AFAP-110 as a cSrc activator in breast cancer

AFAP-110 作为乳腺癌的 cSrc 激活剂

• 批准号: 7740622
• 负责人: Daniel Charles Flynn
• 金额: $ 24.63万
• 依托单位: GEISINGER COMMONWEALTH SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740622
• 关键词: AFAP 110; Address; Adhesions; Affect; Binding; Binding Sites; Breast; Breast Cancer Cell; Cell Adhesion; Cells; Data; Docking; Drug Delivery Systems; Fibronectins; Filament; Fostering; Generations; Integrins; Link; Malignant neoplasm of ovary; Mammary Gland Parenchyma; Mammary gland; Membrane; Microfilaments; Molecular Conformation; Mus; Mutate; Mutation; Neoplasm Metastasis; Ovarian Carcinoma; Pharmaceutical Preparations; Phosphatidic Acid; Phosphatidylinositols; Phospholipids; Phosphorylation; Process; Protein Tyrosine Kinase; Proteins; Role; Signal Transduction; Site; Stress; Structure; Time; Tissues; Transgenic Mice; Variant; Vesicle; Work; crosslink; malignant breast neoplasm; neoplastic cell; overexpression; platelet protein P47; public health relevance; response; restoration; tumor

DESCRIPTION (provided by applicant): As breast and ovarian carcinoma progress the tumor cells develop invasive structures, which provide the cells with a mechanism to cross tissue barriers and metastasize. Activation of the tyrosine kinase cSrc is known to occur in breast and ovarian cancers, will stimulate the formation of invasive structures, and promote invasion. Although cSrc is activated in these tumors, it is not mutated. Rather, cSrc is activated in response to input signals. One example of a cSrc-activating protein is AFAP-110, which has two known functions ? it cross-links actin filaments and can bind to and activate cSrc. AFAP-110 is a substrate of PKCa. Phosphorylation by PKCa results in a conformational change in AFAP-110 that releases 'autoinhibition' and enables it to efficiently cross-link stress filaments and direct cSrc activation. This signaling cascade results in the formation of invasive structures. The question to be addressed in this application is, 'can increased expression of AFAP-110 in mouse breast promote cSrc activation, and if so, how?' Our preliminary data indicate that knockdown of AFAP-110 in breast cancer cells results in reduced cell adhesion. This effect is linked with reduced stress filament cross-linking and decreased invasive potential. Upon MDA-231 cell adhesion to fibronectin, AFAP-110 becomes phosphorylated on Tyr94, a Src target. Thus, in Aim 1, we hypothesize that AFAP-110 promotes cell adhesion by activating cSrc. We also find that the ability of AFAP-110 to activate cSrc requires that the PH1 domain bind to phosphatidic acid (PA). There are two grooves in the PH1 domain that can bind phospholipids. In Aim 2, we predict that binding of PA and PtdIns4P are required for AFAP-110 to activate cSrc and that they bind to separate grooves and work cooperatively to dock AFAP-110 on membranes and subsequently activated cSrc. Interestingly, we identified a polymorphic variant of AFAP-110 that has a nonsynomous SNP in the carboxy terminal pleckstrin homology (PH2) domain. This Ser403?Cys403 change enables AFAP-110403C to independently activate cSrc, but only under conditions of overexpression. We hypothesize that the Cys403 SNP may result in a less efficiently stabilized AFAP-110 multimer, which could weaken autoinhibition and result in cSrc activation. In Aim 3, we will determine how the PH2 domain fosters self-association and if mutations in the opposing PH2 binding site within AFAP-110 also destabilize the multimer and enable cSrc activation. Lastly, we find that AFAP-110 and cSrc are overexpressed together in the same cells in breast and ovarian cancer tissues. We hypothesize that AFAP-110 has the capacity to activate cSrc in breast tissue. In Aim 4, we will create a transgenic mouse and determine if, under conditions of overexpression in mouse mammary tissue, AFAP-110 has the potential to activate cSrc. The significance of these studies is that AFAP-110 may be one protein responsible for activating cSrc. Thus, it may be a legitimate drug target, with a 'drug-able' site in the PH1 domain that could be targeted to disrupt cSrc activation and invasive potential. PUBLIC HEALTH RELEVANCE: This project is relevant to breast cancer in that it will address a mechanism by which cSrc becomes activated in breast cancer. This will be the first time anyone has attempted to determine if cSrc activating proteins (AFAP-110) can direct cSrc activation in mouse breast tissues. As AFAP-110 has a phospholipid binding pocket that appears to be crucial for this function, it may be a drug-able target.

