title: Two nucleus-localized CDK-like kinases with crucial roles for malaria parasite erythrocytic replication are involved in phosphorylation of splicing factor.
abstract: The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Despite extensive research on most plasmodial enzymes, little information is available regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family. In other eukaryotes, CLKs regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Here, we investigate two of the PfCLKs, the Lammer kinase homolog PfCLK-1, and PfCLK-2. Both PfCLKs show homology with the yeast Serine/Arginine protein kinase Sky1p and are transcribed throughout the asexual blood stages and in gametocytes. PfCLK-1/Lammer possesses two nuclear localization signal sites and PfCLK-2 possesses one of these signal sites upstream of the C-terminal catalytic domains. Indirect immunofluorescence, Western blot, and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and PfCLK-2 is further present in the cytoplasm. The two kinases are important for completion of the asexual replication cycle of P. falciparum, as demonstrated by reverse genetics approaches. In vitro kinase assays show substrate phosphorylation by the PfCLKs, including the Sky1p substrate, splicing factor Npl3p, and the plasmodial alternative splicing factor PfASF-1. Mass spectrometric analysis of co-immunoprecipitated proteins indicates assembly of the two PfCLKs with proteins with predicted nuclease, phosphatase, or helicase functions. Our data indicate a crucial role of PfCLKs for malaria blood stage parasites, presumably by participating in gene regulation through the post-transcriptional modification of mRNA.
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Strasse 2, Building D15, 97080 Würzburg, Germany.
authors.forename: Shruti
authors.initials: S
authors.lastname: Agarwal
authors.forename: Selina
authors.initials: S
authors.lastname: Kern
authors.forename: Jean
authors.initials: J
authors.lastname: Halbert
authors.forename: Jude M
authors.initials: JM
authors.lastname: Przyborski
authors.forename: Stefan
authors.initials: S
authors.lastname: Baumeister
authors.forename: Thomas
authors.initials: T
authors.lastname: Dandekar
authors.forename: Christian
authors.initials: C
authors.lastname: Doerig
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pradel
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000201:enzymology; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D008288:Malaria / Q000469:parasitology*; D051379:Mice; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing
chemical_list: C066780:Clk dual-specificity kinases; C433212:psk1 protein, Plasmodium falciparum; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases