The role of protein phosphorylation in the life cycle of malaria parasites is slowly emerging. Here we combine global phospho-proteomic analysis with kinome-wide reverse genetics to assess the importance of protein phosphorylation in Plasmodium falciparum asexual proliferation. We identify 1177 phosphorylation sites on 650 parasite proteins that are involved in a wide range of general cellular activities such as DnA synthesis, transcription and metabolism as well as key parasite processes such as invasion and cyto-adherence. several parasite protein kinases are themselves phosphorylated on putative regulatory residues, including tyrosines in the activation loop of PfGsK3 and PfCLK3; we show that phosphorylation of PfCLK3 Y526 is essential for full kinase activity. A kinome-wide reverse genetics strategy identified 36 parasite kinases as likely essential for erythrocytic schizogony. These studies not only reveal processes that are regulated by protein phosphorylation, but also define potential anti-malarial drug targets within the parasite kinome.

Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) ؊/؊ mice, which lack appreciable cdk5 activity, we observed an increase in the phosphoryla- 1 is a member of the cyclin-dependent protein kinase family (cdc2, CDC28, and other generically cyclin-dependent CDKs). Although cdk5 binds to cyclin D, its activity is not regulated by cyclins and there is little evidence that cdk5 is involved in the progression of the cell cycle (for review see Ref.1; see also Refs. 2 and 3). cdk5 is active mainly in post-mitotic cells such as neurons (4, 5), retinal cells (6), and muscle cells (7), where its activators p35 (or its truncated form p25) (4, 5) and p39 (8 -11) are specifically expressed. cdk5 has been suggested to play important roles in neurite outgrowth (12, 13), neuronal migration (14 -16), dopamine signaling in the striatum (17), exocytosis (18 -21), differentiation of muscle cells (7), and organization of acetylcholine receptors at the neuromuscular junction (22). Although neuronal cytoskeletal proteins were initially identified as the major target substrates (4, 23, 24), the number of cdk5 substrates has expanded considerably (see Table I in Ref. 25). These include DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein involved in dopamine signaling (17), NUDEL (a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE), a protein involved in neuronal migration and axon transport (26), and other proteins involved in cross-talk between protein kinases and phosphatases (27). cdk5 also modulates protein kinase reactions such as the small GTPase-Rac dependent phosphorylation of p21-activated kinase, which results in modification of the actin cytoskeleton (28). By virtue of phosphorylating these diverse substrates, cdk5 plays a multifunctional role in the nervous system. It has been demonstrated that the absence of cdk5 in cdk5 Ϫ/Ϫ mice results in embryonic lethality (16). Although the p35 knockout mice survive longer (14), both cdk5 Ϫ/Ϫ and p35 Ϫ/Ϫ mice exhibit similar defects in cortical neuronal migration and affect the development of the nervous system (14 -16). We observed that in cdk5 Ϫ/Ϫ mice brain stem neurons showed ballooning and hyperphosphorylation of cytoskeletal proteins as detected by the SMI31 antibody (see Fig. 1). Similar observations were obtained from p35 (Ϫ/Ϫ) mice. 2 The antibody cross-reacts with phosphorylated Lys-Ser-Pro (KSP) motifs in neurofilament proteins, tau, and MAPs ...

Polyphosphorylated phosphoinositides (PIPs) are potent second messengers, which trigger a wide variety of signaling and trafficking events in most eukaryotic cells. However, the role and metabolism of PIPs in malaria parasite Plasmodium have remained largely unexplored. Our present studies suggest that PfPI3K, a novel phosphatidylinositol-3-kinase (PI3K) in Plasmodium falciparum, is exported to the host erythrocyte by the parasite in an active form. PfPI3K is a versatile enzyme as it can generate various 3-phosphorylated PIPs. In the parasite, PfPI3K was localized in vesicular compartments near the membrane and in its food vacuole. PI3K inhibitors wortmannin and LY294002 were effective against PfPI3K and were used to study PfPI3K function. We found that PfPI3K is involved in endocytosis from the host and trafficking of hemoglobin in the parasite. The inhibition of PfPI3K resulted in entrapment of hemoglobin in vesicles in the parasite cytoplasm, which prevented its transport to the food vacuole, the site of hemoglobin catabolism. As a result, hemoglobin digestion, which is a source of amino acids necessary for parasite growth, was attenuated and caused the inhibition of parasite growth. (Blood.

Calcium Dependent Protein Kinases are key effectors of calcium signaling in malaria parasite. PfCDPK1 is critical for asexual development of Plasmodium falciparum, but its precise function and substrates remain largely unknown. Using a conditional knockdown strategy, we here establish that this kinase is critical for the invasion of host erythrocytes. Furthermore, using a multidisciplinary approach involving comparative phosphoproteomics we gain insights into the underlying molecular mechanisms. We identify substrates of PfCDPK1, which includes proteins of Inner Membrane Complex and glideosome-actomyosin motor assembly. Interestingly, PfCDPK1 phosphorylates PfPKA regulatory subunit (PfPKA-R) and regulates PfPKA activity in the parasite, which may be relevant for the process of invasion. This study delineates the signaling network of PfCDPK1 and sheds light on mechanisms via which it regulates invasion.

A novel mechanism is described by which neurons reenter the cell cycle, which leads to their apoptosis: β-amyloid peptide activates the MEK-ERK pathway aberrantly, resulting in expression of cyclin D1 and cell death. Cyclin D1, in turn, prevents the activation of neuronal CDK5, which is important for neuronal survival.