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

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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The CSC connects three major axonemal complexes involved in dynein regulation

Molecular Biology of the Cell ◽

10.1091/mbc.e12-05-0357 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3143-3155 ◽

Cited By ~ 51

Author(s):

Thomas Heuser ◽

Erin E. Dymek ◽

Jianfeng Lin ◽

Elizabeth F. Smith ◽

Daniela Nicastro

Keyword(s):

Electron Tomography ◽

Structural Heterogeneity ◽

Flagellar Motility ◽

Wild Type ◽

Localization Model ◽

Cryo Electron Tomography ◽

Radial Spokes ◽

Regulatory Complex ◽

Health And Development ◽

Cilia And Flagella

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

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A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity

Molecular and Cellular Biology ◽

10.1128/mcb.5.7.1772-1779.1985 ◽

1985 ◽

Vol 5 (7) ◽

pp. 1772-1779

Author(s):

M A Snyder ◽

J M Bishop ◽

J P McGrath ◽

A D Levinson

Keyword(s):

Binding Site ◽

Kinase Activity ◽

Atp Binding ◽

Wild Type ◽

Dependent Protein Kinase ◽

Atp Binding Site ◽

Specific Immunoglobulin ◽

Dependent Protein ◽

Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

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

The Journal of Cell Biology ◽

10.1083/jcb.153.2.443 ◽

2001 ◽

Vol 153 (2) ◽

pp. 443-448 ◽

Cited By ~ 88

Author(s):

Anne Roush Gaillard ◽

Dennis R. Diener ◽

Joel L. Rosenbaum ◽

Winfield S. Sale

Keyword(s):

Amino Acids ◽

Complete Loss ◽

Amphipathic Helix ◽

Flagellar Motility ◽

Dependent Protein Kinase ◽

Radial Spoke ◽

Binding Domains ◽

Anchoring Protein ◽

Dependent Protein ◽

Flagellar Axoneme

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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PKA-phosphorylation of PDE4D3 facilitates recruitment of the mAKAP signalling complex

Biochemical Journal ◽

10.1042/bj20040846 ◽

2004 ◽

Vol 381 (3) ◽

pp. 587-592 ◽

Cited By ~ 84

Author(s):

Jennifer J. CARLISLE MICHEL ◽

Kimberly L. DODGE ◽

Wei WONG ◽

Nicole C. MAYER ◽

Lorene K. LANGEBERG ◽

...

Keyword(s):

Protein Kinase ◽

Negative Feedback ◽

Feedback Loop ◽

Negative Feedback Loop ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Anchoring Protein ◽

Dependent Protein

mAKAP (muscle-selective A-kinase-anchoring protein) co-ordinates a cAMP-sensitive negative-feedback loop comprising PKA (cAMP-dependent protein kinase) and the cAMP-selective PDE4D3 (phosphodiesterase 4D3). In vitro and cellular experiments demonstrate that PKA-phosphorylation of PDE4D3 on Ser-13 increases the affinity of PDE4D3 for mAKAP. Our data suggest that activation of mAKAP-anchored PKA enhances the recruitment of PDE4D3, allowing for quicker signal termination.

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Increasing cGMP-dependent protein kinase activity attenuates unilateral ureteral obstruction-induced renal fibrosis

AJP Renal Physiology ◽

10.1152/ajprenal.00657.2013 ◽

2014 ◽

Vol 306 (9) ◽

pp. F996-F1007 ◽

Cited By ~ 17

Author(s):

Wenpeng Cui ◽

Hasiyeti Maimaitiyiming ◽

Xinyu Qi ◽

Heather Norman ◽

Qi Zhou ◽

...

Keyword(s):

