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Originally published as MBC in Press, 10.1091/mbc.E06-02-0095 on March 29, 2006

Vol. 17, Issue 6, 2626-2635, June 2006

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Disruption of the A-Kinase Anchoring Domain in Flagellar Radial Spoke Protein 3 Results in Unregulated Axonemal cAMP-dependent Protein Kinase Activity and Abnormal Flagellar Motility

Anne R. Gaillard*,{dagger}, Laura A. Fox*, Jeanne M. Rhea*, Branch Craige*, and Winfield S. Sale*

*Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322; and {dagger}Department of Biological Sciences, Sam Houston State University, Huntsville, TX 77341

Submitted February 3, 2006; Revised March 17, 2006; Accepted March 21, 2006
Monitoring Editor: Erika Holzbaur

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.

This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-02-0095) on March 29, 2006.

Address correspondence to: Winfield S. Sale ( win{at}cellbio.emory.edu)




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