Pyrophosphoproteomics: extensive protein pyrophosphorylation revealed in human cell lines

Abstract

Reversible protein phosphorylation is a central signaling mechanism in eukaryotic cells. While the identification of canonical phosphorylation sites using mass-spectrometry (MS) based proteomics has become routine, annotation of non-canonical phosphorylation has remained a challenge. Here, we report a tailored pyrophosphoproteomics workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing the first direct evidence of endogenous protein pyrophosphorylation. Detection of protein pyrophosphorylation was reproducible, specific and consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. A predictive workflow based on the MS data set was established to recognize putative pyrophosphorylation sequences, and UBF1, a nucleolar protein incompatible with the proteomics method, was biochemically shown to undergo pyrophosphorylation. When the biosynthesis of PP-InsPs was perturbed in a model cell line, proteins expressed in this background exhibited lower levels of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1, and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation, and should be considered in future phosphoproteomic analyses.

Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-IP7

“…Pyrophosphorylation occurs predominantly on Ser residues that lie in the vicinity of acidic Asp and Glu residues, within an intrinsically disordered region of the substrate protein [9,17].…”

“…Interestingly, the IP6K1 interactome contained 5-IP7 pyrophosphorylation substrates including AP3B1, NOLC1, TCOF and UBF1 (Table 1 and Table S2) [10][11][12]17]. Among the interactors we also observed the catalytic α and α' (CSK21 and CSK22), and regulatory β (CSK2B) subunits of protein kinase CK2 (Table 1 and Table S2), which is the primary kinase responsible for pre-phosphorylation of proteins prior to their pyrophosphorylation by 5-IP7 [11,12,14,15].…”

“…Using radiolabeling methods, 5-IP7 mediated pyrophosphorylation has been demonstrated individually for S. cerevisiae proteins Nsr1, Srp40, Rpa190, Rpa43, Rpa34, and Gcr1, and mammalian proteins AP3B1, DIC2C, MYC, NOLC1, TCOF and UBF1 [10][11][12][13][14][15][16][17]. Recently, 148 sites of pyrophosphorylation were identified in 71 human proteins by a mass-spectrometry based proteomics analysis, of which the nucleolar proteins NOLC1 and TCOF possess the most number of pyrophosphosites [17]. It was shown that endogenous pyrophosphorylation of these proteins is indeed dependent on 5-IP7, as co-expression of active IP6K1 led to increased pyrophosphorylation of these proteins, whereas inactive IP6K1 did not have any effect.…”

“…Although pre-phosphorylation of the target serine residues is brought about by acidophilic Ser/Thr kinases, phosphotransfer of the β-phosphate from the donor PP-IP to the acceptor phosphoserine residue can take place without any enzyme. Using radiolabeling methods, 5-IP7 mediated pyrophosphorylation has been demonstrated individually for S. cerevisiae proteins Nsr1, Srp40, Rpa190, Rpa43, Rpa34, and Gcr1, and mammalian proteins AP3B1, DIC2C, MYC, NOLC1, TCOF and UBF1 [10][11][12][13][14][15][16][17]. Recently, 148 sites of pyrophosphorylation were identified in 71 human proteins by a mass-spectrometry based proteomics analysis, of which the nucleolar proteins NOLC1 and TCOF possess the most number of pyrophosphosites [17].…”

Fluorination Influences the Bioisostery of Myo‐Inositol Pyrophosphate Analogs

“…[2] These properties underlie the ability of PPÀ IPs to regulate target protein function by non-enzymatically phosphorylating a pre-existing phosphorylation site on both Ser and Thr. [3][4][5] The PPÀ IP diphosphate groups also contribute to ligand specificity through allosteric interactions with protein receptors; prominent mammalian examples include the SPX domain of mammalian XPR1, [8,9] and certain pleckstrin homology domains. [10][11][12] Separately, the high negative charge density of PPÀ IPs is also hypothesized to act as an intermolecular electrostatic 'glue' to promote formation of functionally significant macromolecular complexes.…”