Interconversion of Mn2+-Dependent and -Independent Protein Phosphatase 2A from Human Erythrocytes: Role of Zn2+ and Fe2+ in Protein Phosphatase 2A

• Published in: Journal of biochemistry (Tokyo) Vol. 126; no. 3; pp. 632 - 638
• Main Authors: Nishito, Yasumasa, Usui, Hirofumi, Tanabe, Osamu, Shimizu, Masahiro, Takeda, Masao
• Format: Journal Article
• Language: English
• Published: Oxford University Press 01.09.1999
• Subjects: fluoride; protein phosphatase 2A; vanadate; zinc- and iron-metalloenzyme; zinc-metallothionein
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a022495

Human erythrocyte Mn2+-dependent (CA') and -independent (CA) protein-serine/threo-nine phosphatase (PP) 2A are composed of 34-kDa catalytic C and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett 447, 29–33]. The Mn2+-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn2+-dependent form with higher PTP activity. The same NaF treatment had no effect on CA'. Pre-incubation of CA' with ZnCl2, zinc-metallothionein, or FeCl2 activated the Mn2+-independent PP activity, but pre-incubation with FeCl3 did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl2. Pre-incubation of CA' with 5 /iM ZnCl2 and 15 >aM FeCl2 in the presence of 1 mM ascorbate synergistically stimulated the Mn2+-independent PP activity, with concomitant suppression of the Mn2+-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn2+-dependent PP activity of CA' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and CA' with the above mentioned treatments was proved. These results support the notion that Mn2+-independent CA is a Zn2+- and Fe2+-metalloen-zyme, whose apoenzyme is Mn2+-dependent CA'.