title: Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered.
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.
authors.affiliation: Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Franz J J
authors.initials: FJJ
authors.lastname: Rieder
authors.affiliation: Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Marie-Theres
authors.initials: MT
authors.lastname: Kastner
authors.affiliation: Mass Spectrometry Facility, Max F. Perutz Laboratories, Vienna Biocenter, Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Hartl
authors.affiliation: Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Puchinger
authors.affiliation: Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Schneider
authors.affiliation: Center for Pathophysiology, Infectiology and Immunology, Institute of Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Otto
authors.initials: O
authors.lastname: Majdic
authors.affiliation: Department of Pediatrics, Children's Hospital, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Britt
authors.affiliation: Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.|Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinović-Carugo
authors.affiliation: Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Steininger
mesh_terms: D003587:Cytomegalovirus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D014763:Viral Matrix Proteins / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases