Expression of protein phosphatase 2A mutants and silencing of the regulatory Bα subunit induce a selective loss of acetylated and detyrosinated microtubules

Abstract

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the Ba regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation-(L309D) and phosphorylation-(T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B¢, and B¢¢-type regulatory subunits in NIH 3T3 fibroblasts and neuro2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309D, and T304D mutants, which are impaired in their ability to associate with the Ba subunit, induces their loss. Silencing of Ba subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal Ba-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.

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

“…PP2A C Tyr307 phosphorylation might selectively inhibit holoenzyme assembly As stated, the PP2A C Tyr307!Asp [55] or Tyr307!Glu [44] phospho-mimetic mutants cannot be methylated and, therefore, fail to interact with PR55/B [44,51,54,55] or Cdc55p [22,23]; hence, Tyr307 phosphorylation might indirectly affect PP2A T55 assembly. Moreover, these mutations also inhibit interaction of PP2A C with PR61/ B 0 abge [54,55] or Rts1p [23] (which can occur in the absence of methylation), indicating that, in this case, stabilizing contacts with Tyr307 are needed.…”

“…By contrast, a mixture of methylated and demethylated PP2A C is found in immunoprecipitates of endogenous PR61/B 0 d and overexpressed GST fusions of PR61/B 0 a, b, d and e isoforms in addition to GST-PR72/B 00 and GST-PR70/B 00 [55]. Moreover, deletion of Leu309 or a Leu309!Ala substitution in PP2A C abolishes PR55/B binding [44,52,54,55], whereas binding of PR61/B 0 b, PR61/B 0 e [55], PR61/B 0 d and PR72/B 00 subunits is not affected [54,55]. There are conflicting data regarding PR61/B 0 a binding, which might be explained by the different experimental approaches used [54,55].…”

“…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].…”

“…Moreover, deletion of Leu309 or a Leu309!Ala substitution in PP2A C abolishes PR55/B binding [44,52,54,55], whereas binding of PR61/B 0 b, PR61/B 0 e [55], PR61/B 0 d and PR72/B 00 subunits is not affected [54,55]. There are conflicting data regarding PR61/B 0 a binding, which might be explained by the different experimental approaches used [54,55]. Notably, the absence of PP2A C Leu309!Ala mutant binding to PR55/B is not due to lack of methylation [55].…”

“…Moreover, these mutations also inhibit interaction of PP2A C with PR61/ B 0 abge [54,55] or Rts1p [23] (which can occur in the absence of methylation), indicating that, in this case, stabilizing contacts with Tyr307 are needed. Indeed, structural data show a hydrogen bond between the Tyr307 hydroxyl group and the carbonyl group of Val257 in the peptide backbone of PR61/B 0 g1 [36] (Box 1, Figure Ib).…”