Hsp27 phosphorylation is induced in alveolar macrophages exposed to CdO-coated silica particles.
Protein phosphorylation in bovine alveolar macrophages (BAM) activated by quartz dusts and metal oxide-coated silica particles was investigated by means of two-dimensional electrophoresis (2D-PAGE) and densitometric 32[P]-phosphate image analysis. BAM activity was monitored by determining generated superoxide anions and hydrogen peroxide. In vitro stimulation of BAM with cadmium oxide-coated silica particles (LiC-CdO) resulted in characteristic time-dependent changes in 2D-PAGE spot patterns, that were similar to the effects induced by 4ss-phorbol-12-myristate-13-acetate (PMA). Phosphorylation of two proteins with apparent molecular masses of 29 and 42 kDa appeared as main signals in both LiC-CdO and in PMA treated BAM but with different kinetics. Phosphoprotein spot pp29 was identified as an isoelectric form of Hsp27 by microsequence and Western blot analysis. In contrast to PMA stimulation, LiC-CdO-induced Hsp27 and p42 phosphorylation did not correlate with the amount of generated reactive oxygen intermediates. Other potent BAM activators like quartz dust SIKRON F600 or VO-coated silica particles did not show Hsp27 and p42 phosphorylation. LiC-CdO-mediated Hsp27 phosphorylation was inhibited by SB 203580 indicating that p38 MAP kinase is the upstream mediator of the activated signaling pathway(s), while MEK inhibitor PD 98059 had no effect.
Department of Biochemistry and Molecular Biology, University of Hamburg, D-20146 Hamburg, Germany.
M
M
Radloff
M
M
Delling
T
T
Marti
G
G
Gercken
D000818:Animals; D019187:Cadmium Compounds / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D005419:Flavonoids / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000378:metabolism*; D010087:Oxides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D011791:Quartz / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D012822:Silicon Dioxide / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D019187:Cadmium Compounds; D005419:Flavonoids; D006360:Heat-Shock Proteins; D007093:Imidazoles; D010087:Oxides; D010750:Phosphoproteins; D011725:Pyridines; C029663:cadmium oxide; D013481:Superoxides; D011791:Quartz; D012822:Silicon Dioxide; D006861:Hydrogen Peroxide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one