Interaction analysis of the heterotrimer formed by the phosphatase 2A catalytic subunit, alpha4 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. alpha4 and target of rapamycin signaling pathway regulator-like (TIPRL) are the respective mammalian orthologs of Tap42p and Tip41p. alpha4 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. Tauhe TIPRL-interacting region on the catalytic subunit was mapped to residues 210-309 and does not overlap with the alpha4-binding region, as shown by yeast two-hybrid and pull-down assays using recombinant proteins. TIPRL and alpha4 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 alpha4 in human cells. These results suggest the existence of a novel PP2A heterotrimer (alpha4:PP2Ac:TIPRL) in mammalian cells.
Smetana
Juliana H C
JH
Center for Structural Molecular Biology, Brazilian Synchrotron Light Laboratory (LNLS), Campinas, Brazil.
Zanchin
Nilson I T
NI
D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry*; D020134:Catalytic Domain; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D020014:K562 Cells; D008969:Molecular Sequence Data; D054648:Protein Phosphatase 2 / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D001704:Biopolymers; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C444301:TIP41 protein, S cerevisiae; C577326:TIPRL protein, human; D054648:Protein Phosphatase 2