{"gene":"TP53RK","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2002,"finding":"Yeast piD261/Bud32 (ortholog of TP53RK) is a Ser/Thr protein kinase; mutational analysis showed that despite low sequence similarity, invariant residues of protein kinases are conserved. Autophosphorylation at Ser-187 and Ser-189 in the activation loop is required for full catalytic activity; Ser→Ala mutation abolished the upshifted SDS/PAGE band and reduced catalytic activity. Notably, the protein lacks the canonical lysyl residue that interacts with ATP gamma-phosphate, replaced by a threonine.","method":"Recombinant protein expression in E. coli, site-directed mutagenesis, in vitro kinase assay, SDS/PAGE mobility shift","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, multiple orthogonal methods in single rigorous study","pmids":["12023889"],"is_preprint":false},{"year":2002,"finding":"Yeast piD261/Bud32 preferentially phosphorylates acidic substrates in vitro, recognizing seryl residues specified by adjacent carboxylic side chains, distinguishing it from most Ser/Thr kinases that favor basic or proline-directed sites.","method":"In vitro kinase assay with acidic proteins and synthetic peptides","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro assay, single lab, single method","pmids":["12207926"],"is_preprint":false},{"year":2003,"finding":"Human PRPK (TP53RK) phosphorylates p53 at Ser-15 in vitro and interacts with p53 protein; yeast Bud32 also phosphorylates human p53 in vitro and interacts with it, demonstrating functional conservation across distant organisms. PRPK partially complements the growth defect of yeast lacking BUD32.","method":"In vitro kinase assay, protein-protein interaction assay, yeast complementation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro kinase assay with yeast complementation, single lab","pmids":["12914926"],"is_preprint":false},{"year":2003,"finding":"CGI-121 (TPRKB) directly interacts with PRPK (TP53RK) both in vivo and in vitro. Recombinant CGI-121 inhibits the coprecipitation of p53 by recombinant PRPK in vitro, indicating CGI-121 can act as an inhibitor of PRPK–p53 binding.","method":"Yeast two-hybrid, co-immunoprecipitation in vivo and in vitro, recombinant protein competition assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal in vivo and in vitro interaction assays, single lab","pmids":["12659830"],"is_preprint":false},{"year":2004,"finding":"Yeast Bud32 directly phosphorylates the glutaredoxin Grx4 at Ser-134 in vitro. Bud32 also interacts with the putative glycoprotease Kae1 (Ykr038/Kae1) and this interaction is evolutionarily conserved. The phosphotransferase activity of Bud32 is relevant to its in vivo function, but non-catalytic mutants that retain native conformation can partially complement gene deletion, indicating additional non-kinase roles.","method":"Yeast two-hybrid, in vitro kinase assay, phosphosite mapping, complementation of deletion mutant","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro phosphorylation with site identification, two-hybrid interaction, complementation, single lab","pmids":["14519092"],"is_preprint":false},{"year":2006,"finding":"The small Ras-like GTPase Ray/Rab1c (Rab35) directly binds PRPK and redistributes overexpressed PRPK from nucleus to cytosol. Both wild-type Ray and a GTP-binding locked mutant (Ray-Q67L), but not a guanine nucleotide-unstable mutant (Ray-N120I), suppressed PRPK-induced p53 transcriptional activity, indicating GTP binding to Ray is required for this regulatory function.","method":"Co-immunoprecipitation, subcellular localization by immunofluorescence, transcriptional reporter assay with mutant analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP, localization, functional reporter assay with mutant discrimination, single lab","pmids":["16600182"],"is_preprint":false},{"year":2007,"finding":"PRPK (TP53RK) is phosphorylated and activated by Akt/PKB at Ser-250. Recombinant PRPK is phosphorylated in vitro by Akt; co-transfection of Akt with wild-type PRPK but not Ser250Ala mutant increased PRPK phosphorylation. Akt co-expression increased PRPK-dependent p53 Ser-15 phosphorylation, which was abolished by the Akt pathway inhibitor LY294002, establishing Akt as an upstream activating kinase for PRPK.","method":"In vitro kinase assay, phospho-specific antibody, site-directed mutagenesis (Ser250Ala), cell co-transfection, pharmacological inhibition (LY294002)","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, mutagenesis, and pharmacological validation, multiple orthogonal methods, single lab","pmids":["17712528"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of the archaeal Kae1/Bud32 fusion protein MJ1130 revealed that Kae1 (Kae1p) maintains the Bud32 kinase ATP-binding site in an inactive configuration. Yeast Kae1p was shown to repress the kinase activity of yeast Bud32p in vitro. Mutations disrupting the Kae1p/Bud32p interaction in yeast abolished both transcription and telomere homeostasis functions of the EKC/KEOPS complex.","method":"X-ray crystallography, in vitro kinase repression assay, site-directed mutagenesis of protein-protein interface, yeast genetics","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro functional assay and in vivo mutagenesis, multiple orthogonal methods","pmids":["19172740"],"is_preprint":false},{"year":2010,"finding":"siRNA-mediated knockdown of TP53RK accelerated caspase-3/7 activation and cell death after mitotic arrest induced by paclitaxel or PLK1 inhibitor, without affecting mitotic entry kinetics. This places TP53RK as a restrainer of apoptosis specifically after mitotic stress.","method":"siRNA knockdown, caspase-3/7 activation assay, time-lapse microscopy","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean siRNA knockdown with live-cell imaging phenotype, single lab, single method","pmids":["20647325"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of the yeast Bud32/Cgi121 complex with ADP showed ADP bound in the Bud32 catalytic site in a canonical PKA-family manner. The full yeast KEOPS complex exists as a linear heteropentamer (Gon7–Pcc1–Kae1–Bud32–Cgi121), distinct from the archaeal homodimeric Pcc1 arrangement.","method":"X-ray crystallography, analytical ultracentrifugation, structural modeling","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with ADP-bound active site and biochemical validation of complex stoichiometry","pmids":["25735745"],"is_preprint":false},{"year":2018,"finding":"PRPK (TP53RK) is phosphorylated by TOPK (T-LAK cell-originated protein kinase); knockdown of TOPK inhibited PRPK phosphorylation in vivo. Active phospho-PRPK promoted skin carcinogenesis; topical PRPK inhibitors (rocuronium bromide or betamethasone 17-valerate) attenuated TOPK-dependent PRPK signaling and reduced cutaneous SCC development in mice.","method":"siRNA knockdown, in vivo mouse carcinogenesis model, pharmacological inhibition, phosphorylation analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown