Identification of putative PfCDPK1 substrates and interacting proteins. a PfCDPK1-3HA-DD parasites were generated by fusing the C-terminus of PfCDPK1, with an FKBP Death Domain (DD) and HA tag (Supplementary Fig. ). PfCDPK1 possesses a N-terminal myristoylation and palmitoylation motif (red) and a C-terminal calmodulin like domain. The DD domain targets PfCDPK1 to proteasomic degradation in the absence of its ligand Shld-1. b PfCDPK1-3HA-DD parasites were cultured in the presence (+) of Shld-1. At the ring stage, parasites were split and Shld-1 was removed (−) from one half of the cultures. The cultures were harvested at the schizont stage and western blotting was performed using anti-HA antibody to detect the expression of PfCDPK1-3HA-DD. Actin was used as the loading control. Right Panel, Densitometry was performed for PfCDPK1 bands and was normalized with respect to the loading control (Mean±s.e.m., *n=3, p<0.0001, t-test). c Schematic illustrating the work flow followed for quantitative proteomics and phosphoproteomics studies from Shld-1 treated (+) or deprived parasites (−) (for details see Methods). d The phosphorylation ratios of phosphopeptides identified from PfCDPK1-3HA-DD schizonts cultured in the presence or absence of Shld-1 was normalized with respect to total protein. The ratios for all phosphopeptides from various replicates are provided in Supplementary Data . The S-curve for one of the replicates is provided and some of the significantly altered phosphorylation sites belonging to key proteins (Supplementary Data ) are indicated. Two key proteins that were marginally higher than the cut-off in the replicate represented here but were significantly altered in two other replicates (Supplementary Data ) are lettered in red. PfPKA-R-S149, which was altered in only one replicate (Supplementary Data ), is indicated by *. e Proteins that exhibited reduced phosphorylation upon PfCDPK1 depletion were subjected to Gene Ontology enrichment analysis. Biological process of “entry into host cell” that consists of proteins involved in invasion and motility was found to be significantly enriched (Supplementary Data ). f Distribution of amino acids surrounding the S/T in proteins that exhibited significant reduction in phosphorylation (Supplementary Data ). The four major motifs that emerged from Motif-X analysis are indicated. g Protein–protein interaction was predicted between differentially phosphorylated proteins using STRING resource. The analysis exhibited high confidence protein–protein interactions between the candidate genes (d, Supplementary Data )

PfCDPK1 interacts and phosphorylates proteins of IMC and glideosome complex. a The phosphorylation status of PfGAP45 at S103 was assessed after Shld-1 withdrawal from PfCDPK1-3HA-DD parasites. Parasite lysates were prepared from late schizont/segmenter and Western blotting was performed using antibody against PfGAP45 or its form which is phosphorylated at S103. Right Panel, Densitometry was performed for pS103-PfGAP45 bands and was normalized with respect to the WT PfGAP45 (Mean±s.e.m., *n=3, p<0.0001, t-test). Lower Panel, MS/MS spectra for S103 containing phosphopeptide is provided, which indicates differential phosphorylation at this site. b MS/MS spectra of phosphopeptides from IMC1g protein, which was differentially phosphorylated upon PfCDPK1 depletion. c Recombinant 6xHis-PfCDPK1 was used to phosphorylate recombinant 6xHis-IMC1g in a kinase assay using γ^32-P[ATP]. The assay mix was electrophoresed on a SDS-PAGE gel and phosphate incorporation was observed by autoradiography. d PfCDPK1-3HA-DD parasites were synchronized and cultured in the presence or absence of Shld-1. Parasites were labeled with ³²P-orthophosphate and IMC1g was immunoprecipitated. IMC1g-IP was subjected to phosphorimaging and parasite lysate was used for Western blotting with anti-IMC1g. e IFA was performed on PfCDPK1-3HA-DD parasites using anti-HA antibody to detect CDPK1 and anti-IMC1g antisera. Scale bar is 2 µm. f PfCDPK1 was immunoprecipitated from PfCDPK1-3HA-DD or 3D7 (control) parasites using anti-HA antibody. Western blotting was performed on both PfCDPK1-IP and total lysate using anti-IMC1g antisera

PfCDPK1 regulates PfPKA-C by targeting PfPKA-R. a PfPKA-C was immunoprecipitated after Shld-1 removal from PfCDPK1-3HA-DD parasites. PfPKA-C-IP was used to assay kinase activity using Histone H1 as substrate and its phosphorylation was detected by phosphorimaging and quantified by densitometry of the radiolabelled bands (Mean±s.e.m., * n=3, p<0.05, t-test). Western blotting was performed for PfPKA-R and CDPK1-HA on total parasite lysate. b and c The MS/MS spectra indicated that S149 exhibits reduced phosphorylation in the parasite in one of the replicates of phospshoproteomics experiments b PfCDPK1 kinase assays were performed using recombinant PfPKA-R in vitro and kinase assay mix was used for LC–MS/MS analysis, which revealed PfPKA-R to be phosphorylated at S149 in vitro c In addition, several other sites of PfPKA-R were found to be phosphorylated in vitro (Supplementary Fig. ). d PfPKA-R domain architecture indicating the differentially phosphorylated site (S149). The motif encompassing the phosphorylation site is indicated. The Inhibitory Segment (IS) sequence of PfPKA-R is compared with that of human PKA-RIIα. e PfCDPK1-3HA-DD parasites, at late trophozoite stage, were incubated with ³²P-orthophosphate and harvested at late schizont/segmenter stage. PfPKA-R was immunoprecipitated and the IP was electrophoresed and radiolabeled proteins were detected by phosphorimaging. Western blotting with anti-PfPKA-R was performed on parasite lysate. A control IP was also performed using preimmune-sera on Shld-1 treated parasite lysate. f An antibody which specifically recognizes the S149-phosphorylated form of PfPKA-R was generated (Supplementary Fig. ). Western blotting was performed on lysates from PfCDPK1-3HA-DD parasites treated with Shld-1 or left untreated. An antibody, which recognizes both unphosphorylated and phosphorylated form of PKA-R, was used as a control for Western blotting (bottom panel). g Shld-1 was removed from PfCDPK1-3HA-DD parasite cultures as described above and schizont lysates were prepared after ~44 h. PfPKA-C was immunoprecipitated and PfPKA-C-IP was used for western blotting to detect PfPKA-R. Western blotting with indicated antibodies was also performed on total parasite lysates. Right Panel, Fold change in PfPKA-R associated with PfPKA-C was quantitated by densitometry after normalization with respect to PfPKA-R present in total parasite lysate (Mean±s.e.m., *n=4, p<0.05, t-test). h and i 3D7 schizonts were treated with BAPTA-AM to chelate intracellular calcium. Subsequently, PfCDPK1 (h) or PfPKA-C (i) was immunoprecipitated. PfCDPK1-IP and PfPKA-C-IP were immunoblotted with anti-PfPKA-R (h and i). Western blotting was also performed on parasite lysates using indicated antibodies

cAMP-dependent PfPKA-signaling may prevent PfCDPK1 activation via a negative feedback loop. a 3D7 schizonts were treated with DMSO, BAPTA-AM, IBMX or 8Br-cAMP for 3 h following which parasite lysates were prepared and PfCDPK1 was immunoprecipitated. PfCDPK1-IP was used to assay the kinase activity as described above using PfGAP45 as phosphoacceptor substrate. Fold change in activity was determined with respect to DMSO control (Mean±s.e.m., *n=3, p<0.05, t-test). b PfPKA-R-GFP parasites were left untreated (−) or treated with 8Br-cAMP (+) followed by immunoprecipitation with PfCDPK1 or PfPKA-C antisera and kinase assays were performed with the IP, as described above using recombinant PfGAP45 or Histone H1 as phosphoacceptor substrate and phosphorylation was detected by phosphorimaging and quantified by densitometry of the bands from the phosphorimage (right panel) (Mean±s.e.m., *n=3, p<0.05, t-test). c. 3D7 schizont-infected red blood cells were treated with 20 μM myristoylated PKI followed by immunoprecipitation of PfCDPK1 and kinase assay was performed as described above followed by quantitation of CDPK1 activity by densitometry of radiolabeled Histone H1 (Mean±s.e.m., *n=3, p<0.05, t-test). d To assess the effect of PfPKA on PfCDPK1 activity a two step assay was used. Step 1: PfPKA-C-HA was immunoprecipitated from PfPKA-C-HA parasites using anti-HA antibody. PfPKA-C-HA-IP (lane 2 and 3) or a control unrelated IP (lane 1) was incubated with recombinant PfCDPK1 in a kinase assay reaction in the presence (lane 2) or absence (lane 3) of unlabelled ATP. Step 2: Kinase assays were performed for PfCDPK1 obtained after Step 1 by using MBP as the phosphoacceptor substrate and radiolabelled ATP. The phosphorimage of the SDS-PAGE gel revealed that PfPKA-C-HA-IP inhibited PfCDPK1 activity when incubated with ATP (lane 2 vs. lane 3). Lower Panel, Densitometry was performed on radiolabeled bands to quantitate the phosphorylation and fold change in activity with respect to control (lane 1) is indicated. Data presented is Mean±s.e.m. of three independent experiments, *n=3, ANOVA, p<0.05)

