ABSTRACT
Serine (Ser)/threonine (Thr) or tyrosine (Tyr) protein kinases in eukaryotes contain RDxKxxN or RDx(A/R)A(A/R)N sequences, respectively, in the catalytic loop. Myxococcus xanthus DspB is a dual-specificity kinase that contains an atypical sequence, RDVAQKN, in the catalytic loop. The DspB mutant (A165K), which contains the canonical RDxKxxN motif, had an approximate 1.3-fold increase in kinase activity toward myelin basic protein (MBP). Arginine-aspartate (RD) kinases carry a conserved Arg immediately preceding the catalytic Asp that is required for autophosphorylation of the activation loop. DspB belongs to the RD kinase family and contains one Ser residue (Ser-190) and one Thr residue (Thr-194) in the activation loop. Mutation of Ser-190 or Thr-194 to Ala did not significantly affect the kinase activity toward MBP. We previously reported that four M. xanthus eukaryotic-like kinases (EPKs) are autophosphorylated on Tyr residues. These EPKs contain six Tyr residues at homologous positions, and five of those Tyr residues, Y25, Y102, Y145, Y173, and Y205, are conserved in DspB. DspB is mainly autophosphorylated on Y145, and a Y145F mutant has reduced kinase activity, suggesting that autophosphorylation of the Tyr residue of DspB may be required for high-level kinase activity.
Subject(s)
Myxococcus xanthus/enzymology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Catalysis , Eukaryota/enzymology , Humans , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods.
Subject(s)
Gene Expression Regulation, Viral/physiology , Orbivirus/physiology , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Cytopathogenic Effect, Viral , Glycosylation , HEK293 Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Viral Nonstructural Proteins/genetics , Virus Internalization , Virus ReplicationABSTRACT
Myxococcus xanthus has eukaryotic-like protein kinases (EPKs) with different atypical catalytic loop motifs. Seven out of 14 recombinant M. xanthus EPKs containing atypical motifs in the catalytic loop showed protein kinase activity against myelin basic protein and four autophosphorylated EPKs were detected using anti-phosphotyrosine antibody by western blotting.
Subject(s)
Biocatalysis , Eukaryota/enzymology , Myxococcus xanthus/enzymology , Protein Kinases/chemistry , Protein Kinases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Blotting, Western , Catalytic Domain , Molecular Sequence Data , Myelin Basic Protein/chemistry , Myelin Basic Protein/metabolism , Myxococcus xanthus/genetics , Phosphorylation , Phosphotyrosine/analysis , Phosphotyrosine/metabolism , Protein Kinases/analysis , Protein Kinases/genetics , Protein Structure, Tertiary , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The non-structural protein NS3 was investigated in Ibaraki virus (IBAV), an epizootic hemorrhagic disease virus, serotype 2. Degree of NS3 glycosylation, cytopathic effect, and virus release efficiency were compared between mammalian and insect cells. The molecular weight of synthesized NS3 was compared in Western blot analysis following the removal of the glycochain by PNGase F treatment and revealed that glycosylation of NS3 occurred only in mammalian cells. Also, it was revealed that the amount of infectious IBAV in the extracellular fraction continued to increase for insect cells even after 60h post infection without disrupting cells. These results suggested that glycosylation of NS3 controls pathogenicity of IBAV in host cells to protect vector insects by altering the release pathway of assembled progeny viruses.