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

• Published in: Microorganisms (Basel) Vol. 12; no. 5; p. 907
• Main Authors: Maldonado, Edio, Canobra, Paz, Oyarce, Matías, Urbina, Fabiola, Miralles, Vicente J, Tapia, Julio C, Castillo, Christian, Solari, Aldo
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01-05-2024
• Subjects: Acetic acid; Amino acids; Cell division; Cellular signal transduction; Chagas disease; DNA pol β; DNA polymerase; DNA repair; DNA-directed DNA polymerase; Enzymatic activity; Enzymes; Epimastigotes; Fetal calf serum; Gene expression; Hydrogen peroxide; Identification and classification; In vivo methods and tests; Kinases; Mass spectrometry; Mass spectroscopy; Mitochondrial DNA; Monoclonal antibodies; Mutation; Parasites; Phorbol 12-myristate 13-acetate; Phorbol esters; Phosphors; Phosphorylation; Physiological aspects; Polypeptides; Protein expression; Protein kinase C; Protein kinases; Proteins; Protozoa; Residues; Signal transduction; T. cruzi; Trypanosoma cruzi; Trypomastigotes; Vector-borne diseases; Chile; United States > US; Germany; protein kinases; DNA pol β; T. cruzi; signal transduction; phosphorylation
• DOI: 10.3390/microorganisms12050907
• EISSN: 2076-2607

Chagas disease is caused by the single-flagellated protozoan
, 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
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
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
epimastigotes.