title: Ca(2+)-binding proteins of cilia and infraciliary lattice of Paramecium tetraurelia: their phosphorylation by purified endogenous Ca(2+)-dependent protein kinases.
abstract: 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.
authors.affiliation: Department of Oncology, McArdle Lab, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Kwanghee
authors.initials: K
authors.lastname: Kim
authors.forename: Min
authors.initials: M
authors.lastname: Son
authors.forename: Joan B
authors.initials: JB
authors.lastname: Peterson
authors.forename: David L
authors.initials: DL
authors.lastname: Nelson
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002451:Cell Compartmentation / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D002847:Chromatography, Agarose; D002923:Cilia / Q000201:enzymology* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D016805:Paramecium tetraurelia / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation
chemical_list: D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases