Flagellar Radial Spoke Protein 3 Is an a-Kinase Anchoring Protein (Akap)

Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144–180. Amino acids 161–180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

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A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex

The Journal of Cell Biology ◽

10.1083/jcb.201111042 ◽

2012 ◽

Vol 199 (4) ◽

pp. 639-651 ◽

Cited By ~ 22

Author(s):

Priyanka Sivadas ◽

Jennifer M. Dienes ◽

Martin St. Maurice ◽

William D. Meek ◽

Pinfen Yang

Keyword(s):

Membrane Trafficking ◽

Stem Cell Differentiation ◽

Amphipathic Helix ◽

Dependent Protein Kinase ◽

Radial Spoke ◽

Amphipathic Helices ◽

Anchoring Proteins ◽

Anchoring Protein ◽

Dependent Protein

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.

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The Flagellar Motility ofChlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0630 ◽

2006 ◽

Vol 17 (1) ◽

pp. 227-238 ◽

Cited By ~ 23

Author(s):

Chun Yang ◽

Pinfen Yang

Keyword(s):

Regulatory Proteins ◽

Flagellar Motility ◽

Wild Type ◽

Dependent Protein Kinase ◽

Wide Range ◽

Anchoring Protein ◽

Radial Spokes ◽

Dependent Protein ◽

Cilia And Flagella

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.

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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

Molecular Biology of the Cell ◽

10.1091/mbc.e06-02-0095 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2626-2635 ◽

Cited By ~ 42

Author(s):

Anne R. Gaillard ◽

Laura A. Fox ◽

Jeanne M. Rhea ◽

Branch Craige ◽

Winfield S. Sale

Keyword(s):

Protein Kinase ◽

Physiological Role ◽

Binding Domain ◽

Cell Phenotype ◽

Protein Kinase Activity ◽

Flagellar Motility ◽

Dependent Protein Kinase ◽

Radial Spoke ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

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.

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GABAC-Receptor Stimulation Activates cAMP-Dependent Protein Kinase via A-Kinase Anchoring Protein 220

Journal of Pharmacological Sciences ◽

10.1254/jphs.fp0071362 ◽

2008 ◽

Vol 106 (4) ◽

pp. 578-584 ◽

Cited By ~ 7

Author(s):

Li Yang ◽

Yasuhisa Nakayama ◽

Naoki Hattori ◽

Bing Liu ◽

Chiyoko Inagaki

Keyword(s):

Protein Kinase ◽

Receptor Stimulation ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Gabac Receptor ◽

Anchoring Protein ◽

Dependent Protein

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AMY-1 Interacts with S-AKAP84 and AKAP95 in the Cytoplasm and the Nucleus, Respectively, and Inhibits cAMP-dependent Protein Kinase Activity by Preventing Binding of Its Catalytic Subunit to A-kinase-anchoring Protein (AKAP) Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m206387200 ◽

2002 ◽

Vol 277 (52) ◽

pp. 50885-50892 ◽

Cited By ~ 26

Author(s):

Makoto Furusawa ◽

Takahiro Taira ◽

Sanae M. M. Iguchi-Ariga ◽

Hiroyoshi Ariga

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Catalytic Subunit ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Anchoring Protein ◽

Dependent Protein

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The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

Biochemical Journal ◽

10.1042/bj1620411 ◽

1977 ◽

Vol 162 (2) ◽

pp. 411-421 ◽

Cited By ~ 110

Author(s):

S J Yeaman ◽

P Cohen ◽

D C Watson ◽

G H Dixon

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Kinase ◽

Phosphorylation Site ◽

Amino Acid Sequences ◽

Beta Subunit ◽

Alpha Subunit ◽

Phosphorylase Kinase ◽

Dependent Protein Kinase ◽

Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

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Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2931-2940.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2931-2940

Author(s):

S Osawa ◽

L E Heasley ◽

N Dhanasekaran ◽

S K Gupta ◽

C W Woon ◽

...

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

G Proteins ◽

Fold Increase ◽

Alpha Subunit ◽

Wild Type ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Gs Protein ◽

Camp Levels

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.

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