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. 2000:1:2.
doi: 10.1186/1471-2091-1-2. Epub 2000 Sep 5.

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

N G Brown  1 , Nick Morrice D, G Beaud, G Hardie, D P Leader
Affiliations

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

N G Brown et al. BMC Biochem. 2000.

Abstract

Background: Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions.

Results: Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala.

Conclusions: We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

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Figures

Figure 1
Figure 1
HPLC fractionation of V8 peptides from vaccinia virus protein H5R. (a) HPLC fractionation of hydrolysate after passage through a SEP-PAK column using a gradient of 0-50% acetonitrile. The fractions encompassed by the horizontal bar (region 3) were collected for further analysis. (b) Microbore HPLC fractionation of region 3 from (a). A gradient of 15-25% acetonitrile in 0.1% trifluoroacetic acid was used and the fractions encompassed by the horizontal bars (regions I and II) collected for further analysis). (c) Microbore HPLC fractionation of region II from (b). A gradient of 0-50% acetonitrile in 20 mM NaCl (in the absence of trifluoroacetic acid - i.e. at neutral pH) was used and the fractions 28 and 29 collected for sequence analysis). The direction of increasing gradient is from left to right in all three frames; the ultraviolet absorbance trace is labelled as A280, and the radioactivity trace as 32P.
Figure 2
Figure 2
Sequence analysis of H5R phosphopeptide. Fraction 29 from Fig 1c was subjected to automatic Edman degradation, and the 32P radioactivity released at each stage measured. The identity of the phenylthiohydantoin derivative from the first seven cycles was unequivocal and is indicated.
Figure 3
Figure 3
Phosphorylation of synthetic peptides. Peptides were phosphorylated as described in the Experimental section and subjected to electrophoresis on thin-layer cellulose at pH 3.5. The position of the phosphopeptides is indicated by the arrow, the identification being based on the fact that the ninhydrin-stained material (predominantly unphosphorylated and hence with a greater positive charge) migrated just ahead of this. Lane 1 contained RRIEEYHQTTEKN, lane 2: RRIEEYHQATEKN, lane 3: RRIEEYHQTAEKN, and lane 4; no peptide.
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