Monocytic activation of protein tyrosine kinase, protein kinase A and protein kinase C induced by porins isolated from Salmonella enterica serovar Typhimurium.
OBJECTIVE
In the present study a monocytic cell line, U937, was used to investigate the possible involvement of protein tyrosine kinases (NT-PTKs), protein kinase A (PKA) and protein kinase C (PKC) in cell signaling pathways following Salmonella enterica serovar Typhimurium porin stimulation.


METHODS
Different concentrations of porins and lipopolysaccharide (LPS) were analysed to evaluate changes in PTK activity by a non radioactive tyrosine kinase assay and in PKA and PKC phosphorylation by Western blotting analysis. The inhibitors of PTK, PKA and PKC activation used, were: 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase activity; dihychloride (H-89), a selective inhibitor of PKA which is useful to discriminate between the effects of PKC and PKA; diacylglycerol kinase inhibitor II (R59949), which is useful for elucidating roles of PKC; calphostin C, a specific inhibitor of PKC.


RESULTS
Porins of the outer membrane of the ST were isolated to be used as a stimulus in the performed experiments. Following porin treatment, a dose-dependent increase in PTK, PKA and PKC activation was observed. U937 monocytes pretreated with inhibitors induced an evident decrease in PTK activity and PKA and PKC phosphorylation pattern in porin stimulated monocytes.


CONCLUSIONS
Our data support the important role played by NT-PTK, PKA and PKC in transducing the activating signal in macrophages stimulated with porins through the activation of the mitogen-activated protein kinase (MAPK) pathway that participate in the regulation of gene expression.
Galdiero
M
M
Dipartimento di Medicina, Sperimentale, Sezione di Microbiologia e Microbiologia Clinica, facoltà di Medicina e Chiruga, Seconda Università degli Studi di Napoli, Italy. marilena.galdiero@unina2.it
D'Isanto
M
M
Vitiello
M
M
Finamore
E
E
Peluso
L
L
Galdiero
M
M
D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000201:enzymology* / Q000382:microbiology; D009281:Naphthalenes / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D018272:Porins / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology; D052999:Quinazolinones; D012486:Salmonella typhimurium / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013449:Sulfonamides; D020032:Tyrphostins / Q000494:pharmacology; D020298:U937 Cells
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008070:Lipopolysaccharides; D009281:Naphthalenes; D010880:Piperidines; D018272:Porins; D011799:Quinazolines; D052999:Quinazolinones; D013449:Sulfonamides; D020032:Tyrphostins; C058544:R 59949; D007770:L-Lactate Dehydrogenase; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C058819:calphostin C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C060641:tyrphostin A23