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

2012 ◽  
Vol 199 (4) ◽  
pp. 639-651 ◽  
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.

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

2001 ◽  
Vol 153 (2) ◽  
pp. 443-448 ◽  
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.

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

2006 ◽  
Vol 17 (1) ◽  
pp. 227-238 ◽  
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.

scholarly journals The A-kinase-anchoring protein AKAP-Lbc facilitates cardioprotective PKA phosphorylation of Hsp20 on Ser16

2012 ◽  
Vol 446 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Helen V. Edwards ◽  
John D. Scott ◽  
George S. Baillie
Keyword(s):  
Molecular Processes ◽  
Dependent Protein Kinase ◽  
Ischaemia Reperfusion Injury ◽  
Ischaemia Reperfusion ◽  
Anchoring Proteins ◽  
Anchoring Protein ◽  
Extracellular Stimuli ◽  
Dependent Protein ◽  
A Cell ◽  
Apoptotic Effects

Hsp20 (heat-shock protein of 20 kDa; HspB6) is a cardioprotective agent which combats a number of pathophysiological processes in the heart, including hypertrophy, apoptosis and ischaemia/reperfusion injury. The cardioprotective actions of Hsp20 require its phosphorylation by PKA (cAMP-dependent protein kinase) on Ser16. Although the extracellular stimuli that promote cAMP-responsive phosphorylation of Hsp20 are well known, less is understood about the molecular processes that regulate this modification. AKAPs (A-kinase-anchoring proteins) physically compartmentalize PKA to specific locations within a cell to both direct PKA phosphorylation toward selected substrates and to orchestrate downstream signalling events. In the present study we used PKA anchoring disruptor peptides to verify that an AKAP underpins the cardioprotective phosphorylation of Hsp20. Biochemical and immunofluorescence techniques identify the cytosolic protein AKAP-Lbc (AKAP13) as the anchoring protein responsible for directing PKA phosphorylation of Hsp20 on Ser16. Gene silencing and rescue experiments establish that AKAP-Lbc-mediated PKA phosphorylation of Hsp20 is crucial to the anti-apoptotic effects of the Hsp. Thus AKAP-Lbc may serve an ancillary cardioprotective role by favouring the association of PKA with Hsp20.

Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice

2021 ◽  
Vol 13 (600) ◽  
pp. eabc7275
Author(s):  
Xiaoguang Fang ◽  
Zhi Huang ◽  
Kui Zhai ◽  
Qian Huang ◽  
Weiwei Tao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Nuclear Dna ◽  
Stem Cell Differentiation ◽  
Primary Brain Tumor ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Human Gbm ◽  
Stem Cell State

Glioblastoma (GBM), a lethal primary brain tumor, contains glioma stem cells (GSCs) that promote malignant progression and therapeutic resistance. SOX2 is a core transcription factor that maintains the properties of stem cells, including GSCs, but mechanisms associated with posttranslational SOX2 regulation in GSCs remain elusive. Here, we report that DNA-dependent protein kinase (DNA-PK) governs SOX2 stability through phosphorylation, resulting in GSC maintenance. Mass spectrometric analyses of SOX2-binding proteins showed that DNA-PK interacted with SOX2 in GSCs. The DNA-PK catalytic subunit (DNA-PKcs) was preferentially expressed in GSCs compared to matched non–stem cell tumor cells (NSTCs) isolated from patient-derived GBM xenografts. DNA-PKcs phosphorylated human SOX2 at S251, which stabilized SOX2 by preventing WWP2-mediated ubiquitination, thus promoting GSC maintenance. We then demonstrated that when the nuclear DNA of GSCs either in vitro or in GBM xenografts in mice was damaged by irradiation or treatment with etoposide, the DNA-PK complex dissociated from SOX2, which then interacted with WWP2, leading to SOX2 degradation and GSC differentiation. These results suggest that DNA-PKcs–mediated phosphorylation of S251 was critical for SOX2 stabilization and GSC maintenance. Pharmacological inhibition of DNA-PKcs with the DNA-PKcs inhibitor NU7441 reduced GSC tumorsphere formation in vitro and impaired growth of intracranial human GBM xenografts in mice as well as sensitized the GBM xenografts to radiotherapy. Our findings suggest that DNA-PK maintains GSCs in a stem cell state and that DNA damage triggers GSC differentiation through precise regulation of SOX2 stability, highlighting that DNA-PKcs has potential as a therapeutic target in glioblastoma.

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

2004 ◽  
Vol 381 (3) ◽  
pp. 587-592 ◽  
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.

mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes

1999 ◽  
Vol 112 (16) ◽  
pp. 2725-2736 ◽  
Author(s):  
M.S. Kapiloff ◽  
R.V. Schillace ◽  
A.M. Westphal ◽  
J.D. Scott
Keyword(s):  
Protein Kinase ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Dependent Protein Kinase ◽  
Repeat Sequences ◽  
Camp Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
Anchoring Protein ◽  
Dependent Protein ◽  
Green Fluorescent

The compartmentalization of second messenger-activated protein kinases contributes to the fidelity of hormone-mediated signal transduction events. For example, the cAMP-dependent protein kinase is tethered at specific intracellular locations through association with A-kinase anchoring proteins (AKAPs). We now report the cloning of mAKAP, an anchoring protein found predominantly in heart, skeletal muscle and brain, and whose expression is induced in neonatal ventriculocytes by treatment with hypertrophic stimuli. mAKAP is targeted to the nuclear membrane of differentiated myocytes. Analysis of mAKAP-green fluorescent protein (GFP) fusion constructs revealed that nuclear membrane targeting is conferred by two regions of the protein, between residues 772–915 and 915–1065, which contain spectrin-like repeat sequences. Heterologous expression of the mAKAP targeting sequences displaced the endogenous anchoring protein from the nuclear membrane, demonstrating that mAKAP targeting is saturable. Collectively, these data suggest that a domain containing spectrin-like repeats mediates targeting of the anchoring protein mAKAP and the cAMP-dependent protein kinase holoenzyme to the nuclear membrane in response to differentiation signals.

scholarly journals LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
You-hong Wang ◽  
Zhen Guo ◽  
Liang An ◽  
Yong Zhou ◽  
Heng Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nasopharyngeal Cancer ◽  
Noncoding Rnas ◽  
Dependent Protein Kinase ◽  
Damage Repair ◽  
Dependent Protein ◽  
Cancer Tissues

AbstractRadioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

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