THE INTERCONVERSION OF PROTEIN PHOSPHATASE 2A BETWEEN PP2A(1) AND PP2A(0) DURING RETINOIC ACID-INDUCED GRANULOCYTIC DIFFERENTIATION AND A MODIFICATION ON THE CATALYTIC SUBUNIT IN S-PHASE OF HL-60 CELLS

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-indu ced differentiation of HL-60 cells have been investigated. PP2A activi ty of HL-60 cells for phosphorylated myelin basic protein showed a sha rp and transient increase after 18-h treatment with 1 mu M retinoic ac id, which corresponded to G1/S boundary of the cell cycle, This PP2A o f the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 M NaCl, while PP2A from control cells was eluted with 0.23 M NaCl , The phosphorylase phosphatase activity of PP2A in the 0.13 M eluate was greatly enhanced in the presence of protamine compared with that o f the later eluting PP2A. Immunoblot analyses with antisera against B' and B alpha subunits showed that the PP2A in the 0.13 M NaCl eluate f rom 18-h retinoic acid-treated cells was PP2A(0) (AC-B'), whereas the PP2A eluted with 0.23 M NaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A(1) (AC-B alpha). These resul ts strongly suggest that PP2A undergoes a transient and reversible int erconversion of holoenzyme forms during the initial stage of retinoic acid-induced granulocytic differentiation. PP2A activity assayed after dissociation of the catalytic subunit, for phosphorylase as substrate , showed a sharp and transient decrease in S phase of HL 60 cells irre spective of the presence or absence of retinoic acid, Immunoblot analy ses with antisera against C-terminus and N-terminus of the catalytic s ubunit of PP2A suggested that a modification at the C-terminus is resp onsible for the decrease in PP2A activity, Immunoreactivity to the C-t erminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group f rom the C-terminus, These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells. (C) 1997 Academic Press.