RI-alpha/RIAZ on Cell Growth in Breast Cancer

描述（由申请人提供）：cAMP对细胞生长和增殖的影响已被深入研究，但其作用机制尚不完全清楚。cAMP的作用主要由cAMP依赖性蛋白激酶（PKA）介导，PKA由两个不同的亚基，催化(C)和调节(R)组成，形成一个四聚体全酶R2C2。I型调节α (riα)亚基表达与人乳腺组织增生有关，其在人乳腺癌中的过表达与恶性肿瘤和不良预后相关。RI？刺激生长，而C亚基的过表达不会产生这样的后果。最近，RIalpha被证明是卡尼复杂综合征的肿瘤抑制基因。我们之前已经证明了RIalpha与C亚基激酶活性无关的新相互作用。在本应用中，我们通过酵母双杂交互作克隆实验证明RIalpha与一种新的BTB/POZ结构域锌指转录因子（RIalpha-associated zinc finger protein， RIAZ）相结合。我们证明RIAZ是一种camp应答转录激活因子，通过与riα的相互作用以及与camp应答元件结合蛋白（CREB）的潜在合作来调节。我们进一步表明RIAZ在大约15%的人类原发性乳腺癌中异常表达。此外，通过[3H]胸苷摄取测量，RIAZ的过表达会导致生长抑制。cAMP增强了生长抑制作用，但不受PKA抑制剂H-89的影响。我们假设RI？与RIAZ的相互作用可能是cAMP介导生长抑制的一种新的转录机制。在本提案中，我们将：(1)确定国际扶轮的机制？与RIAZ的互动以及cAMP的监管；(2)探讨RIAZ对cAMP的转录反应机制；(3)研究RI？与RIAZ就cAMP进行互动。我们的结果将揭示RIalpha与RIAZ在生长抑制中的这种新的相互作用。

DESCRIPTION (provided by applicant): The effects of cAMP on cell growth and proliferation have been intensely investigated, but its mechanisms of action are not completely understood. The effects of cAMP are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzymes, R2C2. The type I regulatory alpha (RIalpha) subunit expression is associated with hyperproliferation in human breast tissue and its overexpression in human breast cancer correlates with malignancy and poor prognosis. Increased expression of RI? stimulates growth, whereas overexpression of the C subunit does not produce such consequence. Most recently, RIalpha was shown to be a tumor suppressor gene in Carney Complex Syndrome. We have demonstrated previously novel interaction of RIalpha that is independent of the C subunit kinase activity. In this application, we show by yeast two-hybrid interaction cloning experiment that RIalpha associates with a novel BTB/POZ domain zinc finger transcription factor, termed RIalpha-associated zinc finger protein (RIAZ). We demonstrate that RIAZ is a cAMP-responsive transcriptional activator regulated by its interaction with RIalpha and potential cooperation with the cAMP-response element binding protein (CREB). We show further that RIAZ is aberrantly expressed in approximately 15% of human primary breast cancer. Furthermore, overexpression of RIAZ causes growth inhibition measured by [3H]thymidine uptake. The growth inhibition is enhanced by cAMP but not affected by the PKA inhibitor H-89. We hypothesize that RI? interaction with RIAZ may be a novel transcriptional mechanism in growth inhibition transduced by cAMP. In this proposal, we will: (1) determine the mechanisms of RI? interaction with RIAZ and regulation by cAMP; (2) investigate the mechanisms of RIAZ transcriptional response to cAMP; and (3) investigate the mechanisms of growth control by RI? interaction with RIAZ in response to cAMP. Our results will shed light on this novel interaction of RIalpha with RIAZ in growth inhibition.