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 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.
Liu
Matt Y
MY
Department of Carcinogenesis, Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.
Cai
Shengli
S
Espejo
Alexsandra
A
Bedford
Mark T
MT
Walker
Cheryl Lyn
CL
D048948:14-3-3 Proteins; D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D040081:Protein Array Analysis; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity; D000077005:Tuberous Sclerosis Complex 2 Protein; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014446:Tyrosine 3-Monooxygenase / Q000276:immunology / Q000378:metabolism*
D048948:14-3-3 Proteins; D000906:Antibodies; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D014446:Tyrosine 3-Monooxygenase; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt