Cytryńska Małgorzata, Zdybicka-Barabas Agnieszka, Jakubowicz Teresa
Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Skłodowska University, 19 Akademicka St., 20-033 Lublin, Poland.
Comp Biochem Physiol B Biochem Mol Biol. 2007 Sep;148(1):74-83. doi: 10.1016/j.cbpb.2007.04.020. Epub 2007 May 5.
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.
使用特异性肽底物——肯普肽，在大蜡螟（Galleria mellonella）幼虫的血细胞中检测到蛋白激酶A（PKA）活性。该酶在体外可被1微摩尔浓度的环磷酸腺苷（cAMP）、8-溴环磷酸腺苷（8-Br-cAMP）、8-氯环磷酸腺苷（8-Chl-cAMP）和苄基环磷酰胺（BzcMP）激活，而对于环磷酸鸟苷（cGMP），则需要10微摩尔浓度。用细菌对大蜡螟幼虫进行免疫刺激会导致血细胞PKA活性发生变化。革兰氏阳性的藤黄微球菌（M. luteus）比革兰氏阴性的大肠杆菌（E. coli）更能诱导PKA活性。活性变化的动力学取决于所使用的细菌，并且与在水处理昆虫中观察到的情况有很大不同。通过细胞可渗透的特异性抑制剂Rp-8-Br-cAMPS抑制PKA活性，会诱导血细胞形态发生变化，类似于活细菌引起的变化。通过针对PKA磷酸化共有位点的磷酸基序抗体，在未受刺激幼虫的血细胞中识别出四种潜在的155 kDa、44 kDa、40 kDa和22 kDa的PKA底物。在对大蜡螟幼虫进行水处理和免疫刺激后，40 kDa蛋白的修饰水平发生了变化，而155 kDa蛋白的修饰水平仅在注射大肠杆菌和脂多糖（LPS）后发生变化。此外，在受到细菌和LPS刺激的昆虫血细胞中，检测到36 kDa蛋白的瞬时磷酸化。
