Modulation of the Enzymatic Properties of Protein Phosphatase 2A Catalytic Subunit by the Recombinant 65‐kDa Regulatory Subunit PR65α

Abstract

All protein phosphatase 2A (PP2A) holoenzymes contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 6.5 kDa (PR6.5). We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR6Sa expressed in bacteria or insect cells. Bacterially expressed PR6Sa exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system. The association of recombinant PR6.5 with PP2Ac was very tight (&fP = 85 pM) and led to a suppression of PP2A activity, which was maximal (70-80 %) when phosphoproteins were used as substrates. When less-structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30 %. PR6.5 stimulated PP2Ac activity when the assays were performed in the presence of polycations. This indicates that the PR6.5 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac. Furthermore, we identified a site of interaction between PP2Ac and PR6Sa by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416. This produced an almost 100-fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR6.5 and PP2Ac.

Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the Aα subunit gene

“…Therefore, we reason that R418 interacts directly with the catalytic subunit. Mutation of the nearby Lys416 to Glu also causes a defect in C subunit binding (Turowski et al, 1997). Together, our ®ndings further con®rm our model of the A subunit structure and underline the importance of amino acids located in intra-repeat loop regions for binding B and C subunits.…”

Organization and Regulation of Small Conductance Ca2+-activated K+Channel Multiprotein Complexes

“…This previously described mutation decreases the affinity of PR65/A for the catalytic subunit of PP2A by ϳ100-fold (Turowski et al, 1997). Consistent with an effect of PP2A on SK2, the deactivation time constant was 39 Ϯ 2 ms (n ϭ 4) for cells cotransfected with the mutant PP2A, whereas the deactivation time constant from wild-type control cells was significantly larger: 54 Ϯ 2 ms (n ϭ 6) (Fig.…”

Regulation of Protein Phosphatase 2A Activity by Caspase-3 during Apoptosis

“…With respect to core enzyme and free catalytic subunit, the situation is also complex. Depending on the substrate size, type of substrate, and reaction conditions, the A subunit either inhibits or stimulates the catalytic subunit (57).…”