Figure 5: PfCDPK1 is phosphorylated by PfPKG in vitro.

(a) An in vitro kinase reaction with [³²P]-ATP was carried out using a recombinant HIS-tagged PfPKG (1 μg) with histone (2 μg) as a substrate in the presence and absence of cGMP (10 μM). (b) An in vitro kinase reaction with GST-tagged ‘kinase dead’ mutant of PfCDPK1 where asparate-191 was substituted for an asparagine (CDPK1-KD, 1 μg). This construct was used as a substrate for purified recombinant HIS-tagged PfPKG (1 μg) in the presence and absence of cGMP (10 μM). Coomassie-stained gel is shown as a loading control. (c) In vitro PfPKG kinase assay performed with recombinant GST-tagged CDPK1-KD (50 ng) as a substrate or a mutant version of CDPK1-KD where serine-64 was substituted by alanine (CDPK1-KD(S-A); 50 ng). The kinase assay was conducted either in the presence or absence of PfPKG (50 ng) and the resulting reaction probed with a phospho-specific antibody to phosphoserine-64 on CDPK1 (CDPK1-pS64). Blots were stripped and probed with a structural PfCDPK1 antibody as a loading control. The results shown are representative of three independent experiments.