and in vivo pharmacological confirmation, multiple orthogonal methods, single lab","pmids":["29904102"],"is_preprint":false},{"year":2018,"finding":"Active PRPK does not phosphorylate p53 directly (negative result in in vitro kinase assay). Instead, PRPK phosphorylates survivin (Birc5) at Thr-34, which is important for survivin stability, and this promotes colon cancer metastasis.","method":"In vitro kinase assay, knockdown, in vivo metastasis mouse model","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with phosphosite identification and in vivo metastasis model, single lab","pmids":["29483219"],"is_preprint":false},{"year":2019,"finding":"TP53 indirectly mediates degradation of TPRKB (CGI-121); this degradation was rescued by co-expression of PRPK (TP53RK), indicating PRPK stabilizes TPRKB. Depletion of other EKC/KEOPS members had TP53-independent effects, supporting complex-independent functions of TPRKB distinct from those of PRPK.","method":"shRNA knockdown, proteasome inhibition rescue experiment, cell proliferation assay, co-expression rescue","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rescue experiments with proteasome inhibitor and PRPK co-expression, multiple orthogonal methods, single lab","pmids":["31110156"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of the human PRPK–TPRKB complex at 2.53 Å resolution revealed PRPK in an active conformation with AMPPNP coordinated, despite lacking a conventional activation loop. Structural mapping showed the disease-associated mutation PRPK K238Nfs*2 abolishes binding to OSGEP (Kae1), positioning this interaction as critical for complex assembly.","method":"X-ray crystallography, binding assay (PRPK mutant vs. OSGEP), structural modeling of full human KEOPS complex","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure with functional mutant validation, multiple methods, single lab","pmids":["33547416"],"is_preprint":false},{"year":2022,"finding":"Bud32 (TP53RK ortholog) functions primarily as an ATPase rather than a classical protein kinase within the KEOPS complex; its ATPase activity is required for t6A tRNA modification. Bud32 also facilitates tRNA substrate recruitment to KEOPS and helps position tRNA A37 in the Kae1 active site.","method":"In vitro ATPase assay, tRNA binding assay, t6A modification assay, yeast complementation growth assay","journal":"Methods in enzymology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal in vitro and in vivo assays establishing ATPase (not kinase) mechanism, single lab","pmids":["35525560"],"is_preprint":false},{"year":2022,"finding":"A novel homozygous TP53RK missense variant (p.Arg55Gly) causing Galloway-Mowat syndrome was functionally validated: morpholino knockdown of tp53rk in Xenopus laevis caused abnormal eye and head development, rescued by wild-type human TP53RK but not by the p.Arg55Gly mutant or a previously described p.Gly42Asp mutant, demonstrating loss of function for these variants.","method":"Xenopus morpholino knockdown, mRNA rescue experiment with wild-type and mutant TP53RK","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — morpholino knockdown with mutant-specific rescue, multiple constructs tested, single lab","pmids":["36116039"],"is_preprint":false},{"year":2023,"finding":"TP53RK phosphorylates Birc5 (survivin) and facilitates its nuclear translocation; nuclear Birc5 activates PI3K/Akt and MAPK pathways to promote renal fibrosis. Specific deletion of TP53RK in renal tubular cells or fibroblasts in mice mitigated renal fibrosis, and pharmacological inhibition with fusidic acid ameliorated kidney fibrosis.","method":"Conditional gene knockout in mice, in vitro kinase assay, subcellular fractionation, pharmacological inhibition, CKD mouse models","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with cell-type-specific KO in two cell lineages and in vivo pharmacological validation, multiple orthogonal methods","pmids":["37382161"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structures of KEOPS with and without tRNA substrate revealed distinct tRNA flexibility and a conformational change enabling tRNA modification by Kae1. A contact between a flipped-out base of tRNA and an arginine in the C-terminal tail of Bud32 correlates with the tRNA conformational change. Contact surfaces within the KEOPS-tRNA holo-enzyme were identified that are required for Bud32 ATPase regulation and t6A modification activity, clarifying how Bud32 contributes to substrate specificity.","method":"Cryo-electron microscopy, mutagenesis of Bud32 C-terminal tail, t6A modification assay, ATPase assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure combined with mutagenesis and functional assays in single study","pmids":["39639027"],"is_preprint":false},{"year":2025,"finding":"TP53RK stabilizes CDC7 (cell division cycle 7 kinase), ensuring MCM complex phosphorylation and replication fork progression; TP53RK depletion reduced MCM2 enrichment at replication origins and induced DNA replication stress, apoptosis, and cell cycle arrest independent of p53 status.","method":"CRISPR screen, TP53RK knockdown/knockout, MCM ChIP, DNA fiber assay, co-expression analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR validation with multiple orthogonal functional assays, single lab","pmids":["41102525"],"is_preprint":false}],"current_model":"TP53RK (PRPK/Bud32) is an atypical RIO-type Ser/Thr kinase/ATPase that is a core subunit of the evolutionarily conserved KEOPS complex; within KEOPS it functions primarily as an ATPase to facilitate tRNA recruitment and enable Kae1-catalyzed t6A modification of tRNAs, while its kinase activity also phosphorylates substrates including p53 (Ser-15), survivin/Birc5 (Thr-34), and supports CDC7 stability to regulate DNA replication; it is activated by Akt/PKB-mediated phosphorylation at Ser-250 and by TOPK, is repressed by its KEOPS partner Kae1, and interacts with TPRKB/CGI-121, Kae1/OSGEP, and the small GTPase Ray/Rab35, with loss-of-function mutations causing Galloway-Mowat syndrome."