Mechanisms of ERBB receptor activation by radiation

描述(由申请人提供)：受自分泌生长调节的人类肿瘤细胞暴露于治疗剂量的电离辐射可激活ERBB和其他受体酪氨酸激酶。这些反应的细节，包括ERBB受体在细胞上的互补信号的同时激活和沿着主要的现有通路的下游信号，已经由我们的小组描述。ErbB1的激活以Tyr磷酸化的双相增加为特征，最初的反应发生在1-5min内，而后期的反应发生在60min-240min之间，可能是由于初始激活导致转化生长因子-α的释放。相对于生长因子，例如，EGF作为ErbB1的配体，辐射在受体水平上诱导的反应不那么强烈；然而，在细胞质蛋白激酶水平上，信号具有相同的强度。ErbB1的双向激活和所有ERBB受体的同时激活在肿瘤细胞中表达的浓度差异很大，代表了辐射和生长因子诱导的受体激活的根本区别，形成了继续应用的基础。我们希望通过蛋白质组学技术(质谱磷肽鉴定)鉴定ERBBI的Tyr磷酸化模式，以及ErbB1及其异二聚化伙伴ERBB2的激酶和显性负性受体抑制物，来揭示辐射ERBB激活和下游信号传导的独特机制。总体目标分为三个具体目标(SA)。SAI：在辐射和转化生长因子-α暴露后，稳定表达ErbB1 Tyr的CHO.ERBB1细胞中确定了详细的ErbB1 Tyr磷酸化模式。如果可行，ErbB1在血浆和内膜中富含，以检查受体处理过程中Tyr磷酸化的可能变化。通过与ERBB2和显性负性ErbB1-CD533的相互作用的研究，研究了ErbB1 Tyr的磷酸化作为受体同源和异位二聚化的函数。SA2：ErbB1 Tyr磷酸化与受体蛋白信号复合体的形成相关，包括对Tyr-Phe ErbB1突变的研究，以确认单个Tyr残基的功能重要性。SA3.本研究的目的是通过研究已知与ERBBI相互作用的蛋白质Tyr磷酸酶在控制ErbB1的静止稳定状态Tyr磷酸化及其与细胞膜的加工过程中的潜在作用，来研究ERBB受体立即和同时激活的机制。

DESCRIPTION (provided by applicant): The exposure of autocrine growth regulated human tumor cells to therapeutically applied doses of ionizing radiation activates ERBB and other receptor Tyr kinases. Details of these responses, including the simultaneous activation of the compliment of ERBB receptors on a cell and downstream signaling along major existing pathways, have been described by our group. The activation of ERBB1 is characterized by a biphasic increase in Tyr phosphorylation, the primary response occurring within 1-5 min, whereas the later phase is seen between 60 and 240 min, possibly mediated by TGF-alpha release as the result of the initial activation. Relative to growth factors, e.g., EGF as ligand for ERBB1, radiation induces a less intense response at the receptor level; however, at the level of cytoplasmic protein kinases the signals are of identical intensity. The biphasic activation of ERBB1 and the simultaneous activation of all ERBB receptors, expressed at widely varied concentration by tumor cells, represent fundamental differences in radiation- and growth factor-induced receptor activation and form the basis of the continuation application. We expect to unravel the unique mechanisms of radiation ERBB activation and downstream signaling by identifying the Tyr phosphorylation patterns of ERBBI using proteomics technology (mass spectroscopy phosphopeptide identification) and kinase and dominant negative receptor inhibitors of ERBB1 and its heterodimerization partner ERBB2. The overall objectives are addressed in three Specific Aims (SAs). SAI: The detailed ERBB1 Tyr phosphorylation pattern is defined in stably transfected CHO.ERBB1 cells after exposure to radiation and TGF-alpha. If feasible, ERBB1 is enriched in the plasma and the endosomal membranes to examine possible changes in Tyr phoshorylation during receptor processing. ERBB1 Tyr phosphorylation is examined as a function of receptor homo- and heterodimedzation through studies on interactions with ERBB2 and dominant negative ERBB1-CD533. SA2: ERBB1 Tyr phosphorylation is correlated with the formation of receptor protein signaling complexes, including studies with the Tyr-Phe ERBB1 mutation to affirm the functional importance of individual Tyr residues. SA3. This aim addresses the mechanisms underlying the immediate and simultaneous activation of ERBB receptors by investigating the potential role of protein Tyr phosphatases, known to interact with ERBBI, in controlling the quiescent steady state Tyr phosphorylation of ERBB1 and its processing with the cellular membranes.