Abstract

Chimeric phosphatase type 2Aenzymes are substrates for nucleoredoxin

Author(s): Katarzyna Lechward, Magdalena Zychlinska

This report deals with the effects of binding between two enzymatically active proteins: catalytic subunit of protein phosphatase type 2A (PP2Ac) and nucleoredoxin (NRX). We hypothesized that the PP2Ac constitutes a substrate for NRX, as we already detected their interaction in several independent experimental settings[6]. To distinguish the effect of binding of the NRX to its substrate from its action as an enzyme, we measured the phosphatase activity in the presence and absence ofNRX and its artificial cofactor DTT. NRX inhibited activity of recombinant catalytic subunit by 35 to 40% in dose dependent manner, whereas the C terminus of the NRX (NNRX) slightly activated phosphatase activity. Upon addition of DTT the activity of recombinant catalytic subunit of protein phosphatase 2A was inhibited by 60% and in the presence of NRX the inhibitory effect was enhanced up to 80%. A chimera of HA-PP2Ac-PR65/A was rapidly activated (up to 3.2 nmol/minxml) upon exposure to H2O2, whereasHA-PP2APR65/ A-myc-NRX activity was unaffected. In conclusion, we found that the activity of recombinant PP2Ac is lowered by DTT and NRX and that oxidation of PP2Ac upon purification procedure results in formation of internal disulfide bridge, which is a target of NRX in in vitro assays.