},"narrative":{"mechanistic_narrative":"TP53RK (PRPK/Bud32) is an atypical RIO-family Ser/Thr protein kinase that is a core, evolutionarily conserved subunit of the KEOPS complex, where its principal contribution to tRNA t6A modification is enzymatic ATPase rather than classical kinase activity [PMID:12023889, PMID:35525560]. Within KEOPS — a linear heteropentamer (Gon7–Pcc1–Kae1–Bud32–Cgi121) — TP53RK contacts and is held in an inactive kinase configuration by its partner Kae1/OSGEP, while its ATPase activity drives tRNA substrate recruitment and positions the A37 base in the Kae1 active site, with a C-terminal-tail arginine contacting a flipped-out tRNA base to enable the modification-competent conformation [PMID:19172740, PMID:25735745, PMID:35525560, PMID:39639027]. Crystallography of the human PRPK–TPRKB complex shows PRPK in an active, nucleotide-bound conformation and maps the Galloway-Mowat-associated truncation K238Nfs*2 to the OSGEP-binding interface required for complex assembly; loss-of-function TP53RK variants cause Galloway-Mowat syndrome [PMID:33547416, PMID:36116039]. Beyond KEOPS, TP53RK acts as a kinase whose activity is switched on by upstream Akt/PKB phosphorylation at Ser-250 and by TOPK, phosphorylates substrates including p53 at Ser-15 and survivin/Birc5 at Thr-34 to control survivin stability and nuclear translocation, and supports CDC7 stability and replication-fork progression, linking it to apoptosis restraint, cancer progression, renal fibrosis, and DNA replication [PMID:12914926, PMID:17712528, PMID:29904102, PMID:29483219, PMID:37382161, PMID:41102525]. Its substrate preference is unusual among Ser/Thr kinases, favoring acidic, carboxylate-flanked seryl residues [PMID:12207926].","teleology":[{"year":2002,"claim":"Established that the TP53RK ortholog Bud32 is a genuine, if atypical, Ser/Thr protein kinase, defining the catalytic machinery later found to be unconventional.","evidence":"Recombinant yeast Bud32 with site-directed mutagenesis and in vitro kinase/mobility-shift assays","pmids":["12023889"],"confidence":"High","gaps":["Physiological substrates not identified","Functional consequence of lacking the canonical ATP-binding lysine unresolved"]},{"year":2002,"claim":"Defined the kinase's unusual substrate specificity for acidic, carboxylate-flanked sites, distinguishing it from basophilic and proline-directed kinases.","evidence":"In vitro kinase assays on acidic proteins and synthetic peptides","pmids":["12207926"],"confidence":"Medium","gaps":["Specificity determinants on the enzyme not mapped","In vivo relevance of acidic preference untested"]},{"year":2003,"claim":"Linked human PRPK to p53 by showing it phosphorylates p53 Ser-15 and binds p53, with conservation back to yeast Bud32, framing an early tumor-suppressor signaling role.","evidence":"In vitro kinase assay, protein interaction assay, and yeast complementation","pmids":["12914926"],"confidence":"Medium","gaps":["In-cell p53 phosphorylation not demonstrated","Later work reports PRPK does not phosphorylate p53 directly, leaving the relationship unsettled"]},{"year":2003,"claim":"Identified TPRKB/CGI-121 as a direct PRPK partner that can inhibit PRPK–p53 association, introducing a regulatory binding partner.","evidence":"Yeast two-hybrid, reciprocal in vivo and in vitro co-IP, recombinant competition assay","pmids":["12659830"],"confidence":"Medium","gaps":["Mechanism of inhibition (steric vs allosteric) unresolved","Effect on kinase catalysis vs substrate binding not separated"]},{"year":2004,"claim":"Connected Bud32 to the glycoprotease Kae1 and revealed non-kinase functions, foreshadowing the structural/enzymatic partnership at the heart of KEOPS.","evidence":"Yeast two-hybrid, in vitro kinase assay with phosphosite mapping (Grx4 Ser-134), and complementation","pmids":["14519092"],"confidence":"Medium","gaps":["Functional significance of the Grx4 phosphorylation unknown","Nature of the non-catalytic role not defined"]},{"year":2006,"claim":"Showed the Rab-family GTPase Ray/Rab35 binds PRPK and, in a GTP-dependent manner, relocalizes it and suppresses PRPK-driven p53 transcriptional activity, adding spatial regulation.","evidence":"Co-IP, immunofluorescence localization, and transcriptional reporter assays with GTPase mutants","pmids":["16600182"],"confidence":"Medium","gaps":["Physiological trigger for relocalization unknown","Relationship to KEOPS function not addressed"]},{"year":2007,"claim":"Placed PRPK downstream of Akt/PKB by identifying Ser-250 phosphorylation as an activating input, establishing an upstream regulatory kinase.","evidence":"In vitro Akt kinase assay, phospho-specific antibody, Ser250Ala mutant, cell co-transfection, and LY294002 inhibition","pmids":["17712528"],"confidence":"High","gaps":["Whether Akt regulates KEOPS-associated PRPK function untested","Stimuli driving Akt-PRPK signaling not defined"]},{"year":2009,"claim":"Provided the structural basis for KEOPS regulation, showing Kae1 holds the Bud32 kinase site inactive and that the Kae1–Bud32 interface is required for complex function.","evidence":"X-ray crystallography of archaeal Kae1/Bud32 fusion, in vitro kinase repression assay, and yeast interface mutagenesis","pmids":["19172740"],"confidence":"High","gaps":["Catalytic role of Bud32 within KEOPS not yet redefined as ATPase","Mechanism linking kinase site to telomere/transcription functions unclear"]},{"year":2015,"claim":"Defined the architecture and stoichiometry of the eukaryotic KEOPS complex as a linear heteropentamer and captured Bud32 with ADP in a canonical active site.","evidence":"X-ray crystallography of Bud32/Cgi121–ADP and analytical ultracentrifugation","pmids":["25735745"],"confidence":"High","gaps":["Catalytic mechanism (kinase vs ATPase) within the assembled complex not resolved","tRNA-bound state not captured"]},{"year":2018,"claim":"Identified TOPK as an additional upstream kinase activating PRPK and linked active PRPK to skin carcinogenesis, supporting therapeutic targeting.","evidence":"siRNA knockdown, phosphorylation analysis, mouse cutaneous SCC model, and pharmacological inhibitors","pmids":["29904102"],"confidence":"Medium","gaps":["TOPK phosphosite on PRPK not mapped","Relationship between TOPK and Akt inputs unresolved"]},{"year":2018,"claim":"Reassigned a key PRPK substrate by showing it does not phosphorylate p53 directly but phosphorylates survivin/Birc5 at Thr-34 to stabilize it and drive metastasis.","evidence":"In vitro kinase assay (negative for p53), knockdown, and in vivo colon cancer metastasis model","pmids":["29483219"],"confidence":"Medium","gaps":["Contradiction with earlier p53 Ser-15 data not reconciled","How survivin phosphorylation confers stability mechanistically unclear"]},{"year":2019,"claim":"Revealed PRPK stabilizes TPRKB and that TPRKB has TP53-dependent, complex-independent behavior, dissecting subunit-specific functions.","evidence":"shRNA knockdown, proteasome-inhibition and PRPK co-expression rescue, proliferation assays","pmids":["31110156"],"confidence":"Medium","gaps":["Mechanism of PRPK-mediated TPRKB stabilization unknown","Generalizability beyond tested cell models untested"]},{"year":2021,"claim":"Delivered the human PRPK–TPRKB structure in an active conformation and mapped a Galloway-Mowat truncation to the OSGEP-binding interface critical for KEOPS assembly.","evidence":"2.53 Å crystal structure, PRPK-mutant OSGEP binding assay, and human KEOPS modeling","pmids":["33547416"],"confidence":"High","gaps":["Catalytic output of the active conformation in human KEOPS not assayed","How disrupted assembly causes disease phenotype not shown"]},{"year":2022,"claim":"Redefined Bud32's primary KEOPS role as an ATPase required for t6A modification and tRNA recruitment/positioning, resolving the long-standing kinase-vs-enzyme question.","evidence":"In vitro ATPase, tRNA binding, and t6A modification assays plus yeast complementation","pmids":["35525560"],"confidence":"High","gaps":["Whether