Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily

Dahlin, Heather; Zheng, Ning; Scott, John D.

doi:10.1016/j.jbc.2021.100927

Cited by 6 publications

(5 citation statements)

References 72 publications

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“…A PKAR1::mNG signal was still observed in the axoneme of the examined RSP knockouts, including the short axonemes that are characteristic of RSP3 deletion mutants (Supplementary Figure 4F-I). These data suggest that flagellar PKA localisation does not depend on the integrity of the radial spokes in Leishmania, consistent both with the lack of a DD domain in LmxPKARs and the recognition that whilst the RSP3 AKAP domain is homologous to PKA-binding AKAP sequences, its function in the axoneme is docking of non-PKA RII domain proteins (66,67).…”

Section: Localisation Dependenciessupporting

confidence: 57%

Divergent Protein Kinase A contributes to the regulation of flagellar waveform of the motile flagellum ofLeishmania mexicana

Fochler,

Walker,

Wheeler

et al. 2023

Preprint

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Protein kinase A (PKA) is one of the most studied eukaryotic signal transducers and a key target of the second messenger cAMP. Kinetoplastids, a branch of early diverging eukaryotes, possess homologues of catalytic and regulatory PKA subunits that, unusually, are insensitive to cAMP with uncertainty about the endogenous activating ligand. While previous evidence has implicated both cAMP and PKA in the regulation of flagellar motility in kinetoplastids, the specific contributions of the two regulatory (PKAR) and three catalytic (PKAC) subunits of PKA remain unclear. Here we conducted a systematic study combining reverse genetics and microscopy analysis to investigate PKAC and PKAR subcellular localisations, pairing preferences, and the effect of PKA gene deletions on swimming speed and flagellar waveforms inLeishmania mexicanapromastigotes.LmxPKAC1,LmxPKAC2 andLmxPKAR1 are enriched in the flagellum and upon detergent extraction, theLmxPKAR1 andLmxPKAC1 signals remained associated with the cytoskeleton. The flagellarLmxPKAC1 andLmxPKAC2 signals were, however, greatly diminished by removal ofLmxPKAR1, consistent with an anchoring function for the R-subunit. Previous work identified theStreptomycesantimetabolite toyocamycin, activated the divergent PKA ofTrypanosoma brucei. The patterns of sequence divergence within the phosphate binding cassettes ofLmxPKAR1 andLmxPKAR3 suggest both are divergent fromT. brucei. We therefore tested the effect of toyocamycin and a similarly structured compound of the purine metabolism pathway, inosine, on the localisation ofLmxPKAC1 and found that theLmxPKAC1 mNG signal was lost from the cytoskeletal fraction following treatment with either toyocamycin or inosine.LmxPKAC3 andLmxPKAR3 were localised to the cell cortex. Upon removal ofLmxPKAR3,LmxPKAC3 was lost from the cortex and became distributed throughout the cell, including an increased presence in the flagellum. Assessing motility phenotypes, we found this increase ofLmxPKAC3 signal in the flagellum correlated with an increase in swimming speed. Whereas the deletion ofLmxPKAC3 resulted in flagella beating at significantly reduced frequencies and a resulting decrease in population swimming speed. Moreover,LmxPKAC1 null mutants showed a significantly reduced swimming speed and cells were unable to produce symmetric flagellar waves. Taken together these data indicate that the divergent PKA pathway inLeishmaniamodulates flagellar motility.Author SummaryMany cells use whip-like cellular appendages, called flagella, for swimming. Regulating the rhythm and strength of the flagellar beat is important to determine the speed at which cells swim and in what direction they move. How this regulation is accomplished is not fully understood. Here we examined the role of protein kinase A (PKA) in flagellar motility of the unicellular parasite Leishmania. Leishmania have three catalytic PKA proteins, which transduce upstream signals through protein phosphorylation, and two regulatory PKA proteins that serve to anchor the catalytic subunits to specific locations within the cell. We show that two catalytic and one regulatory subunit are part of the flagellar cytoskeleton, while the other regulatory subunit docks its catalytic subunit to the cytoskeleton surrounding the cell body. Removal of individual PKA proteins perturbed the flagellar beat in different ways. We found that cells that lacked the catalytic subunit PKAC1 could only beat their flagella in an uncoordinated manner and as a consequence swam more slowly. These findings suggest that Leishmania PKA proteins are part of a pathway that regulates flagellar beating.

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Section: Localisation Dependenciessupporting

confidence: 57%

Divergent Protein Kinase A contributes to the regulation of flagellar waveform of the motile flagellum ofLeishmania mexicana

Fochler,

Walker,

Wheeler

et al. 2023

Preprint

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“…Notably, all DLBC cases with genetic alteration (~2% frequency) had SPA17 amplification ( Figure 1B ) . However, in general, the frequency of the genetic alteration of SPA17 was not high, which may be related to the high conservation characteristics of SPA17 ( 33 ).…”

Section: Resultsmentioning

confidence: 85%

Sperm Autoantigenic Protein 17 Predicts the Prognosis and the Immunotherapy Response of Cancers: A Pan-Cancer Analysis

Peng

Long

et al. 2022

Front. Immunol.

