Protein kinase A activity and protein phosphorylation in the haemocytes of immune-challenged Galleria mellonella larvae.
Protein kinase A (PKA) activity was detected in the haemocytes of greater wax moth, Galleria mellonella larvae using a specific peptide substrate--kemptide. The enzyme was activated in vitro by 1 microM concentration of cAMP, 8-Br-cAMP, 8-Chl-cAMP and BzcMP, whereas in the case of cGMP 10 microM concentration was necessary. Immune challenge of G. mellonella larvae with bacteria led to changes in haemocyte PKA activity. Gram-positive M. luteus was a better inducer of PKA activity than Gram-negative E. coli. The kinetics of activity changes was dependent on the bacteria used and considerably differed from that observed in water-treated insects. Inhibition of PKA activity by cell-permeable, specific inhibitor, Rp-8-Br-cAMPS, induced changes in haemocyte morphology resembling those caused by live bacteria. Four potential PKA substrates of 155 kDa, 44 kDa, 40 kDa and 22 kDa were recognized in the haemocytes of naive larvae by phospho-motif antibodies for PKA phosphorylation consensus site. The modification level of 40 kDa protein changed after water treatment and immune challenge of G. mellonella larvae, whereas that of 155 kDa protein changed only after E. coli and LPS injections. Additionally, in the haemocytes of bacteria- and LPS-challenged insects a transient phosphorylation of 36 kDa protein was detected.
Cytryńska
Małgorzata
M
Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Skłodowska University, 19 Akademicka St., 20-033 Lublin, Poland. cytryna@biotop.umcs.lublin.pl
Zdybicka-Barabas
Agnieszka
A
Jakubowicz
Teresa
T
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000276:immunology; D006434:Hemocytes / Q000187:drug effects / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism*; D007814:Larva / Q000201:enzymology* / Q000276:immunology / Q000382:microbiology; D016982:Micrococcus luteus / Q000276:immunology; D009036:Moths / Q000201:enzymology* / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry; D010766:Phosphorylation
D004791:Enzyme Inhibitors; D019476:Insect Proteins; D009842:Oligopeptides; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases