Journal of Cell Science, Vol 95, Issue 2 219-230, Copyright © 1990 by Company of Biologists

JOURNAL ARTICLES

Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium

NM Bonini and DL Nelson
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.

Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K = 10(3) Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation. To identify ciliary targets of cyclic nucleotide-dependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[gamma-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the deciliation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22S dynein, and a high molecular weight protein (greater than 300K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.

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