【作者】 陈露；

【导师】 郭行；

【作者基本信息】 浙江大学 ， 细胞生物学， 2020， 博士

【摘要】 26S蛋白酶体负责降解真核细胞中绝大多数的蛋白,与几乎所有的生命活动密切相关。越来越多的证据表明,磷酸化修饰对调控蛋白酶体的功能发挥重要作用。质谱数据显示,人类蛋白酶体上有100多个酪氨酸(pTyr)磷酸化位点,但人们对其功能和调控还知之甚少。在本研究中,我们证明蛋白酶体亚基Rpt2-Tyr439位点的磷酸化抑制蛋白酶体的组装,并意外发现Tyr439的磷酸化依赖于Rpt2 N-肉豆蔻酰化介导的膜定位。多种受体酪氨酸激酶(RTKs)可以通过激活Src上调Rpt2-Y439的磷酸化,影响膜定位蛋白酶体的组装和活性。相反,酪氨酸磷酸酶PTPN2可以去磷酸化Rpt2-pY439并促进蛋白酶体对底物的降解。在Src高度激活的非小细胞肺癌H1975细胞中,Rpt2-Y439F突变明显改变了细胞中多种蛋白对Src抑制剂saracatinib的响应。在体外培养和小鼠成瘤实验中,阻断Rpt2-Y439磷酸化削弱了 saracatinib对癌细胞的生长抑制作用。本研究发现了新的蛋白酶体激酶和磷酸酶,首次揭示了酪氨酸磷酸化参与调控膜定位蛋白酶体的机制,为基于酪氨酸激酶抑制剂的抗癌治疗提供了新的思路。更多还原

【Abstract】 Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease.Over 100 phospho-tyrosine(pTyr)sites of the human proteasome have been detected,and yet their function and regulation remain poorly understood.Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439,a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for proteasome assembly.Unexpectedly,we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation.Multiple receptor tyrosine kinases(RTKs)can trigger Rpt2-Y439 phosphorylation by activating Src,a N-myristoylated tyrosine kinase.Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates proteasome integrity and activity at cellular membranes,which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non receptor type 2(PTPN2).In H1975 lung cancer cells with activated Src,blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model,and caused significant changes of cellular responses to saracatinib at the proteome level.Our study has defined a novel tyrosine kinase-phosphatase pair involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies.更多还原