Editors' ChoiceBiochemistry

Nonenzymatic lysine phosphorylation

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Sci. Signal.  21 Apr 2015:
Vol. 8, Issue 373, pp. ec98
DOI: 10.1126/scisignal.aab3709

Posttranslational modification (PTM) of proteins contributes to cellular complexity and expands the diversity of the encoded genome. Among the hundreds of known PTMs, kinase-mediated phosphorylation—that is, the addition of a phosphate group to serine, threonine, and tyrosine residues—is the best studied. The less studied PTM of pyrophosphorylation, which is the nonenzymatic transfer of a phosphate from inositol pyrophosphate (IP7) to a prephosphosphorylated serine residue, has been reported for the protein Nsr1, which is involved in rRNA synthesis. IP7 can also mediate the production of linear chains of polyphosphate (polyP). Azevedo et al. identified polyP at lysine residues of two proteins involved in rRNA synthesis, nuclear signal recognition (Nsr1) and topoisomerase (Top1), in yeast. Compared with the mobility of Nsr1 from wild-type yeast, the mobility of Nsr1 in a gel shift assay was reduced in yeast lacking inositol 6 kinase (kcs1Δ) or inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase (vip1Δ), enzymes necessary to synthesize IP7. Analysis of the mobility shifts of Nsr1 from yeast lacking vacuolar transporter chaperone (vtcΔ), which cannot produce polyP, indicated that the PTM involved was polyP addition. Furthermore, incubating extracts of wild-type yeast with the exopolyphosphatase Ppx1, which hydrolyzes polyP chains that exceed 3 moieties but not IP7, altered Nsr1 mobility. Purified Nsr1 treated with denatured cellular extracts from wild-type or vip1Δ yeast was phosphorylated and exhibited a mobility shift, indicating the nonenzymatic covalent transfer of the phosphate group. Incubating Top1 with 32P-polyP resulted in a mobility shift of Top1, indicating that Top1 can also undergo nonenzymatic polyP phosphorylation. Unexpectedly, mutational analysis indicated that polyP phosphorylation occurred at a lysine residue in the N-terminal region of both Nsr1 and Top1. Nsr1 and Top1 in wild-type and vip1Δ yeast a diffuse nucleoplasmic localization, whereas in the ksc1Δ and vtc4Δ yeast (with minimal or no polyP phosphorylation, respectively), the two proteins were restricted to nucleolus. Analysis of the ability of Top1 in protein extracts from the yeast mutants to relax supercoiled DNA in vitro revealed that the Top1 from the mutants with the lowest polyP-modifying activity (ksc1Δ and vtc4Δ) had the highest activity, indicating negative regulation by polyP phosphorylation. Thus, nonenzymatic polyP modification on lysine, a residue that is also modified by other PTMs such as acetylation, methylation, ubiquitination, and SUMOylation, represents another form of phosphorylation. How prevalent this modification is and what regulates its occurrence and controls its reversibility are exciting questions for future study.

C. Azevedo,T. Livermore, A. Saiardi, Protein polyphosphorylation of lysine residues by inorganic polyphosphate. Mol. Cell 58, 71–82 (2015). [PubMed]