Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells

Citation
T. Myles et al., Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells, BIOCHEM J, 357, 2001, pp. 225-232
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
0264-6021 → ACNP
Volume
357
Year of publication
2001
Part
1
Pages
225 - 232
Database
ISI
SICI code
0264-6021(20010701)357:<225:AMITCF>2.0.ZU;2-W
Abstract
Members of the phosphoprotein phosphatase (PPP) family of protein serine/th reonine phosphatases. including protein phosphatase (PP)1, PP2A and PP2B, s hare invariant active-site residues that are critical for catalytic functio n [Zhuo, Clemens, Stone and Dixon (1994) J. Biol. Chem, 269, 26234-26238]. Mutation of the active-site residues Asp(88) or His(118) within the human P P2A catalytic subunit (PP2Ac)alpha impaired catalytic activity in vitro; th e D88N and H118N substitutions caused a 9- and 23-fold reduction in specifi c activity respectively, when compared with wild-type recombinant PP2Ac, in dicating an important role for these residues in catalysis. Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Ac alpha, neither substituted for the endogenous PP2Ac enzyme of budding yeast, Relative to wild-type PP2 Ac, the active-site mutants were dramatically overexpressed in High Five((R )) insect cells using the baculovirus system. Milligram quantities of PP2Ac were purified from 1x10(9) High Five cells and the kinetic constants for d ephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notatio n) by PP2Ac (K-m = 337.5 muM; k(eat) = 170 s(-1)) and D88N (K-m = 58.4 muM; k(eat) = 2 s(-1)) were determined. The results show that the substitution impairs catalysis severely without a significant effect on substrate bindin g, consistent with the PPP catalytic mechanism. Combination of the baculovi rus and yeast systems provides a strategy whereby the structure-function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in ripe.