LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

Simona John von Freyend, Heidi Rosenqvist, Annette Fink, Inga Maria Melzer, Joachim Clos, Ole Nørregaard Jensen, Martin Wiese

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.
OriginalsprogEngelsk
TidsskriftInternational Journal for Parasitology
Vol/bind40
Udgave nummer8
Sider (fra-til)969-78
ISSN0020-7519
DOI
StatusUdgivet - 21. feb. 2010

Fingeraftryk

Leishmania mexicana
Staurosporine
Protein Kinase Inhibitors
Tandem Mass Spectrometry
Tyrosine
Amino Acids
Pharmaceutical Preparations
In Vitro Techniques

Bibliografisk note

Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Citer dette

John von Freyend, Simona ; Rosenqvist, Heidi ; Fink, Annette ; Melzer, Inga Maria ; Clos, Joachim ; Jensen, Ole Nørregaard ; Wiese, Martin. / LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5. I: International Journal for Parasitology. 2010 ; Bind 40, Nr. 8. s. 969-78.
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title = "LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5",
abstract = "The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.",
author = "{John von Freyend}, Simona and Heidi Rosenqvist and Annette Fink and Melzer, {Inga Maria} and Joachim Clos and Jensen, {Ole N{\o}rregaard} and Martin Wiese",
note = "Copyright {\circledC} 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.",
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LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5. / John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette; Melzer, Inga Maria; Clos, Joachim; Jensen, Ole Nørregaard; Wiese, Martin.

I: International Journal for Parasitology, Bind 40, Nr. 8, 21.02.2010, s. 969-78.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

AU - John von Freyend, Simona

AU - Rosenqvist, Heidi

AU - Fink, Annette

AU - Melzer, Inga Maria

AU - Clos, Joachim

AU - Jensen, Ole Nørregaard

AU - Wiese, Martin

N1 - Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

PY - 2010/2/21

Y1 - 2010/2/21

N2 - The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.

AB - The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.

U2 - 10.1016/j.ijpara.2010.02.004

DO - 10.1016/j.ijpara.2010.02.004

M3 - Journal article

C2 - 20178803

VL - 40

SP - 969

EP - 978

JO - International Journal for Parasitology

JF - International Journal for Parasitology

SN - 0020-7519

IS - 8

ER -