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Malar J. 2016 Apr 29;15:248. doi: 10.1186/s12936-016-1275-9.

Characterization of an A-kinase anchoring protein-like suggests an alternative way of PKA anchoring in Plasmodium falciparum.

Bandje K1,2,3, Naissant B2,3,4,5, Bigey P2,6, Lohezic M1,2,3, Vayssières M2,7, Blaud M2,7, Kermasson L1,2,3, Lopez-Rubio JJ8, Langsley G2,4,5,9, Lavazec C2,3,4,5,10, Deloron P1,2,3, Merckx A11,12,13.

Author information

1
IRD UMR216-MERIT, Faculté de Pharmacie, Mère et enfant face aux Infections Tropicales, Paris, France.
2
COMUE Sorbonne Paris Cité, Université Paris Descartes, Paris, France.
3
Laboratoire d'Excellence GR-Ex, Paris, France.
4
INSERM U1016, Institut Cochin, Paris, France.
5
CNRS UMR 8104, Faculté de Médecine, Paris, France.
6
Chimie ParisTech, PSL Research University, Paris, France.
7
UMR CNRS 8015, Faculté de Pharmacie, Laboratoire de Cristallographie et RMN Biologiques, Paris, France.
8
CNRS5290, IRD224, University Montpellier 1 & 2, MIVEGEC, Montpellier, France.
9
Laboratoire d'Excellence ParaFrap, Paris, France.
10
Institut Pasteur, Paris, France.
11
IRD UMR216-MERIT, Faculté de Pharmacie, Mère et enfant face aux Infections Tropicales, Paris, France. anais.merckx@parisdescartes.fr.
12
COMUE Sorbonne Paris Cité, Université Paris Descartes, Paris, France. anais.merckx@parisdescartes.fr.
13
Laboratoire d'Excellence GR-Ex, Paris, France. anais.merckx@parisdescartes.fr.

Abstract

BACKGROUND:

The asexual intra-erythrocytic multiplication of the malaria parasite Plasmodium falciparum is regulated by various molecular mechanisms. In eukaryotic cells, protein kinases are known to play key roles in cell cycle regulation and signaling pathways. The activity of cAMP-dependent protein kinase (PKA) depends on A-kinase anchoring proteins (AKAPs) through protein interactions. While several components of the cAMP dependent pathway-including the PKA catalytic and regulatory subunits-have been characterized in P. falciparum, whether AKAPs are involved in this pathway remains unclear. Here, PfAKAL, an open reading frame of a potential AKAP-like protein in the P. falciparum genome was identified, and its protein partners and putative cellular functions characterized.

METHODS:

The expression of PfAKAL throughout the erythrocytic cycle of the 3D7 strain was assessed by RT-qPCR and the presence of the corresponding protein by immunofluorescence assays. In order to study physical interactions between PfAKAL and other proteins, pull down experiments were performed using a recombinant PfAKAL protein and parasite protein extracts, or with recombinant proteins. These interactions were also tested by combining biochemical and proteomic approaches. As phosphorylation could be involved in the regulation of protein complexes, both PfAKAL and Pf14-3-3I phosphorylation was studied using a radiolabel kinase activity assay. Finally, to identify a potential function of the protein, PfAKAL sequence was aligned and structurally modeled, revealing a conserved nucleotide-binding pocket; confirmed by qualitative nucleotide binding experiments.

RESULTS:

PfAKAL is the first AKAP-like protein in P. falciparum to be identified, and shares 23 % sequence identity with the central domain of human AKAP18δ. PfAKAL is expressed in mature asexual stages, merozoites and gametocytes. In spite of homology to AKAP18, biochemical and immunochemical analyses demonstrated that PfAKAL does not interact directly with the P. falciparum PKA regulatory subunit (PfPKA-R), but instead binds and colocalizes with Pf14-3-3I, which in turn interacts with PfPKA-R. In vivo, these different interactions could be regulated by phosphorylation, as PfPKA-R and Pf14-3-3I, but not PfAKAL, are phosphorylated in vitro by PKA. Interestingly, PfAKAL binds nucleotides such as AMP and cAMP, suggesting that this protein may be involved in the AMP-activated protein kinase (AMPK) pathway, or associated with phosphodiesterase activities.

CONCLUSION:

PfAKAL is an atypical AKAP that shares common features with human AKAP18, such as nucleotides binding. The interaction of PfAKAL with PfPKA-R could be indirectly mediated through a join interaction with Pf14-3-3I. Therefore, PfPKA localization could not depend on PfAKAL, but rather involves other partners.

KEYWORDS:

14-3-3 protein; AMP; A‐kinase anchoring protein like (AKAL); Interactome; Nucleotide; Plasmodium falciparum

PMID:
27129434
PMCID:
PMC4850634
DOI:
10.1186/s12936-016-1275-9
[Indexed for MEDLINE]
Free PMC Article

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