Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP7.

Inositol pyrophosphates (PP-InsPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-InsPs can transfer their β-phosphate group to a phosphorylated Ser residue to generate pyrophosphorylated Ser. This unique post-translational modification occurs on Ser residues that lie in acidic stretches within an intrinsically disordered protein sequence. Serine pyrophosphorylation is dependent on the presence of Mg2+ ions, but does not require an enzyme for catalysis. The mechanisms by which cells regulate PP-InsP-mediated pyrophosphorylation are still unknown. We performed mass spectrometry to identify interactors of IP6K1, an enzyme responsible for the synthesis of the PP-InsP 5-InsP7. Interestingly, IP6K1 interacted with several proteins that are known to undergo 5-InsP7-mediated pyrophosphorylation, including the nucleolar proteins NOLC1, TCOF and UBF1, and AP3B1, the β subunit of the AP3 adaptor protein complex. The IP6K1 interactome also included CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We observe the formation of a protein complex between IP6K1, AP3B1, and the catalytic α-subunit of CK2, and show that disrupting IP6K1 binding to AP3B1 lowers its in vivo pyrophosphorylation. We propose that assembly of a substrate-CK2-IP6K complex would allow for coordinated pre-phosphorylation and pyrophosphorylation of the target serine residue, and provide a mechanism to regulate this enzyme-independent modification.

Hamid A ，Ladke JS ，Shah A ，Ganguli S ，Pal M ，Singh A ，Bhandari R ... -  《-》

Inositol pyrophosphates promote MYC polyubiquitination by FBW7 to regulate cell survival.

The transcription factor MYC regulates cell survival and growth, and its level is tightly controlled in normal cells. We report that serine pyrophosphorylation - a posttranslational modification triggered by inositol pyrophosphate signaling molecules - controls MYC levels via regulated protein degradation. We find that endogenous MYC is stabilized and less polyubiquitinated in cells with reduced inositol pyrophosphates. We show that the inositol pyrophosphate 5-IP7 transfers its high-energy beta phosphate moiety to pre-phosphorylated serine residues in the central PEST domain of MYC. Loss of serine pyrophosphorylation in the PEST domain lowers the extent of MYC polyubiquitination and increases its stability. Fusion to the MYC PEST domain lowers the stability of GFP, but this effect is dependent on the extent of PEST domain pyrophosphorylation. The E3 ubiquitin ligase FBW7 can bind directly to the PEST domain of MYC, and this interaction is exclusively dependent on serine pyrophosphorylation. A stabilized, pyrophosphorylation-deficient form of MYC increases cell death during growth stress in untransformed cells. Splenocytes from mice lacking IP6K1, a kinase responsible for the synthesis of 5-IP7, have higher levels of MYC, and show increased cell proliferation in response to mitogens, compared with splenocytes from wild type mice. Thus, control of MYC stability through a novel pyro-phosphodegron provides unexpected insight into the regulation of cell survival in response to environmental cues.

Lolla P ，Shah A ，Unnikannan CP ，Oddi V ，Bhandari R ... -  《-》

Protein pyrophosphorylation by inositol phosphates: a novel post-translational modification in plants?

Inositol pyrophosphates (PP-InsPs) are energy-rich molecules harboring one or more diphosphate moieties. PP-InsPs are found in all eukaryotes evaluated and their functional versatility is reflected in the various cellular events in which they take part. These include, among others, insulin signaling and intracellular trafficking in mammals, as well as innate immunity and hormone and phosphate signaling in plants. The molecular mechanisms by which PP-InsPs exert such functions are proposed to rely on the allosteric regulation via direct binding to proteins, by competing with other ligands, or by protein pyrophosphorylation. The latter is the focus of this review, where we outline a historical perspective surrounding the first findings, almost 20 years ago, that certain proteins can be phosphorylated by PP-InsPs in vitro. Strikingly, in vitro phosphorylation occurs by an apparent enzyme-independent but Mg2+-dependent transfer of the β-phosphoryl group of an inositol pyrophosphate to an already phosphorylated serine residue at Glu/Asp-rich protein regions. Ribosome biogenesis, vesicle trafficking and transcription are among the cellular events suggested to be modulated by protein pyrophosphorylation in yeast and mammals. Here we discuss the latest efforts in identifying targets of protein pyrophosphorylation, pointing out the methodological challenges that have hindered the full understanding of this unique post-translational modification, and focusing on the latest advances in mass spectrometry that finally provided convincing evidence that PP-InsP-mediated pyrophosphorylation also occurs in vivo. We also speculate about the relevance of this post-translational modification in plants in a discussion centered around the protein kinase CK2, whose activity is critical for pyrophosphorylation of animal and yeast proteins. This enzyme is widely present in plant species and several of its functions overlap with those of PP-InsPs. Until now, there is virtually no data on pyrophosphorylation of plant proteins, which is an exciting field that remains to be explored.

Mihiret YE ，Schaaf G ，Kamleitner M 《Frontiers in Plant Science》

A high energy phosphate jump - From pyrophospho-inositol to pyrophospho-serine.

Inositol pyrophosphates (PP-IPs) are a class of energy rich metabolites present in all eukaryotic cells. The hydroxyl groups on these water soluble derivatives of inositol are substituted with diphosphate and monophosphate moieties. Since the discovery of PP-IPs in the early 1990s, enormous progress has been made in uncovering pleiotropic roles for these small molecules in cellular physiology. PP-IPs exert their effect on proteins in two ways - allosteric regulation by direct binding, or post-translational regulation by serine pyrophosphorylation, a modification unique to PP-IPs. Serine pyrophosphorylation is achieved by Mg2+-dependent, but enzyme independent transfer of a β-phosphate from a PP-IP to a pre-phosphorylated serine residue located in an acidic motif, within an intrinsically disordered protein sequence. This distinctive post-translational modification has been shown to regulate diverse cellular processes, including rRNA synthesis, glycolysis, and vesicle transport. However, our understanding of the molecular details of this phosphotransfer from pyrophospho-inositol to generate pyrophospho-serine, is still nascent. This review discusses our current knowledge of protein pyrophosphorylation, and recent advances in understanding the mechanism of this important yet overlooked post-translational modification.

Ganguli S ，Shah A ，Hamid A ，Singh A ，Palakurti R ，Bhandari R ... -  《-》

Back-Pyrophosphorylation Assay to Detect In Vivo InsP(7)-Dependent Protein Pyrophosphorylation in Mammalian Cells.

Chanduri M ，Bhandari R 《-》