Molecular cloning of Trypanosoma brucei CK2 catalytic subunits: the alpha isoform is nucleolar and phosphorylates the nucleolar protein Nopp44/46.
We have demonstrated previously that Nopp44/46, an abundant nucleolar phosphoprotein of Trypanosoma brucei, is associated with a protein kinase. In many organisms multiple nucleolar proteins are phosphorylated by the protein kinase CK2, formerly known as casein kinase II. Here we report the identification of two T. brucei genes, CK2a1and CK2a2, which encode protein kinases bearing signature motifs common to CK2 catalytic subunits. The protein specified by CK2a1, designated CK2alpha, was capable of associating with Nopp44/46 as assessed by yeast two-hybrid analysis. An epitope-tagged version of CK2alpha expressed in T. brucei colocalized with Nopp44/46, with a largely nucleolar localization. This localization contrasts with the predominantly nuclear localization of mammalian CK2. When expressed in Escherichia coli, TbCK2alpha was catalytically active and phosphorylated Nopp44/46. Together these data demonstrate that TbCK2alpha is a Nopp44/46-associated kinase. Competition assays revealed that, unlike most CK2s, TbCK2alpha discriminates highly between ATP and GTP. This distinction may be associated with the substitution of glutamic acid and alanine for the di-asparagine motif thought to participate in purine interaction.
Park
Jeong-Hyun
JH
Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA 98109, USA.
Brekken
Deirdre L
DL
Randall
Amber C
AC
Parsons
Marilyn
M
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D047390:Casein Kinase II; D020134:Catalytic Domain / Q000235:genetics*; D002466:Cell Nucleolus / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006639:Histidine; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015800:Protozoan Proteins; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D007527:Isoenzymes; C100391:NOPP44-46 protein, Trypanosoma brucei; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases