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Ca²⁺-binding proteins of cilia and infraciliary lattice of Paramecium tetraurelia: their phosphorylation by purified endogenous Ca²⁺-dependent protein kinases

¹ Department of Oncology, McArdle Lab, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
² Department of Biochemistry, College of Agricultural and Life Science, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

^* Author for correspondence (e-mail: nelson{at}biochem.wisc.edu )

Accepted 31 January 2002

We purified two small, acidic calcium-binding proteins (Paramecium Ca²⁺-binding proteins, PCBP-25 and PCBP-25ß) from Paramecium tetraurelia by Ca²⁺-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25ß did not react with PCBP-25, and antibodies against centrin from Chlamydomonas reacted with PCBP-25 but not with PCBP-25ß. 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-25ß 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-25 is a Paramecium centrin and that PCBP-25ß is a distinct Ca²⁺-binding protein that confers Ca²⁺ sensitivity on some component of the cilium, ciliary basal body or ICL.

We characterized these proteins and Paramecium calmodulin as substrates for two Ca²⁺-dependent protein kinases purified from Paramecium. PCBP-25 and calmodulin were in vitro substrates for one of the two Ca²⁺-dependent protein kinases (CaPK-2), but only PCBP-25 was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25 and calmodulin are regulated by phosphorylation.

Key words: Centrin, Phosphorylation, Paramecium, Infraciliary lattice, Cilia, Dynein

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