Liu Matt Y, Cai Shengli, Espejo Alexsandra, Bedford Mark T, Walker Cheryl Lyn
Department of Carcinogenesis, Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.
Cancer Res. 2002 Nov 15;62(22):6475-80.
Tuberin, the product of the tuberous sclerosis complex 2 tumor suppressor gene, is a phosphoprotein that negatively regulates phosphatidylinositol 3'-kinase signaling downstream of Akt. Several high stringency 14-3-3 binding sites that overlapped with Akt phosphorylation sites were identified in tuberin in silico. Recognition of tuberin by an alpha-14-3-3 binding site-specific antibody correlated with mitogen-induced phosphorylation of tuberin and recognition of tuberin by an alpha-Akt phosphorylation substrate antibody. Recognition of tuberin by both antibodies was blocked by inhibiting phosphatidylinositol 3'-kinase activity. Using a protein domain microarray, a tuberin peptide containing Ser(939) demonstrated phospho-specific binding to 14-3-3. Glutathione S-transferase pull-down assays with 14-3-3 fusion proteins revealed that all seven 14-3-3 isoforms (beta, gamma, zeta, epsilon, tau, eta, and sigma) could bind tuberin, and this interaction was abrogated by competition with phosphorylated but not unphosphorylated Ser(939) tuberin peptide. Tuberin also coimmunoprecipitated with 14-3-3, confirming the interaction between endogenous 14-3-3 and tuberin. These data establish the presence of functional and overlapping 14-3-3 and Akt recognition site(s) in tuberin.
结节蛋白是结节性硬化症复杂 2 肿瘤抑制基因的产物，是一种磷蛋白，可在 Akt 下游负向调节磷脂酰肌醇 3'-激酶信号传导。通过计算机分析在结节蛋白中鉴定出了几个与 Akt 磷酸化位点重叠的高严格性 14-3-3 结合位点。α-14-3-3 结合位点特异性抗体对结节蛋白的识别与有丝分裂原诱导的结节蛋白磷酸化以及 α-Akt 磷酸化底物抗体对结节蛋白的识别相关。通过抑制磷脂酰肌醇 3'-激酶活性可阻断两种抗体对结节蛋白的识别。使用蛋白质结构域微阵列，含有 Ser(939) 的结节蛋白肽显示出与 14-3-3 的磷酸化特异性结合。用 14-3-3 融合蛋白进行的谷胱甘肽 S-转移酶下拉试验表明，所有七种 14-3-3 异构体（β、γ、ζ、ε、τ、η 和 σ）都可以结合结节蛋白，并且这种相互作用可通过与磷酸化的而非未磷酸化的 Ser(939) 结节蛋白肽竞争而被消除。结节蛋白也与 14-3-3 进行了共免疫沉淀，证实了内源性 14-3-3 与结节蛋白之间的相互作用。这些数据证实了结节蛋白中存在功能性且重叠的 14-3-3 和 Akt 识别位点。
