Downregulation of protein phosphatase 2A activity in HeLa cells at the G2-mitosis transition and unscheduled reactivation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA).
In the cell cycle the transition from G2 phase to cell division (M) is strictly controlled by protein phosphorylation-dephosphorylation reactions effected by several protein kinases and phosphatases. Although much indirect and direct evidence point to a key role of protein phosphatase 2A (PP2A) at the G2/M transition, the control of the enzyme activity prior to and after the transition are not fully clarified. Using synchronized HeLa cells we determined the PP2A activity (i.e. the increment sensitive to inhibition by 2nM okadaic acid) in immunoprecipitates obtained with antibodies raised against a conserved peptide sequence (residues 169-182, Ab(169/182)) of the PP2A catalytic subunit (PP2A C). Two different substrates were offered: the phospho-peptide KR(p)TIRR and histone H1 phosphorylated by means of the cyclin-dependent protein kinase p34(cdc2). The results indicate that in HeLa cells the specific activity of PP2A towards both substrates goes through a minimum in late G2 phase and stays low until metaphase. Treatment of G2 cells with TPA (10(-7) M) caused a reactivation of the downregulated PP2A activity within 20 min, i.e. the same time frame within which TPA was shown earlier to block HeLa cells at the transition from G2 to mitosis [Kinzel et al., 1988. Cancer Res. 48, 1759-1762]. Activation of PP2A was also induced by TPA in mitotic cells. The low activity of PP2A in mitotic cells was accompanied by a strong reaction of mitotic PP2A C with anti-P-Tyr antibodies in Western blots, which was reversed by treatment of mitotic cells with TPA. The results suggest that the activity of cellular PP2A requires downregulation for the transition from G2 phase to mitosis. Unscheduled reactivation of PP2A induced by TPA in late G2 phase appears to inhibit the progress into mitosis.
Klingler-Hoffmann
Manuela
M
Former Department of Pathochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Barth
Holger
H
Richards
James
J
König
Norbert
N
Kinzel
Volker
V
D000906:Antibodies / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D016195:G2 Phase / Q000502:physiology*; D006367:HeLa Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D019319:Okadaic Acid / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D021122:Protein Subunits / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000906:Antibodies; D004791:Enzyme Inhibitors; D010455:Peptides; D021122:Protein Subunits; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D013755:Tetradecanoylphorbol Acetate