Cytokine activation of p38 mitogen-activated protein kinase and apoptosis is opposed by alpha-4 targeting of protein phosphatase 2A for site-specific dephosphorylation of MEK3.
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.
Prickett
Todd D
TD
Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Jefferson Park Avenue, Hospital West Complex, Room 7225, Charlottesville, VA 22908-1400, USA.
Brautigan
David L
DL
D000185:Actinin / Q000235:genetics*; D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D018390:Gene Targeting; D020043:Genes, Essential; D006367:HeLa Cells; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics*; D008868:Microtubule Proteins / Q000378:metabolism; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases
C111453:ACTN4 protein, human; D006388:Hemagglutinins; D053583:Interleukin-1beta; D008840:Microfilament Proteins; D008868:Microtubule Proteins; D009687:Nuclear Proteins; D009842:Oligopeptides; D010455:Peptides; D034741:RNA, Small Interfering; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D000185:Actinin; D013912:Threonine; C085706:FLAG peptide; C109052:MID1 protein, human; D044767:Ubiquitin-Protein Ligases; D048031:JNK Mitogen-Activated Protein Kinases; D048308:Mitogen-Activated Protein Kinase 14; D048371:MAP Kinase Kinase 3; C482530:MAP2K3 protein, human; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D053148:Caspase 3; D053179:Caspase 7; D015739:Nocodazole