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

Abstract

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 ...

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

“…falciparum Cultures-P. falciparum 3D7 strain was cultured at 37°C in RPMI 1640 medium, supplemented either with 0.5% Albumax II (Invitrogen) or 10% ABϩ human serum as described earlier (12,17). For sexual stage studies, 3D7A, a variant of P. falciparum strain 3D7, was used to obtain gametocyte-enriched culture as described previously (18).…”

“…Detailed understanding of calcium-mediated signaling pathways could provide insights into novel molecular mechanisms involved in parasite development. For instance, calcium regulation of a protein kinase B-like enzyme (12) via calmodulin seems to be important for erythrocyte invasion (8). Plasmodium contains calciumdependent protein kinases (CDPKs) (13), which have been found only in plants and some protists but are absent from almost all metazoans (14).…”

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

“…It has also been reported that cytosolic Ca 2ϩ increases in free merozoites in response to the change in K ϩ ion concentration when they are released from the red blood cell (9). This Ca 2ϩ increase and the PfPKB activation are both blocked by the PLC inhibitor U73122 (9,31,32), implying that PLC/IP 3 -dependent Ca 2ϩ signaling may also be active during the late schizont and merozoite stage of the P. falciparum lifecycle, albeit activated by different extracellular signals. In our experiments we were not able to measure Ca 2ϩ signals in intraerythrocytic segmented (late phase) schizonts (Fig.…”

“…It was recently reported that parasite egress from erythrocytes depends on the calcium-dependent protein kinase PfCDPK5 (33) a process that occurs during late schizgony. Moreover, another calcium-dependent kinase, PfPKB, is believed to be involved in the reinvasion of erythrocytes by the released merozoites (31,32). It has also been reported that cytosolic Ca 2ϩ increases in free merozoites in response to the change in K ϩ ion concentration when they are released from the red blood cell (9).…”

Phospholipases during membrane dynamics in malaria parasites

“…At their destination, they support intensified binding of the merozoite to the RBC as well as its reorientation prior to RBC invasion (reviewed in Cowman et al, 2012;Cowman and Crabb, 2006). Inhibition of PI-PLC with the commercial PLC inhibitor U73122 prevents calcium-mediated signaling and thus activation of the plasmodial protein kinase B by calmodulin, in consequence impairing secretion of micronemal proteins (Raabe et al, 2011a(Raabe et al, , 2011bSingh et al, 2010;Vaid et al, 2008;Vaid and Sharma, 2006). The plasmodial PI-PLC is also involved in the signaling pathways initiating gametogenesis, as was shown for P. berghei and P. falciparum (reviewed in Bennink et al, 2016;Kuehn and Pradel, 2010).…”