14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s)

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 αa-14-3-3 binding site-specific antibody correlated with mitogen-induced phosphorylation of tuberin and recognition of tuberin by an α-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 Ser⁹³⁹ demonstrated phospho-specific binding to 14-3-3. Glutathione S-transferase pulldown assays with 14-3-3 fusion proteins revealed that all seven 14-3-3 isoforms (β, γ, ζ, ε, τ, η, and σ) could bind tuberin, and this interaction was abrogated by competition with phosphorylated but not unphosphorylated Ser⁹³⁹ 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.