the kinase activity has any KEOPS-independent in vivo role unsettled","Coupling between ATP hydrolysis and tRNA positioning mechanistically incomplete"]},{"year":2022,"claim":"Functionally validated TP53RK missense variants as loss-of-function causes of Galloway-Mowat syndrome using an in vivo developmental model.","evidence":"Xenopus morpholino knockdown with wild-type vs mutant human TP53RK mRNA rescue","pmids":["36116039"],"confidence":"Medium","gaps":["Tissue-specific basis of the developmental phenotype unknown","Whether variants impair ATPase or assembly not separated"]},{"year":2023,"claim":"Connected TP53RK kinase activity to disease by showing it phosphorylates Birc5 to drive its nuclear translocation and downstream PI3K/Akt and MAPK signaling in renal fibrosis.","evidence":"Conditional cell-type-specific knockout mice, in vitro kinase assay, subcellular fractionation, and pharmacological inhibition","pmids":["37382161"],"confidence":"High","gaps":["Direct mechanism of Birc5 nuclear import via phosphorylation not detailed","Relationship to KEOPS/t6A function unaddressed"]},{"year":2024,"claim":"Provided substrate-bound cryo-EM snapshots showing how a Bud32 C-terminal-tail arginine contacts a flipped-out tRNA base to enable the conformational change required for modification, clarifying its substrate-specificity contribution.","evidence":"Cryo-EM of KEOPS ± tRNA with C-terminal-tail mutagenesis and t6A/ATPase assays","pmids":["39639027"],"confidence":"High","gaps":["Dynamics of ATPase cycle coupled to tRNA flipping not fully resolved","Human-complex equivalence of the tRNA contact untested"]},{"year":2025,"claim":"Linked TP53RK to DNA replication by showing it stabilizes CDC7 to sustain MCM phosphorylation and fork progression, with depletion causing replication stress independent of p53.","evidence":"CRISPR screen, knockdown/knockout, MCM ChIP, DNA fiber assay, co-expression analysis","pmids":["41102525"],"confidence":"Medium","gaps":["Whether stabilization is kinase-dependent or via direct binding unclear","Connection to KEOPS/t6A activity not established"]},{"year":null,"claim":"How TP53RK's two activities — KEOPS-embedded ATPase for tRNA modification versus free kinase phosphorylating survivin/CDC7-related targets — are partitioned in cells, and which underlies Galloway-Mowat pathology, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experiment separates KEOPS-dependent from kinase-dependent phenotypes in disease","Conflicting reports on p53 as a direct substrate not reconciled","Spatial/temporal control of substrate choice undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,11,16]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[14,17]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[14,17]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[14,17]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[14,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,10,16]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[13,15,16]}],"complexes":["KEOPS/EKC complex"],"partners":["OSGEP","TPRKB","RAB35","AKT1","PBK","BIRC5","CDC7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96S44","full_name":"EKC/KEOPS complex subunit TP53RK","aliases":["Atypical serine/threonine protein kinase TP53RK","Nori-2","TP53-regulating kinase","p53-related protein kinase"],"length_aa":253,"mass_kda":28.2,"function":"Component of the EKC/KEOPS complex that is required for the formation of a threonylcarbamoyl group on adenosine at position 37 (t(6)A37) in tRNAs that read codons beginning with adenine (PubMed:22912744, PubMed:27903914). The complex is probably involved in the transfer of the threonylcarbamoyl moiety of threonylcarbamoyl-AMP (TC-AMP) to the N6 group of A37 (PubMed:22912744, PubMed:27903914). TP53RK has ATPase activity in the context of the EKC/KEOPS complex and likely plays a supporting role to the catalytic subunit OSGEP (By similarity). Atypical protein kinase that phosphorylates 'Ser-15' of p53/TP53 protein and may therefore participate in its activation (PubMed:11546806)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96S44/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TP53RK","classification":"Common Essential","n_dependent_lines":1022,"n_total_lines":1208,"dependency_fraction":0.8460264900662252},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"POLR3E","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TP53RK","total_profiled":1310},"omim":[{"mim_id":"617731","title":"GALLOWAY-MOWAT SYNDROME 5; GAMOS5","url":"https://www.omim.org/entry/617731"},{"mim_id":"617730","title":"GALLOWAY-MOWAT SYNDROME 4; GAMOS4","url":"https://www.omim.org/entry/617730"},{"mim_id":"617729","title":"GALLOWAY-MOWAT SYNDROME 3; GAMOS3","url":"https://www.omim.org/entry/617729"},{"mim_id":"610107","title":"O-SIALOGLYCOPROTEIN ENDOPEPTIDASE; OSGEP","url":"https://www.omim.org/entry/610107"},{"mim_id":"608680","title":"TP53RK-BINDING PROTEIN; TPRKB","url":"https://www.omim.org/entry/608680"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TP53RK"},"hgnc":{"alias_symbol":["dJ101A2.2","prpk","Nori-2p","BUD32","TPRKB"],"prev_symbol":["C20orf64"]},"alphafold":{"accession":"Q96S44","domains":[{"cath_id":"3.30.200.20","chopping":"33-115","consensus_level":"high","plddt":94.3677,"start":33,"end":115},{"cath_id":"1.10.510.10","chopping":"121-249","consensus_level":"high","plddt":96.0992,"start":121,"end":249}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96S44","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96S44-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96S44-F1-predicted_aligned_error_v6.png","plddt_mean":91.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TP53RK","jax_strain_url":"https://www.jax.org/strain/search?query=TP53RK"},"sequence":{"accession":"Q96S44","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96S44.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96S44/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96S44"}},"corpus_meta":[{"pmid":"14519092","id":"PMC_14519092","title":"Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin.","date":"2004","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/14519092","citation_count":61,"is_preprint":false},{"pmid":"19172740","id":"PMC_19172740","title":"Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex.","date":"2008","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/19172740","citation_count":55,"is_preprint":false},{"pmid":"25735745","id":"PMC_25735745","title":"Crystal structures of the Gon7/Pcc1 and Bud32/Cgi121 complexes provide a model for the complete yeast KEOPS complex.