Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of CDPK4. This mutant is generated in a non-gametocyte producer clone of P. berghei ANKA (P. berghei ANKA 2.33; see below for more information)
Protein (function)
CDPK4 belongs to an expanded family of Ca2+ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. In plants, CDPKs translate Ca2+ signals generated by external stimuli into cellular responses, thereby regulating cell division and differentiation, the development of tolerance to stress stimuli and the specific defense responses to pathogens.
CDPK4 has a role in male gamete formation: in male gametocytes axoneme formation, formation of mitotic spindles and DNA synthesis is inhibited/blocked (see RMgm-12).
Phenotype
Endogenously tagged CDPK4 localises to the cell periphery of merozoites
The mutant in this study was generated to screen for genetic interactions among protein kinases. In this study a role of CDPK4 during erythrocytic (asexual blood stage) proliferation has been found, which was dependent on a negative interaction with PKG.
From the paper:
'While calcium levels can account for the functional interaction between pkg and cdpk4 mutations, they do not explain how CDPK4 can affect RBC invasion by the merozoite. To address this question, we tagged CDPK4 in PbANKA 2.33, a line unable to produce gametocytes, and confirmed its expression in erythrocytic schizonts. Immunofluorescence localisation shows a signal excluded from the nucleus with a slight enrichment at the merozoite periphery. In CDPK4-3xHA immunoprecipitates following crosslinking, 19 proteins are enriched over wild-type controls, including GAP40, MyoA and GAC, three proteins essential for the IMC biogenesis or gliding motility. In addition to MyoA, CDPK4 also immunoprecipitates an uncharacterised Plasmodium-specific myosin, MyoE (PBANKA_0112200). Altogether, this further suggests that the redundant role of CDPK4 observed in invasion could be linked to the regulation of the molecular machinery that forms the IMC or provides the force for invasion.
Endogenously tagged GAP40 localises to the parasite periphery. Among the 20 proteins coimmunoprecipitated with GAP40-3xHA is CDPK4, in addition to known IMC and glideosome components.
GAP40-3xHA also coprecipitates another member of the CDPK family involved in merozoite invasion, CDPK1, and again, MyoE. Epitope tagged CDPK1 and MyoE are enriched at the cell periphery, and both proteins co-immunoprecipitate multiple glideosome or IMC components. Collectively, these results suggest MyoE may act as an alternative myosin of the motor complex and that both CDPK1 and CDPK4 are at the interface between the glideosome and the IMC. Immunoprecipitation of GAP40 or MyoE recovers multiple peptides from a protein of unknown function which, like GAP40 itself, was one of a small number of hits that emerged from our recent biochemical screen for substrates of CDPK4 (SOC proteins) in parasite lysates. This protein, SOC6 (PBANKA_0707700), has since been shown to interact with the IMC protein Phil1 in P. berghei schizonts and with MyoA in P. falciparum schizonts, and may thus provide a molecular link between CDPK4 and invasion. SOC6 is characterised by a C-terminal stretch of 106 amino acids that are relatively conserved across the syntenic orthologues of different malaria parasites, but lacks obvious homologues in other apicomplexan genomes. SOC6 is further characterised by 4 to 15 tandem amino acid repeats that show sequence and position variability across species. In P. berghei, the serine residue that CDPK4 phosphorylates in vitro lies in one such repeat of 54 amino acids, which has prevented us from mutagenising specifically the phosphosite. Endogenously tagged SOC6-3xHA localises to the cell periphery of merozoites and immunoprecipitates peptides from multiple IMC, glideosomeassociated proteins and glideosome proteins. Altogether, this indicates that SOC6 is also at the interface between the IMC and the glideosome. A SOC6-KO line shows a significant growth defect compared with wild type. While segmented SOC6-KO schizonts display the same number of merozoites as wild type, they show a reduced capacity to transform into ring stage parasites, while no accumulation of circulating SOC6-KO schizonts is observed. This indicates SOC6 is important either at the final stage of schizont maturation or to invade new RBCs. TEM of purified SOC6-KO schizonts reveals a discontinuous IMC as observed for PKGT619Q-3xHA/CDPK4-KO transgenic, suggesting that SOC6 is important for the formation or the stability of the IMC in merozoites. To investigate the function of SOC6 further, we turned to the ookinete stage, which in P. berghei offers a tractable model to study the molecular motor that powers gliding motility. Ookinetes emerge from the zygote that forms after fertilisation of macrogametes by microgametes in the mosquito blood meal. Male gamete formation does not require SOC6, but the SOC6-KO nevertheless fails to form typical banana-shaped ookinetes. Again, TEM of SOC6-KO cells reveals either a discontinuous IMC or the complete absence of an IMC below the plasma membrane, suggesting that SOC6 plays a conserved role to control the IMC formation or stability at multiple stages of the malaria lifecycle.
From the Abstract:
Most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito.
Additional information
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