Raf kinase inhibitor protein affects activity of Plasmodium falciparum calcium-dependent protein kinase 1.
Proteins, such as the raf kinase inhibitory protein (RKIP), serve as modulators of signalling pathways by either promoting or inhibiting the formation of productive signalling complexes through protein-protein interactions. In the present study, the plasmodial RKIP ortholog, PfPE-PB1, was cloned, recombinantly expressed and purified to homogeneity. The purified protein was used to investigate the effect of plasmodial RKIP on the autophosphorylation and substrate phosphorylation activity of Plasmodium falciparum calcium-dependent protein kinase 1, PfCDPK1. Phosphorylation of RKIP by PfCDPK1 in in vitro kinase assays suggests that RKIP may be an in vivo substrate of this kinase, although the specific activity of PfCDPK1 is approximately seven-fold lower when RKIP, instead of casein, an exogenous substrate of this enzyme, is used as a substrate. In addition to the observed phosphorylation of RKIP itself, its presence in the assays greatly enhanced the autophosphorylation capacity of PfCDPK1 by approximately 5.5-fold. This substantial increase in autophosphorylation activity was associated with a diminished substrate phosphorylation activity of PfCDPK1 when casein was used. At the same time, RKIP phosphorylation slightly increased when casein was included into the assays. Thus, RKIP is recognized as a substrate under in vitro conditions and appears to act as a regulator of PfCDPK1 kinase activity, which possibly is one of its actual functions in the parasite.
Kugelstadt
Dominik
D
Universitätsklinikum Heidelberg, Abteilung für Parasitologie, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany.
Winter
Dominic
D
Plückhahn
Kirsten
K
Lehmann
Wolf Dieter
WD
Kappes
Barbara
B
D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D049488:Phosphatidylethanolamine Binding Protein / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D049488:Phosphatidylethanolamine Binding Protein; D010768:Phosphoserine; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases