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.