Roles for beta II-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells.
beta-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O(2)). Neutrophils and neutrophilic HL60 cells contain both betaI and betaII-PKC, isotypes that are derived by alternate splicing. betaI-PKC-positive and betaI-PKC null HL60 cells generated equivalent amounts of O(2) in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of betaII-PKC from betaI-PKC null cells inhibited ligand-initiated O(2) generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47(phox), with betaII-PKC but not with RACK1, a binding protein for betaII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O(2) generation. Therefore, betaII-PKC but not betaI-PKC is essential for activation of O(2) generation and plays a positive role in signaling for NADPH oxidase activation in association with p47(phox). In contrast, RACK1 is involved in negative signaling for O(2) generation. RACK1 binds to betaII-PKC but not with the p47(phox).betaII-PKC complex. RACK1 may divert betaII-PKC to other signaling pathways requiring beta-PKC for signal transduction. Alternatively, RACK1 may sequester betaII-PKC to down-regulate O(2) generation.
Korchak
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HM
Department of Pediatrics, University of Pennsylvania School of Medicine, The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. korchak@email.chop.edu
Kilpatrick
L E
LE
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D015536:Down-Regulation; D018274:Electroporation; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D019255:NADPH Oxidases; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D000075370:Receptors for Activated C Kinase; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D008024:Ligands; D016376:Oligonucleotides, Antisense; D010455:Peptides; D010750:Phosphoproteins; D000075370:Receptors for Activated C Kinase; D011994:Recombinant Proteins; D013481:Superoxides; C070301:peptide I; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D064546:Protein Kinase C beta