1887

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Volume 98, Issue 3

Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered Free

Abstract

Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.

  • Received:
  • Accepted:
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Keyword(s): human cytomegalovirus , intrinsically disordered proteins , phosphatase inhibitors , phosphoprotein 150 , phosphoproteins , pUL32 and tegument proteins
© 2017 The Authors
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2017-03-01
2024-04-24
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Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered
J. Gen. Virol. 98, 471 (2017); https://doi.org/10.1099/jgv.0.000675
/content/journal/jgv/10.1099/jgv.0.000675
/content/journal/jgv/10.1099/jgv.0.000675
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