Mutation of Tyr307 and Leu309 in the Protein Phosphatase 2A Catalytic Subunit Favors Association with the α4 Subunit Which Promotes Dephosphorylation of Elongation Factor-2

Chung, Hyun Kee; Nairn, Angus C.; Murata, Kohei; Brautigan, David L.

doi:10.1021/bi990902g

Cited by 89 publications

(94 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…To further investigate whether the increased phosphorylation of b-catenin is directly linked to the increased phosphorylation of Tyr307 and, thus, inhibition of PP2A, DLD-1 cells were transiently transfected with an N-terminal triple (HA)-tagged PP2A catalytic subunit C construct (Chung et al, 1999). A wild-type catalytic subunit ((HA) 3 -C WT ) construct served as a control and a construct in which Tyr307 of the catalytic subunit is mutated to a phenylalanine ((HA) 3 -C Y307F ) to eliminate this site of phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

“…Transient transfection with PP2A constructs DLD-1 were transfected with an N-terminal triple (HA)-tagged PP2A catalytic subunit C constructs (Chung et al, 1999) or GFP-tagged DNA at 6 Â 10 6 cells/transfection on a nucleofector (Amaxa, Cologne, Germany) using a standard protocol from the supplier and solution V and program T-20. The (HA) 3 -C Y307F construct contains a mutated Tyr307 of the catalytic subunit (tyrosine is mutated to a phenylalanine) to eliminate this site of phosphorylation and one wild-type catalytic subunit ((HA) 3 -C WT ) to serve as control (Chung et al, 1999).…”

Section: Pp2a Activity Determinationmentioning

confidence: 99%

See 1 more Smart Citation

Effect of aspirin on the Wnt/β-catenin pathway is mediated via protein phosphatase 2A

et al. 2006

View full text Add to dashboard Cite

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/b-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of b-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of b-catenin were assessed by immunoblotting, and also the localization of b-catenin was shown by green fluorescent protein-tagged b-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/b-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/b-catenin pathway activity in these cells.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Pp2a Activity Determinationmentioning

confidence: 99%

Effect of aspirin on the Wnt/β-catenin pathway is mediated via protein phosphatase 2A

et al. 2006

View full text Add to dashboard Cite

show abstract

“…PR61/B 0 d and PR72/B 00 recruitment is unaffected by these mutations [54,55], revealing isoform-specific differences within B-type families. Finally, the double Tyr307!Phe, Leu309!Gln mutation favors association with a4 [56], indicating that assembly with a4 might not require stabilizing contacts with the Asp306-Tyr-Phe-Leu309 C-terminal tail. Although the mutational studies are interesting, it remains to be determined whether these phospho-mimetic mutations indeed phenocopy the phosphorylated state.…”

Section: Reviewmentioning

confidence: 99%

“…However, a variety of experimental approaches both in yeast and mammalian cells demonstrate that PP2A C methylation has a crucial role in B-type-subunit binding, specifically in the recruitment of PR55/B subunits [20][21][22][23]44,[51][52][53][54][55]. By contrast, the recruitment of A [44,[51][52][53]55], PR61/B 0 [21][22][23]55], PR72/B 00 [54,55] and striatin [44] does not strictly require PP2A C methylation in vivo, and binding of polyoma middle T [44,51] and a4 [20,56] is increased in the absence of methylation. In yeast, the deletion of PPM1 (protein phosphatase methyltransferase 1, which encodes the yeast LCMT1 ortholog) or the expression of PP2A C harboring a Leu309!Ala substitution, inhibits Cdc55p binding [20][21][22][23], whereas Tpd3p (tRNA-processing-deficient mutant 3; the yeast A subunit homolog) and Rts1p binding is only slightly diminished [20][21][22][23].…”

Section: Trends In Biochemical Sciencesmentioning

confidence: 99%

“…Several site-directed mutagenesis studies have provided additional insights: Thr304!Ala and Thr304!Asp mutants can be methylated in vitro [55] and in vivo [44], so it is likely that Thr304 phosphorylation does not affect PP2A methylation; however, the methylation deficiency of Tyr307!Asp [55] or Tyr307!Glu phospho-mimetic mutants [44] indicates that Tyr307phosphorylation might prevent methylation and, thereby, PP2A T55 assembly. It is possible that the hydroxyl group of the tyrosyl side chain is the determining factor here, as some studies indicate that a Tyr307!Phe mutant cannot be methylated [55,56].…”

