Role of cell signalling in eukaryotic development

Project Associates/Assistants
Priyanka Rathore (since Jan 2004)
Amit Kumar
Justin Vijay
Mukta Belwal (since Jan 2004)
Anindita Bhattacharya (since Jan 2004)

Ph D Students
Ankush Vaid
Ravikant Ranjan (since Jan 2004)

Cells respond to extracellular signals by transmitting intracellular instructions to coordinate appropriate responses. Protein/lipid phosphorylation modulates biochemical machinery of most eukaryotic cells, which is very important for passing on the message encoded in these signals to various compartment of these cells. We are interested in understanding the molecular and cellular mechanisms involving protein and lipid kinases, which regulate specific and important functions like, cell proliferation, differentiation and apoptosis.

One of our 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 (PI) dependent signaling events in Plasmodium are not clear. In most eukaryotes, phosphatidylinositol is found to be phosphorylated at position 3’, 4’ and/or 5’ of the inositol ring. These second messenger phosphoinositides regulate a wide-variety of signaling events. One of the major targets of PIs in most eukaryotes is protein kinase B or AKT. In most mammalian cells 3’-phosphorylated PIs bind to a pleckstrin homology (PH) domain present in PKB that results in membrane translocation and activation of this enzyme. PKB phosphorylates a wide variety of cellular targets and is important for cell survival and proliferation.We have identified a PI-3 kinase (PfPI3K) and Protein kinase B (PfPKB) homologue from Plasmodium falciparum and are interested to investigate (i) how these enzymes are regulated? and (ii) what role do these play in Plasmodium life cycle?

Stage specific expression and localization of PfPKB

In order to decipher the function of PfPKB in P.falciparum life cycle it was important to investigate the expression profile of this enzyme. RT-PCR studies were performed using parasite stage specific RNA, PfPKB expression was detected mainly in the schizont stages. Antisera were raised against peptides derived from two distinct regions of PfPKB. Western-blot analyses performed using these anti-sera suggested that PfPKB protein is synthesized mainly in schizont stages. Results of immunoflluorescence studies complemented the above-mentioned data as only multi-nucleated schizonts exhibited PfPKB expression. PfPKB staining was diffused in schizont stages suggesting its presence in cytoplasm as well as cell surface. Interestingly, it localized at the apical end of the merozoite. It is well known apical end possesses organelles (micronemes, rhoptries, dense granules) which are important for erythrocyte invasion.

Regulation of PfPKB

Studies performed with the catalytic domain of PfPKB (DPfPKB) suggested that its catalytic activity is regulated by autophosphorylation of S271. This S271 is part of the activation loop of PfPKB. PKB and other AGC family kinases possess a hydrophobic motif at the C-terminal end. Phosphorylation of S/T in this hyrdrophobic motif (in addition to phosphorylation of activation loop) results in maximal activation of these enzymes. PfPKB also has a hyrdrophobic motif at its C-terminus with a putative phosphorylation site (S442). Site directed mutagenesis studies suggested that phosphorylation of S442 in addition to S271 may be needed for maximal PfPKB activity.

Full-length PfPKB, containing both the N-terminal region and the catalytic domain, was also expressed. PfPKB (full-length) showed markedly reduced activity in comparison to its N-terminal region (NTR) deleted version, DPfPKB. In addition, when the recombinant NTR was incubated with DPfPKB it resulted in significant inhibition of PfPKB activity. Collectively, these data suggest that N-terminal region negatively regulates the PfPKB activity.

Possible role of PfPKB in P.falciparum life cycle

We were interested in identifying PfPKB inhibitors to help us understand the structure- function relationship of this Plasmodium protein kinase, and its role in parasite growth and development. Since there are no known or available inhibitors of mammalian PKB, we looked at the possibility of using inhibitors of other related kinases as candidates for PfPKB inhibition. As mentioned above, our in-silico analysis suggested that Plasmodium lacks a PKC homologue and the closest enzyme to PKC is PfPKB. Therefore, PKC inhibitors were treated as putative candidates for PfPKB inhibition. We found that Go 6983, an isoform-specific PKC inhibitor, inhibited PfPKB activity with reasonable efficacy.

This inhibitor also arrested P.falciparum growth in a stage-specific manner suggesting that PfPKB may play an important role in parasite development.

Publications

Original peer-reviewed articles

1.     Kumar A, Vaid A, Syin C and Sharma P (2004) PfPKB, a novel protein kinase B like enzyme from Plasmodium falciparum: I. Identification, characterization and possible role in parasite development. J Biol Chem (in press).