèi)特異蛋白的結(jié)合,，阻止磷酸化作用和癌細(xì)胞生成,。
美國(guó)普渡大學(xué)生物化學(xué)分析化學(xué)系副教授、癌癥研究中心成員陶緯國(guó)（W. Andy Tao）是這項(xiàng)研究的領(lǐng)導(dǎo)者,。陶緯國(guó)認(rèn)為在癌癥晚期,，激酶抑制劑無效的原因是因?yàn)榧っ高M(jìn)行了調(diào)整,，尋找到了新的靶標(biāo)蛋白,，形成了新的癌細(xì)胞。他認(rèn)為構(gòu)建出癌細(xì)胞所有潛在路線的圖譜是開發(fā)更好的癌癥治療藥物的一個(gè)關(guān)鍵環(huán)節(jié),。
“我要說的是,，99%的激酶抑制劑都只能在癌癥晚期的數(shù)月時(shí)間內(nèi)有效，隨后癌癥就形成了耐受,。在開始時(shí),，細(xì)胞并不能自我調(diào)節(jié)因此死亡。在晚期,，癌細(xì)胞發(fā)現(xiàn)了一條生存之路,。你阻斷了一條途徑，而它們找到了另一條,，”陶緯國(guó)說,。
陶緯國(guó)構(gòu)建的激酶-蛋白圖譜鑒別了激酶和它們磷酸化的直接蛋白靶標(biāo)，而排除了其他非直接靶向的蛋白,。后者通常是在直接靶向蛋白磷酸化啟動(dòng)的級(jí)聯(lián)反應(yīng)中被磷酸化的,。
陶緯國(guó)將有無激酶的細(xì)胞進(jìn)行了比較,，證實(shí)只有當(dāng)激酶存在時(shí)，磷蛋白才會(huì)被當(dāng)做潛在的靶標(biāo),。首先,，他使這些磷蛋白去磷酸化。隨后重新導(dǎo)入激酶,，這些接受了來自激酶的磷酸基團(tuán)的蛋白就是它們的直接靶標(biāo),。陶緯國(guó)表示有了這些信息，制藥公司就可以研制出阻斷激酶到達(dá)所有潛在的靶標(biāo)的激酶抑制劑,，使藥物更有效,。
“如果你了解這一網(wǎng)絡(luò)，你就可以阻斷所有的途徑來治療癌癥,，”陶緯國(guó)說,。
目前陶緯國(guó)的研究主要集中在與白血病和乳腺癌相關(guān)的SYK激酶。他計(jì)劃進(jìn)一步研究其他的激酶以及突變的激酶,，以了解它們是否具有不同的蛋白靶標(biāo),。（生物谷Bioon.com）
doi:10.1073/pnas.1119418109
PMC:
PMID:
Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates
Liang Xuea, Wen-Horng Wangb, Anton Iliuka, Lianghai Hua, Jacob A. Galana, Shuai Yub, Michael Hansa, Robert L. Geahlenb,c, and W. Andy Taoa,b,c,d,1
Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.
