Role of cell signalling in eukaryotic development

Project Associates/Assistants
Anita Shukla
Garima Verma
Amit Kumar

PhD Students
Ankush Vaid (since Jan 2003)

Understanding the molecular and cellular mechanisms involving protein and lipid kinases, which regulate specific and important functions like, cell proliferation, differentiation and apoptosis is the primary aim of this project. One of the major interests is to understand the role of phosphoinositide mediated cell signaling in malaria parasite-Plasmodium falciparum. Information regarding the role of cell signalling and especially phosphoinositide mediated events in the development of this protozoan parasite is lacking. In-depth understanding of these events may help unravel novel molecular and cellular mechanisms involved in the life cycle of this parasite. It has been shown that hydrolysis of PI (4,5)P2 to IP3 and DAG results in enhanced sexual differentiation in the Plasmodium suggesting that phosphoinositide metabolism may play important role in the life cycle of this parasite. Molecular events guiding this or other phosphoinositide dependent signaling events on Plasmodium are not clear. Our preliminary studies have indicated the presence of enzymes which may be involved in phosphoinositide mediated signaling in P. falciparum.

In order to understand the role of a PI-3 kinase homologue (PfPI3K) and a homologue of Protein Kinase B (PfPKB) in Plasmodium falciparum and other phosphoinositide targets, it is important to characterize these molecules biochemically. These studies would help study the role of these enzymes in P. falciparum life cycle.

Characterization and biochemical analyses of PfPI3K

As stated above, we have identified a PI3 kinase homologue from P. falciparum. At the sequence level, PfPI3K seems to be closest to the class III PI3 kinases. Domain architecture analysis using PFAM and CDART database revealed only two known domains in this enzyme: the anchoring or helical domain and a classical PI3 kinase catalytic domain. It appears that PfPI3K lacks other modular domains like C2, PH, RBD.

In order to characterize this enzyme, we need to over-express it in E. coli or other host systems. So far, expression of the full length PfPI3K enzyme in E. coli has been difficult. However, the anchoring domain and the catalytic domain have been expressed as 6Xhis fusion protein. We have had some success in generating anti-sera against the anchoring domain of PfPI3K. This will be useful in localization, stage specific expression and catalytic activity determination of PfPI3K.

Regulation of PfPKB

PfPKB belongs to the AGC class of protein kinases. Although it shares highest homology to PKB from other eukaryotes, it also has significant sequence homology to another kinase of this family- Protein kinase C. A PKC homologue in P. falciparum has not been yet been identified. Therefore, there it is possible that PfPKB could perform functions of both PKB and PKC.

We have expressed the catalytic domain of PfPKB (DPfPKB) in E. coli as a GST-fusion and 6xHis tagged protein. GST- DPfPKB was able to phosphorylate histones IIA and H1 in vitro. In addition, the recombinant enzyme also phosphorylated a peptide substrate with arginine residues spanning the target serine suggesting that this enzyme prefers to have basic residues in the vicinity of its target S/T. The phosphorylation of activation loops of most protein kinases play important role their catalytic activation. This phosphorylation could be by an upstream kinase or it could even be by autophosphorylation. In some kinases (e.g. PKA) both these events could regulate this activity. In case of mammalian PKB, the phosphorylation of the activation loop is mediated by PI3 dependent kinase 1 (PDK1) is essential for its catalytic activation. The activity of PKB is further enhanced by phosphorylation of a Ser residue located in the C-terminus hydrophobic motif (HM) of this enzyme.

In order to understand the mechanism of catalytic activation of PfPKB, the role of phosphorylation in its activation was investigated. We identified a serine residue (S272) in the activation loop of PfPKB, which could be a putative regulatory site in this enzyme. This residue was replaced to alanine (S272A) and to aspartic acid (S272D) by site-directed mutagenesis. Mutation of S272A resulted in almost complete loss of PfPKB activity suggesting that S272 is crucial for PfPKB activity. S272 was also replaced to D to mimic the negatively charged phosphate on this serine. The loss in activity of S272A was recovered to some extent when S272 was mutated to D suggesting that phosphorylation of this site could regulate PfPKB activity. GST-DPfPKB was found to autophosphorylate itself and mutation of S272A lead to marked decrease in this autophosphorylation. Based on these observations, it can be suggested that S272 phosphorylation is important for its catalytic activation. This is very important information that could prove useful in understanding the role of PfPKB in P. falciparum life cycle.

We have raised antisera against a peptide designed from the C-terminal end of PfPKB. Immunofluorescence and immunoblotting studies were performed on P. falciparum cultures and its lysates respectively. It appears that PfPKB may be predominantly expressed in schizont stage of the life cycle of this parasite.