Molecular analysis of MAP kinase kinase signaling in Leishmania

Student thesis: Doctoral Thesis

Abstract

Mitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated.
Date of Award6 Dec 2019
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SupervisorMartin Wiese (Supervisor) & Katharine Carter (Supervisor)

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