ITPR3 encodes the type 3 inositol 1,4,5-trisphosphate receptor, an endoplasmic-reticulum channel that releases stored calcium when activated by IP3. The receptor participates in calcium-dependent processes including transcription, cell migration, proliferation, apoptosis and homeostasis. Its large cytoplasmic regulatory region contains sites for post-translational modification and interactions with other proteins, making the mapping of individual phosphosites potentially useful for understanding how calcium signaling is coordinated with other cellular systems. Earlier experimental work has suggested that S916, S934 and S1832 do not directly change ITPR3-mediated calcium release, leaving open the possibility that they influence regulatory complexes or downstream signaling instead.

For the study, published by Rai and colleagues in Frontiers in Bioinformatics in October 2026, researchers searched PubMed for human phosphoproteomics studies through May 2024. Their screening yielded 160 relevant publications containing 789 qualitative profiling datasets and 187 quantitative differential datasets. The curated material included label-free, tandem mass tag and SILAC measurements produced with different mass-spectrometry platforms and phosphopeptide-enrichment methods. To retain high-confidence assignments, the team required a localization probability of at least 75% or an A-score above 13, and it filtered differential datasets using p values below 0.05, fold changes of at least 1.3 for increases and no more than 0.76 for decreases.

That harmonized collection contained 24 ITPR3 phosphosites in qualitative profiles and 18 in differential datasets. S1832 was detected most often, appearing in 655 profiling datasets and 99 differential datasets, followed by S2670 with 644 and 69 appearances, S934 with 561 and 74, and S916 with 215 and 24. These four sites were designated as predominant because of their recurrence across independent studies rather than because their biological effects had been demonstrated. S916 and S934 lie between the receptor’s two RYDR domains, S1832 falls between the second RYDR and RIH domains, and S2670 is near the C terminus. All four are outside the IP3-binding and transmembrane channel domains. Sequence comparisons also showed that the positions were not conserved across the three mammalian IP3 receptor isoforms, although they were highly conserved in ITPR3 orthologues from humans, mice, rats and cattle.

The four sites also showed evidence of coordinated phosphorylation. S1832 was positively co-regulated with S2670 in 27 datasets, with S934 in 19 and with S916 in 15; S934 and S916 were positively co-regulated in 11 datasets. More extensive computational comparisons found that S1832 was positively and negatively co-regulated with 165 and 88 phosphosites in other proteins, respectively. The corresponding counts were 241 positive and 108 negative for S934, 412 positive and 61 negative for S2670, and 339 positive and 581 negative for S916. These relationships were not interpreted as direct physical interactions. Instead, they indicate that phosphorylation of ITPR3 at one position often changes alongside phosphorylation elsewhere in the cell under the experimental conditions represented in the datasets.

Functional enrichment separated the four sites into partly overlapping but distinguishable regulatory landscapes. Proteins positively associated with S1832 were enriched for chromatin organization, heterochromatin formation and regulation of DNA-templated and RNA polymerase II transcription. The S2670 network was associated with chromatin remodeling, transcriptional regulation and control of the G2-to-M transition, while proteins positively co-regulated with S916 were enriched for RNA processing and spliceosome-mediated mRNA splicing. The S934 network was associated more prominently with protein localization, small-GTPase signaling, lamellipodium organization, DNA-break localization and cortical microtubules. Negatively co-regulated proteins likewise showed site-specific patterns involving transcription, translation, alternative splicing, organelle organization, the cytoskeleton and anoikis. Hub-protein analysis supported these divisions, with highly connected nodes aligned to the enriched processes.

The analysis also nominated candidate upstream kinases, but did not demonstrate that they phosphorylate the sites in cells. CAMK2D was positively co-regulated with S1832, whereas MELK, RPS6KB2 and PAK4 were negatively associated with it. For S934, RPS6KA4, MARK2 and CAMK2D appeared in the positive network, and PAK4 was identified by both co-regulation analysis and NetworKIN/AKID prediction. PRKCD and PRKCE were predicted as possible upstream kinases for S2670. The S916 network included WNK1, RPS6KA3, MAP3K20, TRPM7, STK10 and PAK2 among negatively associated kinases, with PAK4 and PAK2 also highlighted by sequence-based prediction. These kinase labels therefore combine co-occurrence patterns with computational inference, leaving direct kinase activity and site modification to be tested experimentally.