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25735745","citation_count":38,"is_preprint":false},{"pmid":"12023889","id":"PMC_12023889","title":"Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life.","date":"2002","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/12023889","citation_count":35,"is_preprint":false},{"pmid":"29904102","id":"PMC_29904102","title":"Targeting PRPK and TOPK for skin cancer prevention and therapy.","date":"2018","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/29904102","citation_count":33,"is_preprint":false},{"pmid":"19143597","id":"PMC_19143597","title":"The universal Kae1 protein and the associated Bud32 kinase (PRPK), a mysterious protein couple probably essential for genome maintenance in Archaea and Eukarya.","date":"2009","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/19143597","citation_count":32,"is_preprint":false},{"pmid":"12914926","id":"PMC_12914926","title":"Functional homology between yeast piD261/Bud32 and human PRPK: both phosphorylate p53 and PRPK partially complements piD261/Bud32 deficiency.","date":"2003","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12914926","citation_count":30,"is_preprint":false},{"pmid":"20647325","id":"PMC_20647325","title":"A chemosensitization screen identifies TP53RK, a kinase that restrains apoptosis after mitotic stress.","date":"2010","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/20647325","citation_count":28,"is_preprint":false},{"pmid":"17712528","id":"PMC_17712528","title":"Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB.","date":"2007","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/17712528","citation_count":22,"is_preprint":false},{"pmid":"16600182","id":"PMC_16600182","title":"A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK.","date":"2006","source":"Biochemical and biophysical research 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Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/37382161","citation_count":11,"is_preprint":false},{"pmid":"36116039","id":"PMC_36116039","title":"Functional characterization of a novel TP53RK mutation identified in a family with Galloway-Mowat syndrome.","date":"2022","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/36116039","citation_count":10,"is_preprint":false},{"pmid":"39639027","id":"PMC_39639027","title":"Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39639027","citation_count":3,"is_preprint":false},{"pmid":"36873107","id":"PMC_36873107","title":"Novel TP53RK variants cause varied clinical features of Galloway-Mowat syndrome without nephrotic syndrome in three unrelated Chinese patients.","date":"2023","source":"Frontiers in molecular 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Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41465844","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13925,"output_tokens":4802,"usd":0.056903,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12843,"output_tokens":5309,"usd":0.09847,"stage2_stop_reason":"end_turn"},"total_usd":0.155373,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"Yeast piD261/Bud32 (ortholog of TP53RK) is a Ser/Thr protein kinase; mutational analysis showed that despite low sequence similarity, invariant residues of protein kinases are conserved. Autophosphorylation at Ser-187 and Ser-189 in the activation loop is required for full catalytic activity; Ser→Ala mutation abolished the upshifted SDS/PAGE band and reduced catalytic activity. Notably, the protein lacks the canonical lysyl residue that interacts with ATP gamma-phosphate, replaced by a threonine.\",\n      \"method\": \"Recombinant protein expression in E. coli, site-directed mutagenesis, in vitro kinase assay, SDS/PAGE mobility shift\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"12023889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Yeast piD261/Bud32 preferentially phosphorylates acidic substrates in vitro, recognizing seryl residues specified by adjacent carboxylic side chains, distinguishing it from most Ser/Thr kinases that favor basic or proline-directed sites.\",\n      \"method\": \"In vitro kinase assay with acidic proteins and synthetic peptides\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro assay, single lab, single method\",\n      \"pmids\": [\"12207926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human PRPK (TP53RK) phosphorylates p53 at Ser-15 in vitro and interacts with p53 protein; yeast Bud32 also phosphorylates human p53 in vitro and interacts with it, demonstrating functional conservation across distant organisms. PRPK partially complements the growth defect of yeast lacking BUD32.\",\n      \"method\": \"In vitro kinase assay, protein-protein interaction assay, yeast complementation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro kinase assay with yeast complementation, single lab\",\n      \"pmids\": [\"12914926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CGI-121 (TPRKB) directly interacts with PRPK (TP53RK) both in vivo and in vitro. Recombinant CGI-121 inhibits the coprecipitation of p53 by recombinant PRPK in vitro, indicating CGI-121 can act as an inhibitor of PRPK–p53 binding.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in vivo and in vitro, recombinant protein competition assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal in vivo and in vitro interaction assays, single lab\",\n      \"pmids\": [\"12659830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yeast Bud32 directly phosphorylates the glutaredoxin Grx4 at Ser-134 in vitro. Bud32 also interacts with the putative glycoprotease Kae1 (Ykr038/Kae1) and this interaction is evolutionarily conserved. The phosphotransferase activity of Bud32 is relevant to its in vivo function, but non-catalytic mutants that retain native conformation can partially complement gene deletion, indicating additional non-kinase roles.\",\n      \"method\": \"Yeast two-hybrid, in vitro kinase assay, phosphosite mapping, complementation of deletion mutant\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phosphorylation with site identification, two-hybrid interaction, complementation, single lab\",\n      \"pmids\": [\"14519092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The small Ras-like GTPase Ray/Rab1c (Rab35) directly binds PRPK and redistributes overexpressed PRPK from nucleus to cytosol. Both wild-type Ray and a GTP-binding locked mutant (Ray-Q67L), but not a guanine nucleotide-unstable mutant (Ray-N120I), suppressed PRPK-induced p53 transcriptional activity, indicating GTP binding to Ray is required for this regulatory function.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization by immunofluorescence, transcriptional reporter assay with mutant analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP, localization, functional reporter assay with mutant discrimination, single lab\",\n      \"pmids\": [\"16600182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRPK (TP53RK) is phosphorylated and activated by Akt/PKB at Ser-250. Recombinant PRPK is phosphorylated in vitro by Akt; co-transfection of Akt with wild-type PRPK but not Ser250Ala mutant increased PRPK phosphorylation. Akt co-expression increased PRPK-dependent p53 Ser-15 phosphorylation, which was abolished by the Akt pathway inhibitor LY294002, establishing Akt as an upstream activating kinase for PRPK.\",\n      \"method\": \"In vitro kinase assay, phospho-specific antibody, site-directed mutagenesis (Ser250Ala), cell co-transfection, pharmacological inhibition (LY294002)\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, mutagenesis, and pharmacological validation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"17712528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of the archaeal Kae1/Bud32 fusion protein MJ1130 revealed that Kae1 (Kae1p) maintains the Bud32 kinase ATP-binding site in an inactive configuration. Yeast Kae1p was shown to repress the kinase activity of yeast Bud32p in vitro. Mutations disrupting the Kae1p/Bud32p interaction in yeast abolished both transcription and telomere homeostasis functions of the EKC/KEOPS complex.\",\n      \"method\": \"X-ray crystallography, in vitro kinase repression assay, site-directed mutagenesis of protein-protein interface, yeast genetics\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro functional assay and in vivo mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"19172740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"siRNA-mediated knockdown of TP53RK accelerated caspase-3/7 activation and cell death after mitotic arrest induced by paclitaxel or PLK1 inhibitor, without affecting mitotic entry kinetics. This places TP53RK as a restrainer of apoptosis specifically after mitotic stress.\",\n      \"method\": \"siRNA knockdown, caspase-3/7 activation assay, time-lapse microscopy\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean siRNA knockdown with live-cell imaging phenotype, single lab, single method\",\n      \"pmids\": [\"20647325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of the yeast Bud32/Cgi121 complex with ADP showed ADP bound in the Bud32 catalytic site in a canonical PKA-family manner. The full yeast KEOPS complex exists as a linear heteropentamer (Gon7–Pcc1–Kae1–Bud32–Cgi121), distinct from the archaeal homodimeric Pcc1 arrangement.\",\n      \"method\": \"X-ray crystallography, analytical ultracentrifugation, structural modeling\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with ADP-bound active site and biochemical validation of complex stoichiometry\",\n      \"pmids\": [\"25735745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PRPK (TP53RK) is phosphorylated by TOPK (T-LAK cell-originated protein kinase); knockdown of TOPK inhibited PRPK phosphorylation in vivo. Active phospho-PRPK promoted skin carcinogenesis; topical PRPK inhibitors (rocuronium bromide or betamethasone 17-valerate) attenuated TOPK-dependent PRPK signaling and reduced cutaneous SCC development in mice.\",\n      \"method\": \"siRNA knockdown, in vivo mouse carcinogenesis model, pharmacological inhibition, phosphorylation analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown and in vivo pharmacological confirmation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"29904102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Active PRPK does not phosphorylate p53 directly (negative result in in vitro kinase assay). Instead, PRPK phosphorylates survivin (Birc5) at Thr-34, which is important for survivin stability, and this promotes colon cancer metastasis.\",\n      \"method\": \"In vitro kinase assay, knockdown, in vivo metastasis mouse model\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with phosphosite identification and in vivo metastasis model, single lab\",\n      \"pmids\": [\"29483219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TP53 indirectly mediates degradation of TPRKB (CGI-121); this degradation was rescued by co-expression of PRPK (TP53RK), indicating PRPK stabilizes TPRKB. Depletion of other EKC/KEOPS members had TP53-independent effects, supporting complex-independent functions of TPRKB distinct from those of PRPK.\",\n      \"method\": \"shRNA knockdown, proteasome inhibition rescue experiment, cell proliferation assay, co-expression rescue\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rescue experiments with proteasome inhibitor and PRPK co-expression, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"31110156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of the human PRPK–TPRKB complex at 2.53 Å resolution revealed PRPK in an active conformation with AMPPNP coordinated, despite lacking a conventional activation loop. Structural mapping showed the disease-associated mutation PRPK K238Nfs*2 abolishes binding to OSGEP (Kae1), positioning this interaction as critical for complex assembly.\",\n      \"method\": \"X-ray crystallography, binding assay (PRPK mutant vs. OSGEP), structural modeling of full human KEOPS complex\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure with functional mutant validation, multiple methods, single lab\",\n      \"pmids\": [\"33547416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Bud32 (TP53RK ortholog) functions primarily as an ATPase rather than a classical protein kinase within the KEOPS complex; its ATPase activity is required for t6A tRNA modification. Bud32 also facilitates tRNA substrate recruitment to KEOPS and helps position tRNA A37 in the Kae1 active site.\",\n      \"method\": \"In vitro ATPase assay, tRNA binding assay, t6A modification assay, yeast complementation growth assay\",\n      \"journal\": \"Methods in enzymology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal in vitro and in vivo assays establishing ATPase (not kinase) mechanism, single lab\",\n      \"pmids\": [\"35525560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A novel homozygous TP53RK missense variant (p.Arg55Gly) causing Galloway-Mowat syndrome was functionally validated: morpholino knockdown of tp53rk in Xenopus laevis caused abnormal eye and head development, rescued by wild-type human TP53RK but not by the p.Arg55Gly mutant or a previously described p.Gly42Asp mutant, demonstrating loss of function for these variants.\",\n      \"method\": \"Xenopus morpholino knockdown, mRNA rescue experiment with wild-type and mutant TP53RK\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — morpholino knockdown with mutant-specific rescue, multiple constructs tested, single lab\",\n      \"pmids\": [\"36116039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TP53RK phosphorylates Birc5 (survivin) and facilitates its nuclear translocation; nuclear Birc5 activates PI3K/Akt and MAPK pathways to promote renal fibrosis. Specific deletion of TP53RK in renal tubular cells or fibroblasts in mice mitigated renal fibrosis, and pharmacological inhibition with fusidic acid ameliorated kidney fibrosis.\",\n      \"method\": \"Conditional gene knockout in mice, in vitro kinase assay, subcellular fractionation, pharmacological inhibition, CKD mouse models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with cell-type-specific KO in two cell lineages and in vivo pharmacological validation, multiple orthogonal methods\",\n      \"pmids\": [\"37382161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structures of KEOPS with and without tRNA substrate revealed distinct tRNA flexibility and a conformational change enabling tRNA modification by Kae1. A contact between a flipped-out base of tRNA and an arginine in the C-terminal tail of Bud32 correlates with the tRNA conformational change. Contact surfaces within the KEOPS-tRNA holo-enzyme were identified that are required for Bud32 ATPase regulation and t6A modification activity, clarifying how Bud32 contributes to substrate specificity.