Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178.

Research output: Contribution to Journal/Magazine › Journal article › peer-review

Published

Standard

Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178. / Gomez, Felipe C.; Ali, Nahla O. M.; Brown, Elaine et al.
In: Molecular and Biochemical Parasitology, Vol. 171, No. 2, 06.2010, p. 89-96.

Research output: Contribution to Journal/Magazine › Journal article › peer-review

Harvard

Gomez, FC, Ali, NOM, Brown, E, Walker, RG, Grant, KM & Mottram, JC 2010, 'Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178.', Molecular and Biochemical Parasitology, vol. 171, no. 2, pp. 89-96. https://doi.org/10.1016/j.molbiopara.2010.03.002

APA

Gomez, F. C., Ali, N. O. M., Brown, E., Walker, R. G., Grant, K. M., & Mottram, J. C. (2010). Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178. Molecular and Biochemical Parasitology, 171(2), 89-96. https://doi.org/10.1016/j.molbiopara.2010.03.002

Vancouver

Gomez FC, Ali NOM, Brown E, Walker RG, Grant KM, Mottram JC. Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178. Molecular and Biochemical Parasitology. 2010 Jun;171(2):89-96. doi: 10.1016/j.molbiopara.2010.03.002

Author

Gomez, Felipe C. ; Ali, Nahla O. M. ; Brown, Elaine et al. / Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178. In: Molecular and Biochemical Parasitology. 2010 ; Vol. 171, No. 2. pp. 89-96.

Bibtex

@article{47b758c6ade34049be4f39856ea81d00,

title = "Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178.",

abstract = "The activity of cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, is regulated through post-translational mechanisms, including binding of a cyclin and phosphorylation. Previously studies have shown that Leishmania mexicana CRK3 is an essential CDK that is a functional homologue of human CDK1. In this study, recombinant histidine tagged L. mexicana CRK3 and the cyclin CYCA were combined in vitro to produce an active histone H1 kinase that was inhibited by the CDK inhibitors, flavopiridol and indirubin-3-monoxime. Protein kinase activity was observed in the absence of phosphorylation of the T-loop residue Thr178, but increased 5-fold upon phosphorylation by the CDK activating kinase Civ1 of Saccharomyces cerevisiae. Seven recombinant L. major CRKs (1, 2, 3, 4, 6, 7 and 8) were also expressed and purified, none of which were active as monomers. Moreover, only CRK3 was phosphorylated by Civ1. HA-tagged CYCA expressed in L. major procyclic promastigotes was coprecipitated with CRK3 and exhibited histone H1 kinase activity. These data indicate that in Leishmania CYCA interacts with CRK3 to form an active protein kinase, confirm the conservation of the regulatory mechanisms that control CDK activity in other eukaryotes, but identifies biochemical differences to human CDK1.",

keywords = "Leishmania CRK3 Cell cycle Cyclin dependent kinase CDK Activation loop T-loop",

author = "Gomez, {Felipe C.} and Ali, {Nahla O. M.} and Elaine Brown and Walker, {Roderick G.} and Grant, {K. M.} and Mottram, {Jeremy C.}",

note = "The final, definitive version of this article has been published in the Journal, Molecular and Biochemical Parasitology 171 (2), 2010, {\textcopyright} ELSEVIER.",

year = "2010",

month = jun,

doi = "10.1016/j.molbiopara.2010.03.002",

language = "English",

volume = "171",

pages = "89--96",

journal = "Molecular and Biochemical Parasitology",

issn = "0166-6851",

publisher = "Elsevier",

number = "2",

}

RIS

TY - JOUR

T1 - Recombinant Leishmania mexicana CRK3:CYCA has protein kinase activity in the absence of phosphorylation on the T-loop residue Thr178.

AU - Gomez, Felipe C.

AU - Ali, Nahla O. M.

AU - Brown, Elaine

AU - Walker, Roderick G.

AU - Grant, K. M.

AU - Mottram, Jeremy C.

N1 - The final, definitive version of this article has been published in the Journal, Molecular and Biochemical Parasitology 171 (2), 2010, © ELSEVIER.

PY - 2010/6

Y1 - 2010/6

N2 - The activity of cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, is regulated through post-translational mechanisms, including binding of a cyclin and phosphorylation. Previously studies have shown that Leishmania mexicana CRK3 is an essential CDK that is a functional homologue of human CDK1. In this study, recombinant histidine tagged L. mexicana CRK3 and the cyclin CYCA were combined in vitro to produce an active histone H1 kinase that was inhibited by the CDK inhibitors, flavopiridol and indirubin-3-monoxime. Protein kinase activity was observed in the absence of phosphorylation of the T-loop residue Thr178, but increased 5-fold upon phosphorylation by the CDK activating kinase Civ1 of Saccharomyces cerevisiae. Seven recombinant L. major CRKs (1, 2, 3, 4, 6, 7 and 8) were also expressed and purified, none of which were active as monomers. Moreover, only CRK3 was phosphorylated by Civ1. HA-tagged CYCA expressed in L. major procyclic promastigotes was coprecipitated with CRK3 and exhibited histone H1 kinase activity. These data indicate that in Leishmania CYCA interacts with CRK3 to form an active protein kinase, confirm the conservation of the regulatory mechanisms that control CDK activity in other eukaryotes, but identifies biochemical differences to human CDK1.

AB - The activity of cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, is regulated through post-translational mechanisms, including binding of a cyclin and phosphorylation. Previously studies have shown that Leishmania mexicana CRK3 is an essential CDK that is a functional homologue of human CDK1. In this study, recombinant histidine tagged L. mexicana CRK3 and the cyclin CYCA were combined in vitro to produce an active histone H1 kinase that was inhibited by the CDK inhibitors, flavopiridol and indirubin-3-monoxime. Protein kinase activity was observed in the absence of phosphorylation of the T-loop residue Thr178, but increased 5-fold upon phosphorylation by the CDK activating kinase Civ1 of Saccharomyces cerevisiae. Seven recombinant L. major CRKs (1, 2, 3, 4, 6, 7 and 8) were also expressed and purified, none of which were active as monomers. Moreover, only CRK3 was phosphorylated by Civ1. HA-tagged CYCA expressed in L. major procyclic promastigotes was coprecipitated with CRK3 and exhibited histone H1 kinase activity. These data indicate that in Leishmania CYCA interacts with CRK3 to form an active protein kinase, confirm the conservation of the regulatory mechanisms that control CDK activity in other eukaryotes, but identifies biochemical differences to human CDK1.

KW - Leishmania CRK3 Cell cycle Cyclin dependent kinase CDK Activation loop T-loop

UR - http://www.scopus.com/inward/record.url?scp=77951938344&partnerID=8YFLogxK

U2 - 10.1016/j.molbiopara.2010.03.002

DO - 10.1016/j.molbiopara.2010.03.002

M3 - Journal article

VL - 171

SP - 89

EP - 96

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

SN - 0166-6851

IS - 2

ER -

Research

Electronic data

Links

Text available via DOI:

Keywords