Linking IKKbeta activation to anti-autophagy in viral protein Tax-mediated oncoge

描述（由申请人提供）：T细胞白血病病毒1型（HTLV-1）是成人T细胞白血病和淋巴瘤（ATLL）的致病因子。HTLV-1基因组编码一种病毒转化蛋白Tax 1，其在启动肿瘤发生中起重要作用。Tax 1参与肿瘤发生的关键特征之一是诱导NF-（B.然而，这种活性如何与T细胞转化相关仍然没有解决。我们的工作表明Tax 1是一个关键的脂筏调节剂，可以劫持I（B激酶（IKK）到HTLV-1转化的T细胞中的脂筏微结构域。众所周知，IKK在脂筏微结构域中的富集导致抗原刺激的T细胞中NF-β B的活化。Tax 1能够在脂筏中持续募集IKK，与持续的NF-β B活性相关。Tax 1还以IKK依赖的方式在脂筏中螯合Beclin 1（BECN 1）和Bif-1，这两种自噬介质是必需的。相反，BECN 1通过抑制IKK抑制NF-β的Tax 1活化。由于BECN 1或Bif-1的缺失与自发性肿瘤发生（包括淋巴瘤）有关，我们的研究结果强烈表明Tax 1部分通过其抗自噬活性将IKK（活化）与肿瘤发生联系起来。该提议的基本原理是基于Tax 1-IKK（-BECN 1/Bif-1）轴导致自噬介质的“功能丧失”的假设。这项研究具有创新性和重要性，代表了一个新的概念，填补了我们对Tax如何启动T细胞致癌转化的理解的巨大空白。我们的中心假设是“HTLV-1 Tax通过劫持IKK（脂筏）抑制自噬，导致自噬介质在微结构域中的隔离，从而显著促进肿瘤发生”。我们计划以下列具体目标调查这一假设。目的#1：定义Tax 1的脂筏靶向关键结构域。我们将确定的图案和修饰，如泛素化的Tax 1的脂筏靶向的关键，确定细胞因子在协助Tax 1的脂筏协会，并评估Tax 1的脂筏协会在永生化的原代人CD 4 + T细胞和转化NIH 3 T3细胞的重要性。目标2：研究Tax 1解除自噬调控的潜在机制。我们将研究Tax 1在脂质筏中螯合Bif-1和BECN 1的机制，评估BECN 1对Tax 1激活IKK的抑制作用，并使用MEF（Bif 1 +/+）和（Bif 1-/-）细胞检测Tax 1的抗自噬和致瘤活性。目的3：确定脂筏靶向IKK的致癌潜力和抗自噬功能。我们将评估IKK与Bif-1和BECN 1的物理和功能相互作用，并评估脂筏靶向IKK的抗自噬和体外转化活性。本研究将有助于阐明Tax-IKK（-BECN 1/Bif-1）轴在HTLV-1肿瘤发生中的病理作用。 公共卫生相关性：成人T细胞白血病/淋巴瘤（ATLL）是由人类T细胞白血病病毒1型（HTLV-1）感染引起的，在全球范围内感染了超过2000万患者。HTLV-1编码一种病毒致癌蛋白Tax，其部分通过持续激活NF-（B.我们计划研究Tax在T细胞白血病发生发展过程中介导NF-β B持续活化和抗自噬功能的机制。

DESCRIPTION (provided by applicant): T cell leukemia virus type 1 (HTLV-1) is a causative factor for adult T cell leukemia and lymphoma (ATLL). HTLV-1 genome encodes a viral transforming protein, Tax1, which plays an essential role in initiating oncogenesis. One of the critical features that Tax1 contributes to oncogenesis is induction of persistent activation of NF-(B. Yet, how this activity correlates with T cell transformation remains unsolved. Our work demonstrates that Tax1 is a critical lipid raft modulator that can hijack I(B kinases (IKKs) to the lipid raft microdomains in HTLV-1-transformed T cells. It is well recognized that enrichment of IKKs in the lipid raft microdomains leads to activation of NF-(B in antigen-stimulated T cells. Tax1 is able to recruit IKKs persistently in lipid rafts, correlating with persistent NF-(B activity. We also find that Tax1 sequesters beclin 1 (BECN1) and Bif-1, the essential autophagy mediators, in lipid rafts in IKK(- dependent manner. Conversely, BECN1 represses Tax1 activation of NF-(B by inhibiting IKK(. Since loss of BECN1 or Bif-1 has been linked to spontaneous tumorigenesis including lymphoma, our findings strongly suggest that Tax1 links IKK( activation to oncogenesis partly through its anti-autophagy activity. The rationale for this proposal is based on the assumption that the axis of Tax1-IKK(-BECN1/Bif-1 leads to "loss of function" of the autophagy mediators. The proposed research is both innovative and significant, representing a new concept in filling a giant gap for our understanding about how Tax initiates oncogenic transformation of T cells. Our central hypothesis is that "HTLV-1 Tax suppresses autophagy by hijacking IKK( to lipid rafts leading to sequestration of the autophagy mediators in the microdomains, thereby contributing significantly to oncogenesis". We plan to investigate this hypothesis with following specific aims. Aim#1: Define the domain critical for lipid raft targeting of Tax1. We will determine the motifs and modifications such as ubiquitination crucial for lipid raft targeting of Tax1, identify a cellular factor in assisting lipid raft association of Tax1, and assess the importance of the lipid raft association of Tax1 in immortalizing primary human CD4+ T cells and in transforming NIH3T3 cells. Aim#2: Investigate the underlying mechanism of Tax1 to deregulate autophagy. We will investigate the mechanism of Tax1 in sequestrating Bif-1 and BECN1 in lipid rafts, evaluate the inhibitory effect of BECN1 on Tax1 activation of IKK(, and examine anti-autophagy and tumorigenic activity of Tax1 using MEF (Bif1+/+) and (Bif1-/-) cells. Aim#3: Determine oncogenic potential and anti-autophagy function of lipid raft-targeted IKK(. We will evaluate physical and functional interaction of IKK( with Bif-1 and BECN1 and assess anti-autophagy and in vitro transforming activities of the lipid raft-targeted IKK(. This study will help to decipher the pathological role of the axis of Tax- IKK(-BECN1/Bif-1 in HTLV-1 oncogenesis. PUBLIC HEALTH RELEVANCE: Adult T cell leukemia/lymphoma (ATLL) is caused by infection with human T cell leukemia virus type 1 (HTLV-1), infecting over 20 million patients worldwide. HTLV-1 encodes a viral oncogenic protein Tax, which plays an essential role in initiating malignancy of human T cells partly through persistent activation of NF-(B. We plan to investigate the mechanism that Tax mediates persistent activation of NF-(B and anti-autophagy function during development of T cell leukemia.