Protein kinase A signaling during bidirectional axenic differentiation in Leishmania

Bachmaier, Sabine; Witztum, Ronit; Tsigankov, Polina; Koren, Roni; Boshart, Michael; Zilberstein, Dan

doi:10.1016/j.ijpara.2015.09.003

Cited by 17 publications

(35 citation statements)

References 49 publications

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“…Nevertheless, cell clones can be selected that maintain a basal PKAC1 level sufficient for survival after homozygous deletion of PKAR . The resulting ∆ pkar/ ∆ pkar cell lines 38 used later in this work show a mild growth phenotype with population doubling time (PDT) of 7.5 h versus 5.4 h for wild type cells.…”

Section: Resultsmentioning

confidence: 99%

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

et al. 2019

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Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC 50 ≥ 6.5 nM) and high affinity ligands ( K D ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.

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Section: Resultsmentioning

confidence: 99%

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

et al. 2019

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“…Our previous studies identified protein kinase A (PKA) as a potential key enzyme in this process 7 . Now, we have identified and characterized a highly divergent regulatory subunit of Leishmania PKA (LdPKAR3) as another key player in promastigote-to-amastigote differentiation.…”

Section: Discussionmentioning

confidence: 99%

“…To verify that LdPKAR3 can be categorized as regulatory subunit of PKA, we tested its interaction with the PKA catalytic subunits. Because no antibodies are available for the L. donovani PKAC isoforms, we expressed LdPKAR3-Ty1 under control of a tetracycline repressor in a T. brucei Δpkar1 cell line 7,19 . The absence of TbPKAR1 in this cell line eliminates the possible competition with LdPKAR3 for interaction with TbPKAC subunits.…”

Section: Ldpkar3 Associates With a Catalytic Subunit To Form A Pka Holoenzymementioning

confidence: 99%

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A divergent protein kinase A in the human pathogenLeishmaniais associated with cell cortex microtubules and controls cell shape

Bachmaier

Ramu

et al. 2021

Preprint

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Parasitic protozoa of the genus Leishmania cause human leishmaniasis. They cycle between the phagolysosome of mammalian macrophages, where they reside as round intracellular amastigotes, and the mid-gut of female sand flies, which they colonize as elongated extracellular promastigotes. Shifting promastigotes to a lysosome-like environment (pH 5.5 and 37°C, 5% CO2) initiates their development into amastigotes. Previous studies suggested a role for protein kinase A (PKA) in this differentiation process. Here, we describe a new, divergent, regulatory subunit (PKAR3) found only in a limited member of the family Kinetoplastidae. In L. donovani, phosphorylation of PKAR3 was regulated by the differentiation signal and coincided with parasite morphogenesis during stage development. LdPKAR3 was bound to the subpellicular microtubules cell cortex via a formin homology (FH2)-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET), proteomics analyses, and structural modeling showed that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex. In promastigotes, PKAR3 recruited PKAC3 to the subpellicular microtubules at the central cortex. After exposure to a differentiation signal, PKAR3 homogenously distributed across the entire cortex, in concert with cell rounding. Deleting the genes encoding either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines the normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for a unique PKA complex not present in the host cell.

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“…Despite the importance of phosphosignalling in differentiation, there is little knowledge on the nature of kinases that mediate these transitions. Amongst these, protein kinase A (PKA) activity is known to be required for the promastigote to amastigote transition (Bachmaier et al, ), whereas target of rapamycin (TOR) 3 is known to be associated with the development of the infectious stage (Madeira da Silva & Beverley, ).…”

Section: Introductionmentioning

confidence: 99%

Leishmania dual‐specificity tyrosine‐regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype

Rocha

Dacher

Young

et al. 2020

Molecular Microbiology

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Although the multiplicative and growth-arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re-entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1 -/parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1 -/parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic-enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1 -/growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion).Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity. K E Y W O R D SDYRK1, growth, kinase, Leishmania, stationary

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Protein kinase A signaling during bidirectional axenic differentiation in Leishmania

Cited by 17 publications

References 49 publications

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

A divergent protein kinase A in the human pathogenLeishmaniais associated with cell cortex microtubules and controls cell shape

Leishmania dual‐specificity tyrosine‐regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype

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