title: Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase.
abstract: Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4-10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1.
authors.affiliation: Department of Parasitology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA.
authors.forename: Yueqin
authors.initials: Y
authors.lastname: Chen
authors.forename: Dayadevi
authors.initials: D
authors.lastname: Jirage
authors.forename: Diana
authors.initials: D
authors.lastname: Caridha
authors.forename: April K
authors.initials: AK
authors.lastname: Kathcart
authors.forename: Edison A
authors.initials: EA
authors.lastname: Cortes
authors.forename: Richard A
authors.initials: RA
authors.lastname: Dennull
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Geyer
authors.forename: Sean T
authors.initials: ST
authors.lastname: Prigge
authors.forename: Norman C
authors.initials: NC
authors.lastname: Waters
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
chemical_list: D016213:Cyclins; C484350:PfMrk protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D018844:Cyclin-Dependent Kinases