The Interconversion of Protein Phosphatase 2A between PP2A 1and PP2A 0during Retinoic Acid-Induced Granulocytic Differentiation and a Modification on the Catalytic Subunit in S Phase of HL-60 Cells

Abstract

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-induced differentiation of HL-60 cells have been investigated. PP2A activity of HL-60 cells for phosphorylated myelin basic protein showed a sharp and transient increase after 18-h treatment with 1 μ mretinoic acid, which corresponded to G1/S boundary of the cell cycle. This PP2A of the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 mNaCl, while PP2A from control cells was eluted with 0.23 mNaCl. The phosphorylase phosphatase activity of PP2A in the 0.13 meluate was greatly enhanced in the presence of protamine compared with that of the later eluting PP2A. Immunoblot analyses with antisera against B′ and Bα subunits showed that the PP2A in the 0.13 mNaCl eluate from 18-h retinoic acid-treated cells was PP2A 0(AC-B′), whereas the PP2A eluted with 0.23 mNaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A 1(AC-Bα). These results strongly suggest that PP2A undergoes a transient and reversible interconversion 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 irrespective of the presence or absence of retinoic acid. Immunoblot analyses with antisera against C-terminus and N-terminus of the catalytic subunit of PP2A suggested that a modification at the C-terminus is responsible for the decrease in PP2A activity. Immunoreactivity to the C-terminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group from the C-terminus. These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells.