Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum

Abstract

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.

An evolutionary perspective on the kinome of malaria parasites

“…These cells accumulate in the insect's salivary gland, where they are primed for infection of a novel human host. Stage transitions during this complex life cycle are likely to require efficient signalling pathways, and evidence for the role of protein phosphorylation in life-cycle progression is emerging [18][19][20].…”

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

“…The phospho-null mutant S610A of the PfAMA1 tail was also able to complement (Santos et al, 2011). In the phospho-proteomes published by Treeck et al (2011) andSolyakov et al (2011), S610 was confirmed as well as 4 additional sites in the tail, and two new phosphorylated serine residues were identified in TgAMA1 ( Fig. 1 and Table S1).…”

“…Furthermore, Go 6983, a PKC inhibitor (PfPKB catalytic domain is closely related to PKC) is able to affect in vitro recombinant PfPKB activity and reduce the ability of P. falciparum to invade erythrocytes (Kumar et al, 2004). Consistent with a vital role in parasite development, both PKB and CDPK1 are refractory to gene disruption in P. berghei and P. falciparum (Kato et al, 2008;Solyakov et al, 2011;Tewari et al, 2010). Using a broad-range serine/threonine kinase inhibitor (staurosporine), a marked reduction of phosphorylation of PfGAP45 was observed (Jones et al, 2009).…”

“…Recent advances in phosphopeptide enrichment coupled with high-throughput mass spectrometry have considerably increased the power of global phosphoproteomic analysis. Phosphoproteomes for "intracellular" and "extracellular" T. gondii tachyzoites as well as for P. falciparum schizont stage have been recently reported (Solyakov et al, 2011;Treeck et al, 2011). Additionally, a phosphoproteome with thousands of phosphorylation sites in P. falciparum erythrocytic stages will be released shortly (Lasonder et al, 2012).…”

“…In Solyakov et al (2011), a kinome-wide reverse genetics approach was conducted in parallel to identify the essential malaria kinases (Solyakov et al, 2011). Some of the predicted essential kinases were previously implicated in motility such as cAMPdependent protein kinase A (PKA) (Kurokawa et al, 2011;Leykauf et al, 2010), calcium-dependant protein kinase 1 (CDPK1) (Green et al, 2008;Kato et al, 2008;Winter et al, 2009), protein kinase B (PKB) (Vaid et al, 2008), and protein kinase G (PKG) (Hopp et al, 2012).…”