title: Protein phosphorylation during Plasmodium berghei gametogenesis.
abstract: Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies.
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Avenida Instituto Politécnico Nacional # 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, C.P. 07360, México, D.F., México.
authors.forename: Alberto
authors.initials: A
authors.lastname: Alonso-Morales
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Avenida Instituto Politécnico Nacional # 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, C.P. 07360, México, D.F., México.
authors.forename: Lorena
authors.initials: L
authors.lastname: González-López
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Avenida Instituto Politécnico Nacional # 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, C.P. 07360, México, D.F., México.
authors.forename: Febe Elena
authors.initials: FE
authors.lastname: Cázares-Raga
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Avenida Instituto Politécnico Nacional # 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, C.P. 07360, México, D.F., México.
authors.forename: Leticia
authors.initials: L
authors.lastname: Cortés-Martínez
authors.affiliation: Centro Regional de Investigación en Salud Pública, Instituto Nacional de Salud Pública, Avenida 19 Poniente esquina 4a Norte s/n, Colonia Centro, C.P. 62100 Tapachula, Chiapas, Mexico.
authors.forename: Jorge Aurelio
authors.initials: JA
authors.lastname: Torres-Monzón
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, USA.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Gallegos-Pérez
authors.affiliation: Centro de Investigación Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Avenida Universidad # 655, Colonia Santa María Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, México.
authors.forename: Mario Henry
authors.initials: MH
authors.lastname: Rodríguez
authors.affiliation: Departments of Molecular Biology and Biochemistry, and Microbiology and Molecular Genetics, University of California, Irvine, CA 92697, USA.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: James
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Avenida Instituto Politécnico Nacional # 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, C.P. 07360, México, D.F., México. Electronic address: cruzcruz@cinvestav.mx.
authors.forename: Fidel de la Cruz
authors.initials: Fde L
authors.lastname: Hernández-Hernández
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D005718:Gametogenesis / Q000187:drug effects / Q000502:physiology*; D006918:Hydroxyurea / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010962:Plasmodium berghei / Q000502:physiology*; D015800:Protozoan Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000494:pharmacology
chemical_list: D010750:Phosphoproteins; D015800:Protozoan Proteins; C009495:titanium dioxide; D014025:Titanium; D006918:Hydroxyurea
keywords: 2-DE immunoblotting; Gametogenesis; Mass spectroscopy; Plasmodium berghei; Protein phosphorylation