title: Protein kinase C isotypes and signaling in neutrophils. Differential substrate specificities of a translocatable calcium- and phospholipid-dependent beta-protein kinase C and a phospholipid-dependent protein kinase which is inhibited by long chain fatty acyl coenzyme A.
abstract: Neutrophils possess a classical Ca2+, phosphatidyl serine (PS) and diglyceride (DG)-dependent protein kinase C (beta-PKC) which was translocatable from cytosol to membrane in response to elevated Ca2+ in the physiologic range or to pretreatment with phorbol myristate acetate (PMA). The translocatable beta-PKC was purified from neutrophil membranes prepared in the presence of Ca2+, eluted with EGTA and subjected to hydroxyapatite chromatography. An 80-kDa protein possessing Ca/DG/PS-dependent histone phosphorylating activity was recognized by a monoclonal antibody to beta-PKC but not to alpha-PKC or gamma-PKC. A cytosolic kinase activity remaining after Ca(2+)-induced translocation of beta-PKC was dependent on PS and DG but did not require Ca2+. This novel Ca(2+)-independent, PS/DG-dependent kinase, termed nPKC, eluted from hydroxyapatite between alpha-PKC and beta-PKC, ran as a 76-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was reactive to a polyclonal consensus antibody but not to monoclonal antibodies to alpha-PKC, beta-PKC, or gamma-PKC. Long chain fatty acyl-CoA, but not the corresponding free fatty acids, inhibited nPKC in the 1-10 microM range. The chemotactic peptide fMet-Leu-Phe triggered prompt but transient increases in neutrophil long chain fatty acid acyl-CoA, suggesting that nPKC is regulated by fatty acyl-CoA as well as DG during neutrophil activation. Purified beta-PKC phosphorylated a number of cytosolic proteins in a Ca(2+)-dependent manner, including a major 47-kDa cytosolic protein, which may be implicated in superoxide anion generation. In contrast, nPKC did not phosphorylate the 47-kDa protein, but phosphorylated numerous cytosolic proteins in a Ca(2+)-independent manner, including a 66-kDa protein which was not phosphorylated by beta-PKC. Differences in location, substrate specificity, and cofactor dependence between nPKC and beta-PKC suggest these kinases may play selective roles in the activation sequence of the neutrophil.
authors.affiliation: Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia 19104.
authors.forename: S
authors.initials: S
authors.lastname: Majumdar
authors.forename: M W
authors.initials: MW
authors.lastname: Rossi
authors.forename: T
authors.initials: T
authors.lastname: Fujiki
authors.forename: W A
authors.initials: WA
authors.lastname: Phillips
authors.forename: S
authors.initials: S
authors.lastname: Disa
authors.forename: C F
authors.initials: CF
authors.lastname: Queen
authors.forename: R B
authors.initials: RB
authors.lastname: Johnston
authors.forename: O M
authors.initials: OM
authors.lastname: Rosen
authors.forename: B E
authors.initials: BE
authors.lastname: Corkey
authors.forename: H M
authors.initials: HM
authors.lastname: Korchak
mesh_terms: D000214:Acyl Coenzyme A / Q000494:pharmacology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000502:physiology; D002451:Cell Compartmentation; D003065:Coenzyme A / Q000378:metabolism; D004075:Diglycerides / Q000502:physiology; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D010718:Phosphatidylserines / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000145:classification / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000214:Acyl Coenzyme A; D000911:Antibodies, Monoclonal; D004075:Diglycerides; D005230:Fatty Acids, Nonesterified; D010455:Peptides; D010718:Phosphatidylserines; D010750:Phosphoproteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D003065:Coenzyme A; D002118:Calcium