Identification of a PP2A-interacting protein that functions as a negative regulator of phosphatase activity in the ATM/ATR signaling pathway

Abstract

Protein serine/threonine phosphatase 2A (PP2A) activity must be tightly controlled to maintain cell homeostasis. Here, we report the identification of a previously uncharacterized mammalian protein, type 2A-interacting protein (TIP), as a novel regulatory protein of PP2A and the PP2A-like enzymes PP4 and PP6. TIP is a ubiquitously expressed protein and parallels the distribution of the PP2A catalytic subunit. Unlike its role in yeast, TIP does not interact with the mammalian homolog of type 2A-associated protein of 42 kDa (Tap42), a4, but instead associates with PP2A, PP4 and PP6 catalytic subunits independently of mammalian target of rapamycin kinase activity. Interestingly, the 20 kDa TIP splice variant TIP_i2, which lacks amino acids 173-272 of TIP's C-terminus, does not interact with PP2A; this finding indicates that residues 173-272 are important for the assembly of the TIP . phosphatase complex. In contrast to purified PP2A holoenzymes, TIP . PP2A complexes are devoid of phosphatase activity. Furthermore, alterations in the cellular levels of TIP influence the phosphorylation state of a specific protein substrate of ataxia-telangiectasia mutated (ATM)/ATM-and Rad3-related (ATR) kinases. Elevated levels of TIP result in an increase in the phosphorylation state of this protein substrate, whereas TIP-depleted cells exhibit a significant decrease in this protein's phosphorylation state, which is reversed by treatment with the PP2A inhibitor okadaic acid. These results indicate TIP is a novel inhibitory regulator of PP2A and implicate a role for TIP . PP2A complexes within the ATM/ATR signaling pathway controlling DNA replication and repair.

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.…”

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Similarly, in mammalian cells, the inhibitory protein TIP (type-2A-interacting protein) interacts directly with the C subunit in the absence of the A subunit. Its yeast homolog, Tip41p (Tap42p-interacting protein of 41 kDa), however, interacts with and inhibits Tap42p activity [67]. Another non-classical PP2A complex comprises the C subunit, an atypical cyclin, G1 or G2, and a PR61/B 0 subunit [68,69].…”

Activation and regulation of ATM kinase activity in response to DNA double-strand breaks

“…This suggests that autophosphorylation of ATM is regulated by PP2A and that PP2A may constitutively dephosphorylate ATM in human cells. Recently, McConnell et al (2007) identified a type 2A interacting protein (TIP), which interacts with the catalytic subunit of PP2A and inhibits PP2A phosphatase activity. They also showed that overexpression of TIP causes an increase in phosphorylation of ATM/ATR substrates, and depletion of TIP by siRNA causes a reduction in phosphorylation of ATM substrates.…”