\",\n      \"method\": \"Cryo-electron microscopy, mutagenesis of Bud32 C-terminal tail, t6A modification assay, ATPase assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure combined with mutagenesis and functional assays in single study\",\n      \"pmids\": [\"39639027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TP53RK stabilizes CDC7 (cell division cycle 7 kinase), ensuring MCM complex phosphorylation and replication fork progression; TP53RK depletion reduced MCM2 enrichment at replication origins and induced DNA replication stress, apoptosis, and cell cycle arrest independent of p53 status.\",\n      \"method\": \"CRISPR screen, TP53RK knockdown/knockout, MCM ChIP, DNA fiber assay, co-expression analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR validation with multiple orthogonal functional assays, single lab\",\n      \"pmids\": [\"41102525\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TP53RK (PRPK/Bud32) is an atypical RIO-type Ser/Thr kinase/ATPase that is a core subunit of the evolutionarily conserved KEOPS complex; within KEOPS it functions primarily as an ATPase to facilitate tRNA recruitment and enable Kae1-catalyzed t6A modification of tRNAs, while its kinase activity also phosphorylates substrates including p53 (Ser-15), survivin/Birc5 (Thr-34), and supports CDC7 stability to regulate DNA replication; it is activated by Akt/PKB-mediated phosphorylation at Ser-250 and by TOPK, is repressed by its KEOPS partner Kae1, and interacts with TPRKB/CGI-121, Kae1/OSGEP, and the small GTPase Ray/Rab35, with loss-of-function mutations causing Galloway-Mowat syndrome.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TP53RK (PRPK/Bud32) is an atypical RIO-family Ser/Thr protein kinase that is a core, evolutionarily conserved subunit of the KEOPS complex, where its principal contribution to tRNA t6A modification is enzymatic ATPase rather than classical kinase activity [#0, #14]. Within KEOPS — a linear heteropentamer (Gon7–Pcc1–Kae1–Bud32–Cgi121) — TP53RK contacts and is held in an inactive kinase configuration by its partner Kae1/OSGEP, while its ATPase activity drives tRNA substrate recruitment and positions the A37 base in the Kae1 active site, with a C-terminal-tail arginine contacting a flipped-out tRNA base to enable the modification-competent conformation [#7, #9, #14, #17]. Crystallography of the human PRPK–TPRKB complex shows PRPK in an active, nucleotide-bound conformation and maps the Galloway-Mowat-associated truncation K238Nfs*2 to the OSGEP-binding interface required for complex assembly; loss-of-function TP53RK variants cause Galloway-Mowat syndrome [#13, #15]. Beyond KEOPS, TP53RK acts as a kinase whose activity is switched on by upstream Akt/PKB phosphorylation at Ser-250 and by TOPK, phosphorylates substrates including p53 at Ser-15 and survivin/Birc5 at Thr-34 to control survivin stability and nuclear translocation, and supports CDC7 stability and replication-fork progression, linking it to apoptosis restraint, cancer progression, renal fibrosis, and DNA replication [#2, #6, #10, #11, #16, #18]. Its substrate preference is unusual among Ser/Thr kinases, favoring acidic, carboxylate-flanked seryl residues [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that the TP53RK ortholog Bud32 is a genuine, if atypical, Ser/Thr protein kinase, defining the catalytic machinery later found to be unconventional.\",\n      \"evidence\": \"Recombinant yeast Bud32 with site-directed mutagenesis and in vitro kinase/mobility-shift assays\",\n      \"pmids\": [\"12023889\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates not identified\", \"Functional consequence of lacking the canonical ATP-binding lysine unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the kinase's unusual substrate specificity for acidic, carboxylate-flanked sites, distinguishing it from basophilic and proline-directed kinases.\",\n      \"evidence\": \"In vitro kinase assays on acidic proteins and synthetic peptides\",\n      \"pmids\": [\"12207926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specificity determinants on the enzyme not mapped\", \"In vivo relevance of acidic preference untested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Linked human PRPK to p53 by showing it phosphorylates p53 Ser-15 and binds p53, with conservation back to yeast Bud32, framing an early tumor-suppressor signaling role.\",\n      \"evidence\": \"In vitro kinase assay, protein interaction assay, and yeast complementation\",\n      \"pmids\": [\"12914926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In-cell p53 phosphorylation not demonstrated\", \"Later work reports PRPK does not phosphorylate p53 directly, leaving the relationship unsettled\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified TPRKB/CGI-121 as a direct PRPK partner that can inhibit PRPK–p53 association, introducing a regulatory binding partner.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal in vivo and in vitro co-IP, recombinant competition assay\",\n      \"pmids\": [\"12659830\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of inhibition (steric vs allosteric) unresolved\", \"Effect on kinase catalysis vs substrate binding not separated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Connected Bud32 to the glycoprotease Kae1 and revealed non-kinase functions, foreshadowing the structural/enzymatic partnership at the heart of KEOPS.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro kinase assay with phosphosite mapping (Grx4 Ser-134), and complementation\",\n      \"pmids\": [\"14519092\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional significance of the Grx4 phosphorylation unknown\", \"Nature of the non-catalytic role not defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed the Rab-family GTPase Ray/Rab35 binds PRPK and, in a GTP-dependent manner, relocalizes it and suppresses PRPK-driven p53 transcriptional activity, adding spatial regulation.\",\n      \"evidence\": \"Co-IP, immunofluorescence localization, and transcriptional reporter assays with GTPase mutants\",\n      \"pmids\": [\"16600182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger for relocalization unknown\", \"Relationship to KEOPS function not addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed PRPK downstream of Akt/PKB by identifying Ser-250 phosphorylation as an activating input, establishing an upstream regulatory kinase.