Section: Reviewmentioning

confidence: 99%

See 1 more Smart Citation

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

Janssens

Longin

Goris

2008

Trends in Biochemical Sciences

366

424

View full text Add to dashboard Cite

The α4‐containing form of protein phosphatase 2A in liver and hepatic cells

Yoo

Jimenez

Sanders

et al. 2008

J of Cellular Biochemistry

Self Cite

View full text Add to dashboard Cite

The Ser/Thr phosphatase PP2A is a set of multisubunit enzymes that regulate many cellular processes. In yeast, the PP2A regulatory subunit Tap42 forms part of the Target of Rapamycin (TOR) signaling pathway that links nutrient and energy availability to cell growth. The physiological intersection between the mammalian orthologs of Tap42 and TOR, α4 and mTOR, has not been fully characterized. We used two in vivo models of liver growth in the rat, late gestation fetal development and regeneration after partial hepatectomy, to explore the regulation of the α4-containing form of PP2A. The α4/PP2A catalytic subunit (α4/PP2A-C) complex was present in both fetal and adult liver extracts. There was a trend towards higher levels of α4 protein in fetal liver, but the complex was more abundant in adult liver. Fractionation of extracts by ion exchange chromatography and transient transfection of the AML12 mouse hepatic cell line indicated that α4 associates with PP2A-C but that these complexes have low catalytic activity with both peptide and protein substrates. α4 was able to associate with forms of PP2A-C that were both methylated and non-methylated at the carboxy-terminus. The mTOR inhibitor rapamycin did not block the formation of α4/PP2A-C in liver or hepatic cells, nor did it appear to modulate PP2A activity. Furthermore, sensitivity to the growth inhibitory effects of rapamycin among a panel of hepatic cell lines did not correlate with levels of α4 or α4/PP2A-C. Our results indicate that the yeast Tap42/TOR paradigm is not conserved in hepatic cells. KeywordsProtein phosphorylation; mTOR; rapamycin; regeneration; cell growth PP2A is a major Ser/Thr phosphatase that plays a significant role in the regulation of many cellular processes, including metabolism, transcription, translation, cell cycle progression, cell growth and apoptosis [Janssens and Goris, 2001;Mumby and Walter, 1993;Sontag, 2001;Zolnierowicz, 2000]. The activity of such a versatile enzyme must therefore be tightly controlled in vivo. Indeed, the catalytic subunit of PP2A (PP2A-C) is regulated at many levels. The C-terminus of PP2A-C is post-translationally modified by phosphorylation at Y307 and methyl esterification at L309. Phosphorylation results in inhibition of PP2A activity [Chen et al., 1992;Guo and Damuni, 1993] while methylation is thought to play an important function in regulatory subunit binding [Bryant et al., 1999;Tolstykh et al., 2000;Wu et al., 2000;Yu et al., 2001]. More specifically, PP2A-C methylation can influence the association of PP2A-C with specific regulatory subunits, thereby serving to determine the NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript composition, subcellular localization and function of PP2A-C holoenzymes in a variety of cell types [Gentry et al., 2005;Longin et al., 2007;Nunbhakdi-Craig et al., 2007].The prototypical PP2A enzyme is a heterodimer that consists of the catalytic C subunit and scaffolding A subunit. The substrate specificity and subcellular localization of PP2A are...

show abstract

Mutation of Tyr307 and Leu309 in the Protein Phosphatase 2A Catalytic Subunit Favors Association with the α4 Subunit Which Promotes Dephosphorylation of Elongation Factor-2

Cited by 89 publications

References 47 publications

Effect of aspirin on the Wnt/β-catenin pathway is mediated via protein phosphatase 2A

Effect of aspirin on the Wnt/β-catenin pathway is mediated via protein phosphatase 2A

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

The α4‐containing form of protein phosphatase 2A in liver and hepatic cells

Contact Info

Product

Resources

About