A new p21waf1/cip1 isoform is an early event of cell response to oxidative stress.
p21waf1/cip1 mRNA and protein accumulate in intact cells exposed to oxidizing agents through a p53-independent, MAPK-dependent mechanism. Treatment with oxidizing agents also yields a second form of this protein (FM p21), characterized by a faster migration on SDS-PAGE. This phenomenon depends on the modification of intracellular redox conditions induced by diethylmaleate, a glutathione-depleting agent, being prevented by the pretreatment with the glutathione precursor N-acetylcysteine. The appearance of this FM p21 form is very early, being observed 5 min after exposure to diethylmaleate, long before the already observed accumulation of p21 induced by oxidative stress. Furthermore, experiments with dominant negative mutants of MEK demonstrate that, in contrast with that observed for the oxidative stress-induced accumulation of p21 mRNA and protein, the appearance of FM p21 form is not dependent from the activation of the MAPK pathway. It was previously observed (Tchou et al, 1996) that in some lung carcinoma cells long exposure to high doses of phorbol esters also induces the appearance of a faster-migrating p21 electrophoretic band and it was suggested that this could result from a different phosphorylation or from a proteolytic processing at the C-terminus of the protein. The latter is not the case for the diethylmaleate-induced FM p21 whose C-terminus is intact, as demonstrated by the expression of a C-terminus tagged p21 cDNA. On the contrary, the observed migration shift seems to be dependent on the hypophosphorylation of the protein; in fact, a pretreatment of cells with okadaic acid, an inhibitor of (serine/threonine) phosphatases, inhibits the oxidation-dependent appearance of the FM p21 and the block of protein synthesis, caused by cycloeximide, does not affect the appearance of FM p21, that thus could derive from the dephosphorylation of preexisting protein.
Esposito
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F
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy.
Cuccovillo
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Russo
L
L
Casella
F
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Russo
T
T
Cimino
F
F
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008298:Maleates / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics* / Q000502:physiology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017931:DNA Primers; D008298:Maleates; D012333:RNA, Messenger; C014476:diethyl maleate; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate