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

Abstract

Type 2A phosphatases are part of the PPP subfamily that is formed by PP2A, PP4 and PP6, the mammalian orthologs of yeast Pph21 ⁄ 22, Pph3 and Sit4, respectively. These are serine ⁄ threonine phosphatases with a wide range of substrates acting in a variety of cellular processes such as transcription, translation, regulation of the cell cycle, signal transduction and apoptosis [1][2][3][4]. PP2A has been described as a holoenzyme formed by a catalytic (C), a regulatory (B, B¢ or B¢¢) and a scaffolding (PR65 ⁄ A) subunit [1][2][3][4].Although dimers formed by AC subunits have been described in vivo, the prevalent form of the PP2A holoenzyme is the trimeric A:B:C complex. The number of B-type subunits is still growing with new members continuously being discovered. The subunit composition of the holoenzyme determines its subcellular localization, activation state and substrate specificity [1][2][3][4]. PP4 forms either a heterotrimer with the subunits PP4R2 and PP4R3 or a heterodimer with PP4R1 [5], and specific subunits of PP6 (PP6R1, 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. a4 and target of rapamycin signaling pathway regulator-like (TIPRL) are the respective mammalian orthologs of Tap42p and Tip41p. a4 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. The TIPRL-interacting region on the catalytic subunit was mapped to residues 210-309 and does not overlap with the a4-binding region, as shown by yeast two-hybrid and pull-down assays using recombinant proteins. TIPRL and a4 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 a4 in human cells. These results suggest the existence of a novel PP2A heterotrimer (a4:PP2Ac:TIPRL) in mammalian cells.Abbreviations 3-AT, 3-amino-triazol; GST, glutathione S-transferase; RBCC, ring finger B-box coiled coil; TIPRL, TOR signaling pathway regulator-like; TOR, target of rapamycin.

Control of mTORC1 signaling by the Opitz syndrome protein MID1

“…Similarly, in mammalian cells, small molecule inhibition of PP2A activity blocks rapamycin-induced dephosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 (18)(19)(20)(21)(22), suggesting that PP2A directly dephosphorylates mTORC1 substrates when mTORC1 activity is inhibited by rapamycin, or that mTORC1 constrains PP2A activity. However, with the latter scenario, it is unclear whether the analogous regulatory paradigm with the Tip41L/α4/PP2A axis in mammals is conserved from yeast (37)(38)(39). Our data are consistent with a regulatory loop involving the MID1/α4/PP2A degradation pathway, where PP2A acts on mTOR itself as opposed to acting on downstream mTORC1 substrates.…”

Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae

“…2A). TIP41 was reported to be involved in the regulation of yeast type 2A phosphatases (43). However, the functional relationship between TIP41 and fRMsr is not clear.…”

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit

“…DNA Cloning and Sequencing-The clone 3.4 cDNA from the novel PP2Ac␣2 isoform cloned into pACT2 and pGEX-5X-2, the ␣4 cDNA cloned into pET28a, and the TIPRL cDNA cloned into pET-TEV (a pET28a derivative) were already available in our laboratory from previous studies (12,20). The PP2Ac␣ cDNA (from the gene located on chromosome 5q31.1, GRCh37.p5, NC_000005.9) was amplified by RT-PCR from several immortalized human cell lines and from peripheral blood mononuclear cells (PBMCs) and cloned using the pGEM-T-easy cloning system (Promega) according to the manufacturer's instructions.…”

“…Protein expression was induced with isopropyl 1-thio-␤-D-galactopyranoside (0.5 mM), and the cells were incubated at 16°C for 16 h or at 25°C for 4 h. Cell lysis and interaction assays were performed as described previously (12).…”

“…Both can interact simultaneously constituting a trimeric complex (12). ␣4 has been recognized as a major regulator of PP2A complex assembly and PP2Ac degradation (13,14).…”