Interaction analysis of the heterotrimer formed by the phosphatase 2A catalytic subunit, α4 and the mammalian ortholog of yeast Tip41 (TIPRL)

Type 2A serine/threonine phosphatases are part of the PPP subfamily that is formed by PP2A, PP4 and PP6, and participate in a variety of cellular processes including transcription, translation, regulation of the cell cycle, signal transduction and apoptosis. PP2A is found predominantly as a heterotrimer formed by the catalytic subunit (C) and by a regulatory (B, B' or B'') and a scaffolding (A) subunit. Yeast Tap42p and Tip41p are regulators of type 2A phosphatases, playing antagonistic roles in the target of rapamycin signaling pathway. alpha 4 and target of rapamycin signaling pathway regulator-like (TIPRL) are the respective mammalian orthologs of Tap42p and Tip41p. alpha 4 has been characterized as an essential protein implicated in cell signaling, differentiation and survival; by contrast, the role of mammalian TIPRL is still poorly understood. In this study, a yeast two-hybrid screen revealed that TIPRL interacts with the C-terminal region of the catalytic subunits of PP2A, PP4 and PP6. Tau he TIPRL-interacting region on the catalytic subunit was mapped to residues 210-309 and does not overlap with the alpha 4-binding region, as shown by yeast two-hybrid and pull-down assays using recombinant proteins. TIPRL and alpha 4 can bind PP2Ac simultaneously, forming a stable ternary complex. Reverse two-hybrid assays revealed that single amino acid substitutions on TIPRL including D71L, I136T, M196V and D198N can block its interaction with PP2Ac. TIPRL inhibits PP2Ac activity in vitro and forms a rapamycin-insensitive complex with PP2Ac and alpha 4 in human cells. These results suggest the existence of a novel PP2A heterotrimer (alpha 4:PP2Ac:TIPRL) in mammalian cells.