Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium.
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.
Bonini
N M
NM
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects*; D002923:Cilia / Q000187:drug effects*; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004398:Dyneins / Q000502:physiology; D010247:Paramecium; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D010750:Phosphoproteins; C016276:8-bromocyclic GMP; D000242:Cyclic AMP; D011494:Protein Kinases; D004398:Dyneins; D006152:Cyclic GMP; D002118:Calcium