Mapping co-regulated proteins onto established signaling resources placed the ITPR3 sites within a network extending beyond the receptor’s immediate calcium-release function. S1832 was positively associated with calcium/calmodulin-dependent protein kinase II delta and epidermal growth factor receptor, while S934 was positively associated with histamine receptor H1, VDAC1, PHKB, PHKA2 and CAMK2D. PLCD3, a component of IP3 generation, appeared in the positive S2670 network, while STIM2, involved in store-operated calcium entry, appeared in the negative S916 network. Other associated proteins included the receptors EGFR and MET and the calcium-responsive kinase PTK2B. Complex-interaction networks were enriched for pathways including calcium signaling, Rap1 signaling, focal adhesion, adherens and tight junctions, endocytosis, axon guidance, neurotrophin signaling and proteoglycans in cancer. These pathway associations connect ITPR3 phosphorylation with receptor signaling, endoplasmic-reticulum calcium release, store-operated entry and potentially mitochondrial calcium transfer, but do not establish the direction or mechanism of those effects.

Restricting the curated differential data to cancer-derived cell lines showed that each predominant site was repeatedly observed, though at different frequencies. S1832 appeared in 82 of 98 cancer-cell-line datasets, S934 in 65 of 76, S2670 in 55 of 63 and S916 in 17 of 24. In the broader collection, S1832 was generally increased in skin, blood and cervical cancer datasets but decreased in lung cancer datasets. S934 showed different directions among cancer types, including decreases in lung and liver and increases in skin, colon, breast, cervical and blood cancers. A separate cProSite analysis provided tumor-level log2 fold changes and found particularly strong differences for individual sites: S1832 was upregulated in pancreatic ductal adenocarcinoma at log2 fold change 1.2909 with p below 0.0001; S2670 in breast cancer at 1.4838 with p equal to 0.0004; and S934 in ovarian cancer at 2.4224 with p below 0.0001. S916 was downregulated in kidney cancer at −0.8818 with p equal to 0.0013.

The results generate a site-specific map for future experiments, but the evidence is exploratory. The underlying studies used different cell types, treatments, mass-spectrometry platforms, quantification methods and data-processing pipelines. The researchers could not uniformly normalize or correct batch effects because raw data were not available for every study and a common reprocessing workflow was lacking. Even though fold-change thresholds were applied within individual datasets, differences in dynamic range among quantification strategies could influence the compiled patterns. Functional enrichment, interaction networks, pathway assignments and kinase relationships are computational associations, and the cancer findings do not show that ITPR3 phosphorylation causes tumor growth, changes calcium flux or predicts clinical outcomes. The authors accordingly call for phosphosite-directed mutagenesis, kinase assays and cellular functional studies. Until those tests are performed, the strongest conclusion is that four conserved ITPR3 phosphosites recur in cancer datasets and occupy distinct, testable regulatory networks associated with calcium handling, transcription, RNA processing, cytoskeletal organization and receptor signaling.

Subject of Research: Computational mapping of recurrent ITPR3 phosphosites, calcium-signaling associations and cancer-cell-line phosphorylation patterns

Article Title: Decoding the phosphorylation landscape of ITPR3 across calcium signaling and regulatory networks in cancer

Article References: Rai, S. B., Sujina, A., Kammarambath, S. R., Mahin, A., Subair, S., Sambreena, A., Pavithra, S., Raju, R., & Palollathil, A. (2026). Decoding the phosphorylation landscape of ITPR3 across calcium signaling and regulatory networks in cancer. Frontiers in Bioinformatics, 6, Article 1972498. https://doi.org/10.3389/fbinf.2026.1972498

Image Credits: AI Generated

DOI: 10.3389/fbinf.2026.1972498

Keywords: ITPR3, phosphoproteomics, protein phosphorylation, co-regulation, calcium signaling, cancer cell lines, kinase prediction, endoplasmic reticulum, bioinformatics, interaction networks, Decoding, phosphorylation