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BackgroundSperm autoantigen protein 17 (SPA17) is a highly conserved mammalian protein that participates in the acrosome reaction during fertilization and is a recently reported member of the cancer-testicular antigen (CTA) family. It has been reported that the SPA17 expression is limited in adult somatic tissues and re-expressed in tumor tissues. Recently, studies have found that SPA17 regulates the progression of various cancers, but its role in cancer immunotherapy is not clear.MethodsThe pan-cancer and normal tissue transcriptional data were acquired from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) datasets. We explored the SPA17 pan-cancer genomic alteration analysis in the cBioPortal webtool. The Human Protein Atlas (HPA) and ComPPI websites were used to mine the SPA17 protein information. We performed a western blotting assay to validate the upregulated SPA17 expression in clinical glioblastoma (GBM) samples. The univariate Cox regression and Kaplan–Meier method were used to assess the prognostic role of SPA17 in pan-cancer. Gene Set Enrichment Analysis (GSEA) was used to search the associated cancer hallmarks with SPA17 expression in each cancer type. TIMER2.0 was the main platform to investigate the immune cell infiltrations related to SPA17 in pan-cancer. The associations between SPA17 and immunotherapy biomarkers were performed by Spearman correlation analysis. The drug sensitivity information from the Connectivity Map (CMap) dataset was downloaded to perform SAP17-specific inhibitor sensitivity analysis.FindingsSPA17 was aberrantly expressed in most cancer types and exhibited prognosis predictive ability in various cancers. In addition, our results also show that SPA17 was significantly correlated with immune-activated hallmarks (including pathways and biological processes), immune cell infiltrations, and immunoregulator expressions. The most exciting finding was that SPA17 could significantly predict anti-PDL1 and anti-PD1 therapy responses in cancer patients. Finally, specific inhibitors, like irinotecan and puromycin, which correlate with SPA17 expression in different cancer types, were also screened using Connectivity Map (CMap).ConclusionsOur results reveal that SPA17 was abnormally expressed in cancer tissues, and this expression pattern could be associated with immune cell infiltrations in tumor microenvironments. Clinically, SPA17 not only acted as a potent prognostic factor to predict the clinical outcomes of cancer patients but was also a promising immunotherapy predictive biomarker for cancer patients treated with immune-checkpoint inhibitors (ICIs).

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“…Tpk isoenzymes phosphorylate the same basophilic recognition sequence (RRXS*/T* motif) but phosphoproteomics has revealed that the isoforms target different substrates, raising the question as to how substrate specificity arises [ 40 ]. In mammalian cells, substrate specificity can be driven by scaffold proteins called A-kinase anchoring proteins (AKAPs), which are responsible for determining the subcellular localisation of the holoenzyme and subsequent access to its activators and substrates [ 41 ]. Unlike the single yeast R protein, there are four mammalian R protein isoforms which display differential affinities with AKAPs.…”

Section: The Mechanics Of Pka Enzymatic Control By Camp and Regulator...mentioning

confidence: 99%

Yeast Protein Kinase A Isoforms: A Means of Encoding Specificity in the Response to Diverse Stress Conditions?

et al. 2022

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Eukaryotic cells have developed a complex circuitry of signalling molecules which monitor changes in their intra- and extracellular environments. One of the most widely studied signalling pathways is the highly conserved cyclic AMP (cAMP)/protein kinase A (PKA) pathway, which is a major glucose sensing circuit in the yeast Saccharomyces cerevisiae. PKA activity regulates diverse targets in yeast, positively activating the processes that are associated with rapid cell growth (e.g., fermentative metabolism, ribosome biogenesis and cell division) and negatively regulating the processes that are associated with slow growth, such as respiratory growth, carbohydrate storage and entry into stationary phase. As in higher eukaryotes, yeast has evolved complexity at the level of the PKA catalytic subunit, and Saccharomyces cerevisiae expresses three isoforms, denoted Tpk1-3. Despite evidence for isoform differences in multiple biological processes, the molecular basis of PKA signalling specificity remains poorly defined, and many studies continue to assume redundancy with regards to PKA-mediated regulation. PKA has canonically been shown to play a key role in fine-tuning the cellular response to diverse stressors; however, recent studies have now begun to interrogate the requirement for individual PKA catalytic isoforms in coordinating distinct steps in stress response pathways. In this review, we discuss the known non-redundant functions of the Tpk catalytic subunits and the evolving picture of how these isoforms establish specificity in the response to different stress conditions.

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Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily

Cited by 6 publications

References 72 publications

Divergent Protein Kinase A contributes to the regulation of flagellar waveform of the motile flagellum ofLeishmania mexicana

Divergent Protein Kinase A contributes to the regulation of flagellar waveform of the motile flagellum ofLeishmania mexicana

Sperm Autoantigenic Protein 17 Predicts the Prognosis and the Immunotherapy Response of Cancers: A Pan-Cancer Analysis

Yeast Protein Kinase A Isoforms: A Means of Encoding Specificity in the Response to Diverse Stress Conditions?

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