Shubhra Ganguli

The inositol pyrophosphate 5-InsP ₇ , composed of an inositol ring substituted with five monophosphates and one diphosphate, modulates diverse cellular functions by protein pyrophosphorylation, during which its β-phosphate moiety is transferred to a pre-phosphorylated serine residue on the target protein. In mammals, the synthesis of 5-InsP ₇ from its precursor InsP ₆ is catalyzed by a family of enzymes called IP6Ks. We report that during recovery from genotoxic stress, cells lacking the IP6K isoform IP6K1 exhibit prolonged persistence of DNA damage foci marked by the homologous recombination repair protein RAD51. Expression of catalytically active but not inactive IP6K1 reverses this defect, implying that 5-InsP ₇ supports the dissolution of RAD51 foci. Upon DNA damage, we observe an increase in IP6K1 activity, contingent on its phosphorylation by the protein kinases CK2 and CDK1. IP6K1 is recruited to sites of DNA damage, and interacts with RAD51, CDK1, and the C-terminal domain (CTD) of BRCA2. Disruption of binding between RAD51 and BRCA2-CTD is known to support the disassembly of RAD51 foci. We show that 5-InsP ₇ can pyrophosphorylate RAD51, and that the presence of 5-InsP ₇ diminishes RAD51 binding to BRCA2-CTD. Our findings provide a mechanism by which 5-InsP ₇ synthesized by IP6K1 facilitates the removal of RAD51 from sites of DNA repair. Summary statement: Inositol hexakisphosphate kinase 1, an enzyme that catalyses the synthesis of the inositol pyrophosphate 5-InsP ₇ , localises to DNA double strand breaks, and engages in interactions with proteins involved in homologous recombination (HR)-mediated DNA repair. 5-InsP ₇ disrupts the interaction between RAD51 and the C-terminus of BRCA2, promoting dislodgement of RAD51 from DNA damage sites post-repair.

Reversible protein phosphorylation is a central signaling mechanism in eukaryotes. Although mass-spectrometry-based phosphoproteomics has become routine, identification of non-canonical phosphorylation has remained a challenge. Here we report a tailored workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing, to our knowledge, the first direct evidence of endogenous protein pyrophosphorylation. We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. Detection was consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). When the biosynthesis of PP-InsPs was perturbed, proteins expressed in this background exhibited no signs 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.

Inositol pyrophosphates (PP-IPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-IPs 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 Mg ²⁺ ions, but does not require an enzyme for catalysis. The mechanisms by which cells can regulate this enzyme-independent modification are still unknown. Here, we show that IP6K1, an enzyme responsible for the synthesis of the PP-IP 5-IP7, interacts with several proteins that undergo 5-IP7 mediated pyrophosphorylation, and with CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We characterized the interaction of IP6K1 with AP3B1, the β subunit of the AP3 adaptor protein complex, which is a known pyrophosphorylation substrate. 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.