描述(申请人提供)：随着乳腺癌和卵巢癌的发展，肿瘤细胞形成侵袭性结构，这为细胞提供了跨越组织屏障和转移的机制。酪氨酸激酶CSRC的激活已知发生在乳腺癌和卵巢癌中，将刺激侵袭性结构的形成，并促进侵袭性。虽然中国证监会在这些肿瘤中被激活，但它不会发生突变。相反，中国证监会是根据输入信号而激活的。中国证监会激活蛋白的一个例子是AFAP-110，它有两个已知的功能？它能交联肌动蛋白细丝，并能与中国证监会结合并激活。AFAP-110是PKCA的底物。PKCA的磷酸化导致AFAP-110的构象变化，从而释放“自我抑制”，使其能够有效地使应激丝交联并直接激活CSRC。这种信号级联导致侵袭性结构的形成。在这项应用中需要解决的问题是，‘AFAP-110在小鼠乳房中的表达增加能否促进CSRC的激活，如果是，又是如何促进的？’我们的初步数据表明，AFAP-110在乳腺癌细胞中的敲除导致细胞黏附减少。这种作用与减少应力纤维的交联性和降低侵袭潜能有关。vt.在.的基础上 当MDA-231细胞与纤维连接蛋白黏附时，AFAP-110在Src靶标Tyr94上被磷酸化。因此，在目标1中，我们假设AFAP-110通过激活CSRC促进细胞黏附。我们还发现AFAP-110激活CSRC的能力需要PH1结构域与磷脂酸(PA)结合。在PH1结构域上有两个凹槽可以结合磷脂。在目标2中，我们预测PA和PtdIns4P的结合是AFAP-110激活CSRC所必需的，它们结合到单独的凹槽并协同工作，将AFAP-110对接在膜上，随后激活CSRC。有趣的是，我们发现了AFAP-110的一个多态变体，它在羧基末端Pleckstrin同源(PH2)结构域具有非同源SNP。此Ser403-Cys403更改启用 AFAP-110403C独立激活中国证监会，但仅在过度表达的条件下。我们推测，Cys403 SNP可能导致AFAP-110多聚体不太稳定，从而削弱自身抑制并导致CSRC激活。在目标3中，我们将确定PH2结构域如何促进自结合，以及AFAP-110中相反的PH2结合位点的突变是否也破坏了多聚体的稳定并使CSRC激活。最后，我们发现AFAP-110和CSRC在乳腺癌和卵巢癌组织中的同一细胞中同时过表达。我们推测AFAP-110具有激活乳腺组织中CSRC的能力。在目标4中，我们将创建一只转基因小鼠，并确定在小鼠乳腺组织过表达的条件下，AFAP-110是否具有激活CSRC的潜力。这些研究的意义在于，AFAP-110可能是一个负责激活中国证监会的蛋白质。因此，它可能是一个合法的药物靶点，在PH1区域有一个可以药物的部位，可以作为靶点来干扰中国证监会的激活和侵袭潜力。公共卫生相关性：这个项目与乳腺癌相关，因为它将解决中国证监会在乳腺癌中变得活跃的机制。这将是第一次有人试图确定CSRC激活蛋白(AFAP-110)是否可以指导小鼠乳房组织中CSRC的激活。由于AFAP-110有一个磷脂结合口袋，这似乎是这一功能的关键，它可能是一个药物靶点。