Distinguishing functions of trypanosomatid protein kinases

Cayla, Mathieu; Nievas, Y. Romina; Matthews, Keith R.; Mottram, Jeremy C.

doi:10.1016/j.pt.2022.08.009

Cited by 9 publications

(7 citation statements)

References 87 publications

(116 reference statements)

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“…In this parasite, the second messenger system can regulate cell growth and differentiation by modulating protein phosphorylation of a set of protein substrates essential for these processes [11]. Several protein kinases play key roles in trypanosomatid protozoa, and disrupting their activity harms the parasite [41,43]. It is well known that reversible protein phosphorylation is one of the most important mechanisms for quickly regulating adaptive responses to intraand extracellular signals in several organisms [44].…”

Section: Discussionmentioning

confidence: 99%

In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes

Maldonado,

Canobra,

Oyarce

et al. 2024

Microorganisms

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Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite’s growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in T. cruzi epimastigotes.

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Section: Discussionmentioning

confidence: 99%

In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes

Maldonado,

Canobra,

Oyarce

et al. 2024

Microorganisms

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“…Trypanosomatids are evolutionarily divergent eukaryotes and exhibit unique characteristics in their cell cycle, many of which will be regulated by protein kinases. 32 This includes its unique apparatus for chromosome segregation and the regulatory processes that govern the highly orchestrated duplication and segregation of single-copy organelles. 32 Therefore, it is important to know the state of the art on this topic.…”

Section: Introductionmentioning

confidence: 99%

“… 32 This includes its unique apparatus for chromosome segregation and the regulatory processes that govern the highly orchestrated duplication and segregation of single-copy organelles. 32 Therefore, it is important to know the state of the art on this topic. 32 …”

Section: Introductionmentioning

confidence: 99%

GSK-3 kinase a putative therapeutic target in trypanosomatid parasites

Diaz,

Canal,

Valdés

et al. 2024

The Brazilian Journal of Infectious Diseases

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“…These pathways are obviously uncoupled from the cAMP unresponsive PKA and seem to use alternative and novel cAMP effectors (Bachmaier et al 2023). PKA in these parasites is essential and important for cell division (Bachmaier et al 2019; Baker et al 2021; Cayla et al 2022) and in T. brucei has been identified as candidate member of a quorum sensing pathway and implicated in stage development (Mony et al 2014; Toh et al 2021). The genome of T. brucei encodes one regulatory and three catalytic subunits that all have syntenic orthologs in T. cruzi and Leishmania .…”

Section: Introductionmentioning

confidence: 99%

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens

Ober,

Githure,

Santos

et al. 2023

Preprint

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Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signalling proteins, including PKA and PKG. PKA of phylogenetically distantTrypanosomais the first exception as it is cyclic nucleotide independent and responsive to nucleoside analogues (Bachmaier et al. 2019). Here we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens T. brucei,T. cruziandLeishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of 2-3 amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligandin vivoinT. bruceibloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signalling.

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Distinguishing functions of trypanosomatid protein kinases

Cited by 9 publications

References 87 publications

In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes

In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes

GSK-3 kinase a putative therapeutic target in trypanosomatid parasites

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens

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