Abstract
alpha-4 is an essential gene and is a dominant antiapoptotic factor in various tissues that is a regulatory subunit for type 2A protein phosphatases. A multiplexed phosphorylation site screen revealed that knockdown of alpha-4 by small interfering RNA (siRNA) increased p38 mitogen-activated protein kinase (MAPK) and c-Jun phosphorylation without changes in JNK or ERK. FLAG-alpha-4 coprecipitated hemagglutinin-MEK3 plus endogenous protein phosphatase 2A (PP2A) and selectively enhanced dephosphorylation of Thr193, but not Ser189, in the activation loop of MEK3. Overexpression of alpha-4 suppressed p38 MAPK activation in response to tumor necrosis factor alpha (TNF-alpha). The alpha-4 dominant-negative domain (DND) (residues 220 to 340) associated with MEK3, but not PP2A, and its overexpression sensitized cells to activation of p38 MAPK by TNF-alpha and interleukin-1beta, but not by ansiomycin or sorbitol. The response was diminished by nocodazole or by siRNA knockdown of the Opitz syndrome protein Mid1 that binds alpha-4 to microtubules. Interference by alpha-4 DND or alpha-4 siRNA increased caspase 3/7 activation in response to TNF-alpha. Growth of transformed cells in soft agar was enhanced by alpha-4 and suppressed by alpha-4 DND. The results show that alpha-4 targets PP2A activity to MEK3 to suppress p38 MAPK activation by cytokines, thereby inhibiting apoptosis and anoikis.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Actinin / genetics*
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Animals
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Apoptosis*
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COS Cells
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Caspase 3 / metabolism
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Caspase 7 / metabolism
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Cell Line
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Chlorocebus aethiops
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Dose-Response Relationship, Drug
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Enzyme Activation / drug effects
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Gene Targeting
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Genes, Essential
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HeLa Cells
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Hemagglutinins / metabolism
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Humans
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Interleukin-1beta / pharmacology
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JNK Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 3 / chemistry
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MAP Kinase Kinase 3 / metabolism*
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Microfilament Proteins / genetics*
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Microtubule Proteins / metabolism
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Mitogen-Activated Protein Kinase 14 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 14 / metabolism*
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Nocodazole / pharmacology
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Nuclear Proteins / metabolism
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Oligopeptides
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Peptides / immunology
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Phosphoprotein Phosphatases / antagonists & inhibitors
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Phosphoprotein Phosphatases / genetics
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Phosphoprotein Phosphatases / metabolism*
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Phosphorylation
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Promoter Regions, Genetic
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Protein Phosphatase 2
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Protein Structure, Tertiary
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RNA, Small Interfering / pharmacology
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Threonine / metabolism
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Transcription Factors / metabolism
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Tumor Necrosis Factor-alpha / pharmacology
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Ubiquitin-Protein Ligases
Substances
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ACTN4 protein, human
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Hemagglutinins
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Interleukin-1beta
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Microfilament Proteins
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Microtubule Proteins
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Nuclear Proteins
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Oligopeptides
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Peptides
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RNA, Small Interfering
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Transcription Factors
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Tumor Necrosis Factor-alpha
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Actinin
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Threonine
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FLAG peptide
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MID1 protein, human
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Ubiquitin-Protein Ligases
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinase 14
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MAP Kinase Kinase 3
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MAP2K3 protein, human
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Phosphoprotein Phosphatases
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Protein Phosphatase 2
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Caspase 3
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Caspase 7
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Nocodazole