Dissection of Mechanisms Involved in the Regulation of Plasmodium falciparum Calcium-dependent Protein Kinase 4

Ravikant Ranjan,Anwar Ahmed,Samudrala Gourinath,Pushkar Sharma

doi:10.1074/jbc.m900656200

Abstract

Recent studies have demonstrated that calcium-dependent protein kinases (CDPKs) are used by calcium to regulate a variety of biological processes in the malaria parasite Plasmodium. CDPK4 has emerged as an important enzyme for parasite development, because its gene disruption in rodent parasite Plasmodium berghei causes major defects in sexual differentiation of the parasite ( Billker, O., Dechamps, S., Tewari, R., Wenig, G., Franke-Fayard, B., and Brinkmann, V. (2004) Cell 117, 503-514 ). Despite these findings, it is not very clear how PfCDPK4 or any other PfCDPK is regulated by calcium at the molecular level. We report the biochemical characterization and elucidation of molecular mechanisms involved in the regulation of PfCDPK4. PfCDPK4 was detected on gametocyte periphery, and its activity in the parasite was regulated by phospholipase C. Even though the Junction Domain (JD) of PfCDPK4 shares moderate sequence homology with that of the plant CDPKs, it plays a pivotal role in PfCDPK4 regulation as previously reported for some plant CDPKs. The regions of the J-domain involved in interaction with both the kinase domain and the calmodulin-like domain were mapped. We propose a model for PfCDPK regulation by calcium, which may also prove useful for design of inhibitors against PfCDPK4 and other members of the PfCDPK family.

Highlights

Sequencing of reverse transcription (RT)-PCR product confirmed that PfCDPK4 contains an intron of 347 nucleotides (Fig. 1A), which was similar to the predictions made by PlasmoDb
The deduced amino acid sequence suggested that PfCDPK4 possesses a calmodulin-like domain at the C terminus and an N-terminal serine/threonine kinase domain, which are characteristic of calciumdependent protein kinases (CDPKs) [14]
PfCDPK4 catalytic domain possesses all 11 sub-domains that are representative of most protein kinases

Summary

Introduction

Biochemical studies done on plant CDPKs have revealed that the JD regulates CDPK activity by interacting with both the kinase domain and the CLD [14, 15]. J-domain Is Responsible for PfCDPK4 Activation—Deletion and truncation mutants of PfCDPK4 domains were created to understand its regulation (Fig. 5A), and the activity of recombinant mutant proteins was determined by performing in vitro kinase assays.

Results

Conclusion