Transgenic Mice ◽

Renal Fibrosis ◽

Cell Function ◽

Wild Type ◽

Dependent Protein Kinase ◽

Tubular Cells ◽

Cgmp Dependent Protein Kinase ◽

Proximal Tubular ◽

Dependent Protein

Our previous studies support the protective effect of cGMP and cGMP-dependent protein kinase I (PKG-I) pathway on the development of renal fibrosis. Therefore, in the present studies, we determined whether pharmacologically or genetically increased PKG activity attenuates renal fibrosis in a unilateral ureteral obstruction (UUO) model and also examined the mechanisms involved. To increase PKG activity, we used the phosphodiesterase 5 inhibitor sildenafil and PKG transgenic mice. UUO model was induced in wild-type or PKG-I transgenic mice by ligating the left lateral ureteral and the renal fibrosis was observed after 14 days of ligation. Sildenafil was administered into wild-type UUO mice for 14 days. In vitro, macrophage and proximal tubular cell function was also analyzed. We found that sildenafil treatment or PKG transgenic mice had significantly reduced UUO-induced renal fibrosis, which was associated with reduced TGF-β signaling and reduced macrophage infiltration into kidney interstitial. In vitro data further demonstrated that both macrophages and proximal tubular cells were important sources of UUO-induced renal TGF-β levels. The interaction between macrophages and tubular cells contributes to TGF-β-induced renal fibrosis. Taken together, these data suggest that increasing PKG activity ameliorates renal fibrosis in part through regulation of macrophage and tubular cell function, leading to reduced TGF-β-induced fibrosis.

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Regulation of Flagellar Dynein by Phosphorylation of a 138-kD Inner Arm Dynein Intermediate Chain

The Journal of Cell Biology ◽

10.1083/jcb.136.1.167 ◽

1997 ◽

Vol 136 (1) ◽

pp. 167-176 ◽

Cited By ~ 136

Author(s):

Geoffrey Habermacher ◽

Winfield S. Sale

Keyword(s):

Genetic Analysis ◽

Regulatory Proteins ◽

Flagellar Motility ◽

Wild Type ◽

Functional Assays ◽

Dynein Intermediate Chain ◽

In Vitro Reconstitution ◽

Experimental Approaches ◽

Intermediate Chain

One of the challenges in understanding ciliary and flagellar motility is determining the mechanisms that locally regulate dynein-driven microtubule sliding. Our recent studies demonstrated that microtubule sliding, in Chlamydomonas flagella, is regulated by phosphorylation. However, the regulatory proteins remain unknown. Here we identify the 138-kD intermediate chain of inner arm dynein I1 as the critical phosphoprotein required for regulation of motility. This conclusion is founded on the results of three different experimental approaches. First, genetic analysis and functional assays revealed that regulation of microtubule sliding, by phosphorylation, requires inner arm dynein I1. Second, in vitro phosphorylation indicated the 138-kD intermediate chain of I1 is the only phosphorylated subunit. Third, in vitro reconstitution demonstrated that phosphorylation and dephosphorylation of the 138-kD intermediate chain inhibits and restores wild-type microtubule sliding, respectively. We conclude that change in phosphorylation of the 138-kD intermediate chain of I1 regulates dynein-driven microtubule sliding. Moreover, based on these and other data, we predict that regulation of I1 activity is involved in modulation of flagellar waveform.

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Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G

mBio ◽

10.1128/mbio.02011-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 18

Author(s):

Abhisheka Bansal ◽

Kayode K. Ojo ◽

Jianbing Mu ◽

Dustin J. Maly ◽

Wesley C. Van Voorhis ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Kinase ◽

Protein Kinase G ◽

Wild Type ◽

Dependent Protein Kinase ◽

Calcium Dependent ◽

Dependent Protein ◽

Reduced Activity ◽

Calcium Dependent Protein Kinase

ABSTRACT We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 ( Pf CDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes. IMPORTANCE Protein kinases of Plasmodium falciparum are being actively pursued as drug targets to treat malaria. However, compensatory mechanisms may reverse the drug activity against a kinase. In this study, we show that replacement of the wild-type threonine gatekeeper residue with methionine reduces the transphosphorylation activity of CDPK1. Mutant parasites with methionine gatekeeper residue compensate the reduced activity of CDPK1 through the action of PKG possibly by upregulation of CDPK6 or some other kinase. This study highlights that targeting one enzyme may lead to changes in transcript expression of other kinases that compensate for its function and may select for mutants that are less dependent on the target enzyme activity. Thus, inhibiting two kinases is a better strategy to protect the antimalarial activity of each, similar to artemisinin combination therapy or malarone (atovaquone and proguanil).

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The CSC is required for complete radial spoke assembly and wild-type ciliary motility

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0271 ◽

2011 ◽

Vol 22 (14) ◽

pp. 2520-2531 ◽

Cited By ~ 52

Author(s):

Erin E. Dymek ◽

Thomas Heuser ◽

Daniela Nicastro ◽

Elizabeth F. Smith

Keyword(s):

Calcium Binding ◽

Critical Role ◽

Artificial Microrna ◽

Structural Studies ◽

Wild Type ◽

Radial Spoke ◽

Radial Spokes ◽

Dynein Arms ◽

Cilia And Flagella

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.

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