PfPKB, a Novel Protein Kinase B-like Enzyme from Plasmodium falciparum

Self Cite

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 calmodulinlike 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.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Ranjan

Ahmed

Gourinath

et al. 2009

Self Cite

“…The resulting cell debris was removed by centrifugation at 20,000 ϫ g for 40 min at 4°C. Fusion proteins from the cell lysates were affinity-purified using glutathione-Sepharose resin as described previously (22,25). Briefly, the resin was washed with lysis buffer, and bound proteins were eluted with 50 mM Tris, pH 8.0, with 10 mM glutathione.…”

Section: Expression and Purification Of Recombinant Proteins-mentioning

confidence: 99%

PfSRPK1, a Novel Splicing-related Kinase from Plasmodium falciparum

Dixit

Singh

Sharma

et al. 2010

Self Cite

Even though it is increasingly evident that post-transcriptional events like mRNA processing and splicing may regulate gene expression and proteome diversity of malaria parasite Plasmodium, molecular mechanisms that regulate events like mRNA splicing in malaria parasite are poorly understood. Protein kinases control a wide variety of cellular events in almost all eukaryotes, including modulation of mRNA splicing, transport, and stability. We have identified a novel splicing-related protein kinase from Plasmodium falciparum, PfSRPK1. PfSRPK1 when incubated with parasite nuclear extracts inhibited RNA splicing, suggesting that it may control mRNA splicing in the parasite. PfSR1, a putative splicing factor from P. falciparum, was identified as a substrate of PfSRPK1. PfSR1 interacts with RNA and PfSRPK1 modulates its RNA binding. Early in the parasite development, PfSRPK1 and PfSR1 are present in the nucleus. These studies provide useful insights into the function of two potentially key components of P. falciparum mRNA splicing machinery.Malaria is one of the most serious infectious diseases and causes several million deaths and clinical illness in hundreds of millions of people every year (1). One of the major problems of recent times has been the emergence of new drugresistant strains of the malaria parasite Plasmodium falciparum. After invasion of the erythrocytes, the parasite can propagate asexually, giving rise to several new merozoites that invade fresh erythrocytes, which serve as hosts for the parasite to propagate. The blood stage infection is the cause of malaria pathogenesis. The parasite also undergoes sexual differentiation, which leads to the formation of male and female gametocytes. Upon ingestion of gametocytes by the Anopheles mosquito, further development continues inside the vector host. It is well known that signal transduction events mediated by protein kinases regulate diverse cellular processes. The importance of protein kinases in malaria parasite has been highlighted by a series of recent reports (2, 3), as these enzymes have been implicated in wide ranging events in both asexual and sexual stages of the parasite life cycle and, therefore, are considered as potential drug targets. Despite these reports, the information regarding signaling networks in the parasite is very limited.Although it is clear that metazoan protein kinase cascades control gene expression by regulating transcriptional or posttranscriptional events, this area in P. falciparum biology is largely unexplored. Plasmodium displays a high degree of developmental control of gene expression (4). Strikingly, only a few transcription factors have been identified in the parasite leading to the speculation that post-transcriptional events may be pivotal in regulating gene expression in the parasite (5). Translational repression of some genes (6) and control of gene expression by antisense RNA are some of the post-transcriptional (7) events that have been implicated in regulating gene expression in the parasite. mRNA splicing is on...

“…Despite sharing significant sequence homology (ϳ70%) with the catalytic domain of PKB, PfPKB lacks a pleckstrin homology (PH) domain present at the N terminus of the mammalian enzyme. The N-terminal region (NTR) of PfPKB is inhibitory as its deletion results in PfPKB catalytic activation (1). The NTR does not exhibit similarity with any other protein in the non-redundant protein data base.…”

mentioning

confidence: 99%

PfPKB, a Protein Kinase B-like Enzyme from Plasmodium falciparum

Vaid¹,

Sharma

2006

Self Cite

Intracellular cell signaling cascades of protozoan parasitePlasmodium falciparum are not clearly understood. We have reported previously (Kumar, A., Vaid, A., Syin, C., and Sharma, P. (2004) J. Biol. Chem. 279, 24255-24264) the identification and characterization of a protein kinase B-like enzyme in P. falciparum (PfPKB). PfPKB lacks the phosphoinositide-interacting pleckstrin homology domain present in mammalian protein kinase B. Therefore, the mechanism of PfPKB regulation was expected to be different from that of the host and had remained unknown. We have identified calmodulin (CaM) as the regulator of PfPKB activity. A CaM binding domain was mapped in the N-terminal region of PfPKB. CaM, in a calcium-dependent manner, interacts with this domain and activates PfPKB. CaM associates with PfPKB in the parasite and regulates its activity. Furthermore phospholipase C acts as an upstream regulator of this cascade as it facilitates the release of calcium from intracellular stores. This is one of the first multicomponent signaling pathways to be dissected in the malaria parasite.Plasmodium falciparum is responsible for most cases of human malaria worldwide. This parasite invades both hepatocytes as well as erythrocytes in human host, but it is the erythrocytic phase of its life cycle that causes severe pathogenesis of malaria. After invading erythrocytes, the parasite undergoes well defined developmental changes inside the erythrocyte host. The parasite adopts a ringlike morphology and acquires necessary nutrients from the host during the trophozoite stages. Subsequently nuclear division gives rise to multinucleated schizont containing ϳ24 merozoites. These merozoites when released after schizont rupture invade fresh erythrocytes to start another cycle of asexual development. Although it is known that Plasmodium can utilize host G-protein signaling (2) and alters phosphorylation of erythrocyte cytoskeletal proteins during infection (3), parasite signaling pathways have remained largely uncharacterized. Given the importance of cell signaling cascades in proliferation and differentiation of eukaryotic cells, dissection of signal transduction mechanisms may provide useful insights about the development of this protozoan parasite. Plasmodium genome analysis revealed that there are close to 65 protein kinases, major mediators of cell signaling, in P. falciparum (4, 5). Apart from a few of these kinases (6 -10), the function and mechanism of regulation and identity of cellular targets of most of these enzymes is largely unknown.We recently identified a protein kinase B-like enzyme in P. falciparum (PfPKB) 3 . Despite sharing significant sequence homology (ϳ70%) with the catalytic domain of PKB, PfPKB lacks a pleckstrin homology (PH) domain present at the N terminus of the mammalian enzyme. The N-terminal region (NTR) of PfPKB is inhibitory as its deletion results in PfPKB catalytic activation (1). The NTR does not exhibit similarity with any other protein in the non-redundant protein data base. PKB binds phosphoinositides ...