Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2AC, PP4C, and PP6C) and analysis of the interaction of PP2AC with alpha4 protein

Kloeker, Susanne; Reed, Robin B.; McConnell, Jamie L.; Chang, Dennis Hsu-Tung; Tran, Kim C.; Westphal, Ryan S.; Law, Brian K.; Colbran, Roger J.; Kamoun, Malek; Campbell, Kerry S.; Wadzinski, Brian E.

doi:10.1016/s1046-5928(03)00141-4

Cited by 62 publications

(67 citation statements)

References 88 publications

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“…Although the mammalian homolog of Tap42, a4, has been shown to interact with the catalytic subunits of PP2A family members (PP2Ac, PP4c and PP6c), the effect of a4 on phosphatase activity remains controversial and appears to be substratedependent, as it has been shown to both enhance and inhibit PP2A (Murata et al, 1997;Inui et al, 1998). Moreover, the stability of Tap42 Á phosphatase complexes is dependent on rapamycin-sensitive TOR kinase activity, whereas mammalian target of rapamycin (mTOR)-dependent regulation of the a4 Á phosphatase complex is in question (Murata et al, 1997;Chen et al, 1998;Kloeker et al, 2003). Indeed, the role of a4 Á phosphatase complexes as downstream effectors of mTOR is itself dubious.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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“…Some results derived from immunoprecipitation using different antibodies suggest that the dimeric (AC) form of PP2A may constitute up to a third of the total PP2A pool [22]. Previous studies on the expression of hepatic PP2A components in the adult rat [23][24][25] have identified both dimeric and heterotrimeric PP2A complexes by using affinity purified antibodies specific for PP2A subunits [26]. Limited data are available on the forms of PP2A in fetal liver [27].…”

Section: Introductionmentioning

confidence: 99%

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)

Yoo¹,

Boylan²,

Brautigan³

et al. 2007

Archives of Biochemistry and Biophysics

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The prototypical form of the Ser/Thr phosphatase PP2A is a heterotrimeric complex consisting of catalytic subunit (C), A and B regulatory subunits. C-terminal methylation of PP2A-C influences holoenzyme assembly. Using late gestation development in the rat as an in vivo model of liver growth, we found that PP2A-C protein and activity levels were higher in fetal compared to adult liver extracts. However, unmethylated PP2A-C was much higher in the adult extracts. In MonoQ fractionation, unmethylated C eluted separately from methylated C, which was present predominantly in ABC heterotrimers. Gel filtration chromatography revealed that some unmethylated C was present as free catalytic subunit. In addition, a significant proportion of PP2A was in inactive forms that may involve novel regulatory subunits. Our results indicate that methylation of PP2A-C appears to be a primary determinant for the biogenesis of PP2A heterotrimers.

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“…38 The phosphatase activity affecting the phosphorylation state of downstream targets of mTOR appears to be dependent on its association with the a4 protein, 36,37,39 an integral component of the rapamycin-sensitive-signaling pathway. 1 The complex a4/ phosphatase and its dephosphorylating activity on S6K is disrupted in the presence of phosphatase inhibitors, 40,41 whereas the effects of rapamycin are controversial. 35,36,39,41 The decreased cell growth rate and the progressive reduction of cell size accompanying or promoted by S6K1 deactivation that follows the inhibition of the mTOR kinase depress cell proliferation 20,29,30,42,43 (with wide variations among cell types) by a complex mechanism still poorly understood.…”

Section: Introductionmentioning

confidence: 99%

“…1 The complex a4/ phosphatase and its dephosphorylating activity on S6K is disrupted in the presence of phosphatase inhibitors, 40,41 whereas the effects of rapamycin are controversial. 35,36,39,41 The decreased cell growth rate and the progressive reduction of cell size accompanying or promoted by S6K1 deactivation that follows the inhibition of the mTOR kinase depress cell proliferation 20,29,30,42,43 (with wide variations among cell types) by a complex mechanism still poorly understood. Whether these changes in cell growth (and size) affect cell survival by inhibition or promotion of cell death is still controversial and no specific experiments have been addressed to evaluate their proapoptotic or antiapoptotic effects on the different death pathways.…”

Section: Introductionmentioning

confidence: 99%

Cell size reduction induced by inhibition of the mTOR/S6K-signaling pathway protects Jurkat cells from apoptosis

et al. 2005

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In Jurkat cells, the decreased cell growth rate associated with a long-lasting deactivation of the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K)-signaling pathway generates a cell population of progressively reduced cellular mass and size. When promoted by rapamycin as prototype inhibitor, the mTOR deactivation-dependent cell size reduction was associated with slowed, but not suppressed, proliferation. Small-size cells were significantly protected from apoptosis induced by Fas/Apo-1 deathreceptor activation (as shown by reduced procaspase cleavage and decreased catalytic activity of relevant caspases) or by stress signals-dependent mitochondrial perturbation (as shown by reduced cleavage of caspase-2, lower dissipation of mitochondrial membrane potential and decreased release of cytochorome c and apoptosis-inducing factor from mitochondria). Protection faded when reactivation of the mTOR/S6K pathway promoted the cell recovery to normal size. These results suggest that cells induced to reduce their mass by the mTOR deactivation-dependent inhibition of cell growth become more resilient to lethal assaults by curbing the cell's suicidal response.

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Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2AC, PP4C, and PP6C) and analysis of the interaction of PP2AC with alpha4 protein

Cited by 62 publications

References 88 publications

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

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

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)

Cell size reduction induced by inhibition of the mTOR/S6K-signaling pathway protects Jurkat cells from apoptosis

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