The Plasmodium falciparum Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling Edwin Lasonder,* ,† Judith L. Green, ‡ Grazia Camarda, § Hana Talabani, ∥ Anthony A. Holder, ‡ Gordon Langsley, ∥ and Pietro Alano § † Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands ‡ Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom § Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanita ̀ , Viale Regina Elena n.299, 00161 Rome, Italy ∥ Laboratoire de Biologie Cellulaire Comparative des Apicomplexes, Institut Cochin, Inserm U1016, CNRS UMR 8104, Faculte ́ de Me ́ decine, Universite ́ Paris Descartes Sorbonne Paris Cite ́ , 27, rue du Faubourg-Saint-Jacques, 75014 Paris, France * S Supporting Information ABSTRACT: The asexual blood stages of Plasmodium falciparum cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of P. falciparum schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion. KEYWORDS: malaria, phosphoproteome, schizonts, inositol/PKA signalling pathways ■ INTRODUCTION The life cycle of the Apicomplexan malaria parasite Plasmodium falciparum is complex and composed of several developmental stages in the mosquito and human hosts. Asexual blood stage development causes the mortality and morbidity associated with malaria. After red blood cell invasion by a single merozoite, the parasite develops within a parasitophorous vacuole (PV), from ring stage to trophozoite, and then undergoes 4 to 5 rounds of DNA synthesis, mitosis and nuclear division that result in a schizont with 16−22 nuclei, which produces and releases new invasive merozoites to continue the cycle. 1 Merozoite release and reinvasion requires signal transduction and secretion of organellar contents at egress, and the formation of a moving junction between parasite and host cell surface at invasion that involves transfer of some parasite proteins to the newly invaded erythrocyte: processes that may be controlled in part by calcium flux and calcium-dependent phosphorylation. 2 Fine-tuning of the eukaryotic cellular machinery is regulated by several mechanisms involving transcriptional control, post- translational control, and post-translational modification of proteins (PTM). It has become evident that protein kinases and signal transduction pathways are integral to regulation of the Plasmodium parasite life cycle. 3,4 In other organisms inter- and intracellular signals trigger the release of secondary messenger molecules that activate protein kinases, which transmit signals to the nucleus via a cascade of phosphorylation events to regulate the transcription machinery of the cell. P. falciparum is considered in this respect an “insensitive beast”, because there is a relatively small number of known external stimuli, 5 and because it apparently lacks the tyrosine kinase signaling pathways found in most eukaryotes that are activated by signals from outside the cell (e.g., growth factors). Phylogenetic studies 6,7 revealed that the kinome of P. falciparum (approx 100 eukaryotic protein kinases (ePKs)) diverges profoundly from those of mammalian species, with a large proportion of kinases classified either as semiorphan ePKs Received: June 20, 2012 Published: October 1, 2012 Article pubs.acs.org/jpr © 2012 American Chemical Society 5323 dx.doi.org/10.1021/pr300557m | J. Proteome Res. 2012, 11, 5323−5337