\",\n      \"evidence\": \"In vitro Akt kinase assay, phospho-specific antibody, Ser250Ala mutant, cell co-transfection, and LY294002 inhibition\",\n      \"pmids\": [\"17712528\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Akt regulates KEOPS-associated PRPK function untested\", \"Stimuli driving Akt-PRPK signaling not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Provided the structural basis for KEOPS regulation, showing Kae1 holds the Bud32 kinase site inactive and that the Kae1–Bud32 interface is required for complex function.\",\n      \"evidence\": \"X-ray crystallography of archaeal Kae1/Bud32 fusion, in vitro kinase repression assay, and yeast interface mutagenesis\",\n      \"pmids\": [\"19172740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic role of Bud32 within KEOPS not yet redefined as ATPase\", \"Mechanism linking kinase site to telomere/transcription functions unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the architecture and stoichiometry of the eukaryotic KEOPS complex as a linear heteropentamer and captured Bud32 with ADP in a canonical active site.\",\n      \"evidence\": \"X-ray crystallography of Bud32/Cgi121–ADP and analytical ultracentrifugation\",\n      \"pmids\": [\"25735745\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism (kinase vs ATPase) within the assembled complex not resolved\", \"tRNA-bound state not captured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified TOPK as an additional upstream kinase activating PRPK and linked active PRPK to skin carcinogenesis, supporting therapeutic targeting.\",\n      \"evidence\": \"siRNA knockdown, phosphorylation analysis, mouse cutaneous SCC model, and pharmacological inhibitors\",\n      \"pmids\": [\"29904102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TOPK phosphosite on PRPK not mapped\", \"Relationship between TOPK and Akt inputs unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Reassigned a key PRPK substrate by showing it does not phosphorylate p53 directly but phosphorylates survivin/Birc5 at Thr-34 to stabilize it and drive metastasis.\",\n      \"evidence\": \"In vitro kinase assay (negative for p53), knockdown, and in vivo colon cancer metastasis model\",\n      \"pmids\": [\"29483219\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Contradiction with earlier p53 Ser-15 data not reconciled\", \"How survivin phosphorylation confers stability mechanistically unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed PRPK stabilizes TPRKB and that TPRKB has TP53-dependent, complex-independent behavior, dissecting subunit-specific functions.\",\n      \"evidence\": \"shRNA knockdown, proteasome-inhibition and PRPK co-expression rescue, proliferation assays\",\n      \"pmids\": [\"31110156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of PRPK-mediated TPRKB stabilization unknown\", \"Generalizability beyond tested cell models untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Delivered the human PRPK–TPRKB structure in an active conformation and mapped a Galloway-Mowat truncation to the OSGEP-binding interface critical for KEOPS assembly.\",\n      \"evidence\": \"2.53 Å crystal structure, PRPK-mutant OSGEP binding assay, and human KEOPS modeling\",\n      \"pmids\": [\"33547416\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic output of the active conformation in human KEOPS not assayed\", \"How disrupted assembly causes disease phenotype not shown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Redefined Bud32's primary KEOPS role as an ATPase required for t6A modification and tRNA recruitment/positioning, resolving the long-standing kinase-vs-enzyme question.\",\n      \"evidence\": \"In vitro ATPase, tRNA binding, and t6A modification assays plus yeast complementation\",\n      \"pmids\": [\"35525560\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the kinase activity has any KEOPS-independent in vivo role unsettled\", \"Coupling between ATP hydrolysis and tRNA positioning mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Functionally validated TP53RK missense variants as loss-of-function causes of Galloway-Mowat syndrome using an in vivo developmental model.\",\n      \"evidence\": \"Xenopus morpholino knockdown with wild-type vs mutant human TP53RK mRNA rescue\",\n      \"pmids\": [\"36116039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific basis of the developmental phenotype unknown\", \"Whether variants impair ATPase or assembly not separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected TP53RK kinase activity to disease by showing it phosphorylates Birc5 to drive its nuclear translocation and downstream PI3K/Akt and MAPK signaling in renal fibrosis.\",\n      \"evidence\": \"Conditional cell-type-specific knockout mice, in vitro kinase assay, subcellular fractionation, and pharmacological inhibition\",\n      \"pmids\": [\"37382161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism of Birc5 nuclear import via phosphorylation not detailed\", \"Relationship to KEOPS/t6A function unaddressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided substrate-bound cryo-EM snapshots showing how a Bud32 C-terminal-tail arginine contacts a flipped-out tRNA base to enable the conformational change required for modification, clarifying its substrate-specificity contribution.\",\n      \"evidence\": \"Cryo-EM of KEOPS ± tRNA with C-terminal-tail mutagenesis and t6A/ATPase assays\",\n      \"pmids\": [\"39639027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of ATPase cycle coupled to tRNA flipping not fully resolved\", \"Human-complex equivalence of the tRNA contact untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked TP53RK to DNA replication by showing it stabilizes CDC7 to sustain MCM phosphorylation and fork progression, with depletion causing replication stress independent of p53.\",\n      \"evidence\": \"CRISPR screen, knockdown/knockout, MCM ChIP, DNA fiber assay, co-expression analysis\",\n      \"pmids\": [\"41102525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether stabilization is kinase-dependent or via direct binding unclear\", \"Connection to KEOPS/t6A activity not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TP53RK's two activities — KEOPS-embedded ATPase for tRNA modification versus free kinase phosphorylating survivin/CDC7-related targets — are partitioned in cells, and which underlies Galloway-Mowat pathology, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experiment separates KEOPS-dependent from kinase-dependent phenotypes in disease\", \"Conflicting reports on p53 as a direct substrate not reconciled\", \"Spatial/temporal control of substrate choice undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 11, 16]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [14, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 10, 16]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [13, 15, 16]}\n    ],\n    \"complexes\": [\"KEOPS/EKC complex\"],\n    \"partners\": [\"OSGEP\", \"TPRKB\", \"RAB35\", \"AKT1\", \"PBK\", \"BIRC5\", \"CDC7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}