Possible involvement of cAMP and protein phosphorylation in the cell signaling pathway for resting cyst formation of ciliated protozoan Colpoda cucullus

摘要

It has been suggested that encystment of Colpoda cucullus is mediated by intracellular Ca~(2+)-activated signaling pathways involving an increase in the cAMP concentration. In the present study, cAMP enzyme immunoassay (EIA) and chemiluminescence detection for phosphorylated proteins using anti-phosphoserine antibody, anti-phosphothreonine antibody and biotinylated phosphate-binding tag molecules (Phos-tag) showed that the intracellular cAMP concentration in Colpoda cells was raised and the phosphorylation level of serine/ threonine residues was elevated in many proteins prior to the cyst formation. Such encystment induction and protein phosphorylation were suppressed by the addition of an intracellular Ca ~(2+) chelating reagent (BAPTA-AM) or the encystment inhibitor, chlorophyllin in all or most of these proteins, respectively. The phosphorylation level of some proteins was slightly elevated by the addition of IBMX, which tended to promote encystment induction, and tended to be slightly suppressed by the addition of H-89 (PKA inhibitor), which also suppressed encystment induction. These results suggest that Ca~(2+)-activated signaling pathway involving cAMP/PKA-dependent protein phosphorylation may be responsible for the encystment induction of C. cucullus.