PfCDPK1 regulates invasion of host erythrocytes by P. falciparum. a Depletion of PfCDPK1 results in reduced growth of PfCDPK1-3HA-DD parasites. Synchronized PfCDPK1-3HA-DD parasites were cultured either in the presence or absence of Shld-1. % parasitemia was determined by counting at least 500 erythrocytes from Giemsa-stained thin blood smears which were prepared after every cycle, which indicated that parasites were mostly in ring forms. b PfCDPK1 may play a role in host cell invasion by P. falciparum. Schizonts±Shld-1 were incubated with fresh erythrocytes and after indicated time blood smears were prepared and the number of schizonts and rings were counted and represented as % infected erythrocytes. Shld-1 removal lead to a significant decrease in the number of rings, which were counted from thin blood smears (Mean ± s.e.m., *, t-test, n=4, P<0.05). Right Panel, % invasion was also determined by flow cytometry (Mean±s.e.m., n=4, *P<0.05, t-test). c PfCDPK1-3HA-DD parasites±Shld-1 were used for attachment assays performed using cytochalasin D (Cyt D). The number of attached merozoites were counted and data represents fold change in attachment with respect to Shld-1 treated parasites (100%) (Mean±s.e.m., n=3 *, p<0.05). d The release of EBA-175 by PfCDPK1-3HA-DD parasites±Shld-1 was determined by performing western blotting of culture supernatant. EBA-175 secretion was significantly reduced upon Shld-1 removal, which was quantitated by densitometry of the Western blot (lower panel, Mean±s.e.m., n=3 *, t-test, p<0.05). Actin blot was performed on the corresponding parasite lysate and used for normalization. e and f Shld-1 was withdrawn from parasites and invasion assays were performed using schizonts as described above except that in one case RBCs treated with either neuraminidase e or with chymotrypsin f were used for the assay. The number of rings formed, which reflected successful invasion, were determined. Data represents % invasion with respect to Shld-1 treated parasites (100%) (Mean±s.e.m., n=3 *, ANOVA, p<0.05, **p>0.05, NS)

Regulation of invasion by PfCDPK1 and PfPKA. a The invasion of PKA-R-GFP schizonts in the absence (squares) or presence (triangles) of 8Br-cAMP was assessed by counting the number of rings and schizonts on Giemsa smears after indicated time as described above. A representative of three independent experiments is shown. b Shld-1 was withdrawn from the parasites and invasion assays were performed using schizonts as described above. In one set of experiments PKI was added for 3 h before addition of fresh RBCs for invasion. The number of rings formed were counted from Giemsa stained thin blood smears, which reflected successful invasion. Data represents % invasion with respect to Shld-1 treated parasites (100%) (Mean±s.e.m., n=2 *, ANOVA, p<0.05, **p>NS). c Model of PfCDPK1 mediated signaling in malaria parasite: PLC-mediated calcium release results in the activation of PfCDPK1 (Pushkar Sharma, unpublished results), which triggers phosphorylation of key substrates like IMC-glideosome proteins that include IMC1g, IMC1c, and PfGAP45. PfCDPK1 facilitates the activation of PfPKA-C by promoting its dissociation from PfPKA-R. PfCDPK1 may be turned off in the parasite via a negative feedback loop regulated by PfPKA. These events and the regulation of exocytosis of EBA-175 by PfCDPK1 signaling may contribute to the process of invasion