Ca(2+)-binding proteins of cilia and infraciliary lattice of Paramecium tetraurelia: their phosphorylation by purified endogenous Ca(2+)-dependent protein kinases.

We purified two small, acidic calcium-binding proteins (Paramecium Ca(2+)-binding proteins, PCBP-25alpha and PCBP-25beta) from Paramecium tetraurelia by Ca(2+)-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25beta did not react with PCBP-25alpha, and antibodies against centrin from Chlamydomonas reacted with PCBP-25alpha but not with PCBP-25beta. Like the centrins described previously, both PCBPs were associated with the infraciliary lattice (ICL), a fibrillar cytoskeletal element in Paramecium. Both were also present in isolated cilia, from which they could be released (with dynein) by a high-salt wash, and both PCBPs cosedimented with dynein in a sucrose gradient. PCBP-25beta was especially prominent in cilia and in the deciliation supernatant, a soluble fraction released during the process of deciliation. The results of immunoreactivity and localization experiments suggest that PCBP-25alpha is a Paramecium centrin and that PCBP-25beta is a distinct Ca(2+)-binding protein that confers Ca(2+) sensitivity on some component of the cilium, ciliary basal body or ICL. We characterized these proteins and Paramecium calmodulin as substrates for two Ca(2+)-dependent protein kinases purified from Paramecium. PCBP-25alpha and calmodulin were in vitro substrates for one of the two Ca(2+)-dependent protein kinases (CaPK-2), but only PCBP-25alpha was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25alpha and calmodulin are regulated by phosphorylation.