Activation of several MAP kinases upon stimulation of rat alveolar macrophages: role of the NADPH oxidase.
Zymosan-activated serum (ZAS), a source of C5a, stimulates the rat alveolar macrophages (AM) to release superoxide anion. Here we show that treatment of rat AM with ZAS induced a time-dependent increase in the tyrosine phosphorylation of several proteins (116, 105-110, 82-78, 66-72, 62, 45, 42, and 38 kDa). This increase was sensitive to genistein, a tyrosine kinase inhibitor. ZAS stimulated the tyrosine phosphorylation and activation of three members of a family of serine/threonine kinases known as the mitogen-activated protein kinases (MAPK), i.e., ERK1 and ERK2, as assessed by immunoblotting, immunoprecipitation, and phosphotransferase activity, and p38 MAPK, as determined by immunoblotting with phospho-specific antibodies. In addition, ZAS induced the tyrosine phosphorylation of the SHC proteins and their association with GRB2, suggesting a role for this complex in the activation of the ERK pathway. Addition of extracellular catalase during ZAS stimulation significantly reduced the tyrosine phosphorylation response and the activation of ERK1 and ERK2 and their activator MEK1/2 while it did not affect that of p38 MAPK and MKK3/MKK6. Superoxide dismutase marginally increased the response to ZAS, supporting a role for hydrogen peroxide. In contrast to the results with AM, stimulation of human neutrophils with ZAS in the presence of catalase minimally altered the activation of ERK1 and ERK2. These data show that, in ZAS-stimulated rat AM, activation of the respiratory burst and production of hydrogen peroxide via superoxide dismutation are largely responsible for the activation of the ERK pathway through an upstream target.
Torres
M
M
Department of Pediatrics, Childrens Hospital Los Angeles Research Institute, School of Medicine, California 90027, USA. mtorres@chla.usc.edu
Forman
H J
HJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006861:Hydrogen Peroxide / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D019255:NADPH Oxidases / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D015054:Zymosan / Q000494:pharmacology; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D010750:Phosphoproteins; D011506:Proteins; D019000:Phosphotyrosine; D015054:Zymosan; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D066246:ErbB Receptors; D017871:Calcium-Calmodulin-Dependent Protein Kinases