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.
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.
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Anne R
AR
Gaillard
Laura A
LA
Fox
Jeanne M
JM
Rhea
Branch
B
Craige
Winfield S
WS
Sale
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D017124:Conserved Sequence; D005407:Flagella / Q000502:physiology*; D008969:Molecular Sequence Data; D010940:Plant Proteins; D015800:Protozoan Proteins / Q000502:physiology*; D012038:Regeneration
D048868:Adaptor Proteins, Signal Transducing; D010940:Plant Proteins; D015800:Protozoan Proteins; C060324:radial spoke protein, Chlamydomonas