ALPK3 is a muscle-enriched alpha-kinase-family protein that organizes sarcomeric M-band proteins and supports the development and maintenance of functional cardiomyocytes. Human cell studies place ALPK3 at the M band and, in tagged immature cardiomyocytes, at the nuclear envelope, where its loss disrupts myomesin organization. Interactions with M-band proteins connect ALPK3 to sarcomeric protein homeostasis. Its intrinsic protein kinase activity remains disputed: positive recombinant-domain phosphorylation assays coexist with negative catalytic-domain and cellular mutant studies. Loss-of-function variants cause severe pediatric and adult cardiomyopathy.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | UNDECIDED | Summary: Intrinsic ALPK3 serine/threonine kinase activity is experimentally disputed. Reason: The InterPro/EC inference is plausible from family membership but cannot settle ALPK3-specific catalysis. PMID:39196058 reports positive recombinant-domain phosphoproteomics and a SQSTM1 immunoprecipitate assay with heat-denatured-domain and phosphatase controls. PMID:36321451 reports negative cellular catalytic-cleft tests, and PMID:38048395 reports a negative mouse-domain assay with an active TRPM7 control. The latter does not test every full-length human condition, and the positive experiment does not prove that all observed phosphorylation is intrinsic to ALPK3. Retain the unresolved conflict rather than declaring the positive result an artifact or accepting family activity as measured human catalysis. Propagation Review Root cause: UNRESOLVED Sources checked: InterPro:IPR004166 UNRESOLVED Source identifier is retained; the target-specific evidence and limits are described in the reason. EC:2.7.11.1 UNRESOLVED Source identifier is retained; the target-specific evidence and limits are described in the reason. Supporting Evidence: PMID:36321451 Our phosphoproteomic studies of ALPK3dIC/dIC hiPSC-CMs, which altered the conformation of the kinase domain, ALPK3sIC/sIC, which targeted the predicted aspartate residue critical for catalysis, and massive overexpression of ALPK3 in HEK293T cells indicated that none of these perturbations significantly affected phosphorylation across the proteome. PMID:38048395 However, our experimental results revealed the absence of detectable kinase activity in ALPK3 under the tested conditions (Figure 1B). PMID:39196058 Before the assay, lysates were treated with an irreversible pan-kinase inhibitor to block endogenous kinase activity32,33. When recombinant ALPK3 kinase was applied to lysates, we identified 500 phosphosites that were significantly elevated compared to controls. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | UNDECIDED | Summary: Direct ATP binding remains unresolved independently of catalytic activity. Reason: This InterPro mapping predicts ATP binding from the alpha-kinase domain. The 2022 and 2023 negative studies question catalysis, but pseudokinase status does not itself imply loss of ATP binding. Conversely, ATP supplied to the disputed phosphorylation assays is not a direct binding measurement. No target-specific affinity or ligand-bound structure was established in the inspected papers, so neither acceptance solely from the fold nor removal solely from lack of catalysis is justified. Propagation Review Root cause: UNRESOLVED Sources checked: InterPro:IPR004166 UNRESOLVED Source identifier is retained; the target-specific evidence and limits are described in the reason. Supporting Evidence: PMID:36321451 Our phosphoproteomic studies of ALPK3dIC/dIC hiPSC-CMs, which altered the conformation of the kinase domain, ALPK3sIC/sIC, which targeted the predicted aspartate residue critical for catalysis, and massive overexpression of ALPK3 in HEK293T cells indicated that none of these perturbations significantly affected phosphorylation across the proteome. PMID:38048395 However, our experimental results revealed the absence of detectable kinase activity in ALPK3 under the tested conditions (Figure 1B). PMID:39196058 Before the assay, lysates were treated with an irreversible pan-kinase inhibitor to block endogenous kinase activity32,33. When recombinant ALPK3 kinase was applied to lysates, we identified 500 phosphosites that were significantly elevated compared to controls. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Retain nuclear association with a resolved nuclear-envelope context. Reason: The mouse donor is experimentally grounded: MGI links nuclear IDA to J:71661, the original mouse Midori paper PMID:11418590, whose tagged protein localizes to COS-cell nuclei. Independent human support comes from PMID:36321451 Figure 6: transient ALPK3-FLAG in hiPSC-derived cardiomyocytes localizes mainly to the nuclear envelope within the nuclear-associated pool. This supports the broad nucleus term without asserting ubiquitous nucleoplasmic residence. PMID:40135575 found no endogenous nuclear signal in its neonatal/adult mouse cardiomyocytes; the developmental, species and tagging differences are retained rather than treating one model as a universal localization veto. The exact PTN002931915 ancestral IBD placement was not recovered locally; the biological retention rests on the positive donor and human evidence, not donor count or an invented node history. The endogenously tagged human ALPK3 study PMID:39196058 likewise did not detect a nuclear pool, so the positive human nuclear-envelope evidence is specifically from transiently tagged cells. Propagation Review Root cause: NO FAILURE CORE Sources checked: MGI:MGI:2151224 SUPPORTS TRANSFER Source identifier is retained; the target-specific evidence and limits are described in the reason. PANTHER:PTN002931915 UNRESOLVED Ancestral assertion is preserved as supplied by GOA; exact IBD/tree placement not independently recovered. The positive mouse IDA and human localization support the target term. Supporting Evidence: PMID:11418590 The MIDORI protein was localized in the nucleus PMID:36321451 Depth-encoded projections of confocal z-slices confirmed that the majority of nuclear ALPK3 signal was at the nuclear envelope, as opposed to within the nucleus (Figure 6D). PMID:40135575 we did not observe the nuclear localization of endogenous ALPK3. PMID:39196058 In three-dimensional (3D) reconstructions of live ALPK3โtdTomato hPSC CMs, ALPK3 formed striated patterns consistent with sarcomere localization; no nuclear localization was observed (Supplementary Videos 2 and 3). |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Retain nuclear association with a resolved nuclear-envelope context. Reason: This composite electronic annotation combines mouse ALPK3/Q924C5 and Ensembl mouse-protein transfer with the UniProt subcellular-location vocabulary mapping SL-0191. These are supporting identifiers for the same broad location, not three independent experiments. The actual mouse nuclear IDA traces to J:71661/PMID:11418590 and tagged mouse Midori expressed in COS cells. Independent human support in PMID:36321451 Figure 6 localizes transient ALPK3-FLAG mainly to the nuclear envelope within the nuclear-associated pool. GO:0005635 nuclear envelope is part_of GO:0005634 nucleus, so the broader nucleus location is compatible with that result. The human endogenous-tag study PMID:39196058 and endogenous-tagged mouse study PMID:40135575 did not detect nuclear localization in their cardiomyocytes. Retain the observed nuclear association with species, developmental and construct boundaries rather than asserting ubiquitous nucleoplasmic residence. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q924C5 SUPPORTS TRANSFER Mouse ALPK3 ortholog; its nuclear evidence traces to tagged mouse Midori expressed in COS cells, not endogenous human cardiomyocytes. Independent human nuclear-envelope localization supports the broad target term. ensembl:ENSMUSP00000102971 SUPPORTS TRANSFER Mouse protein transfer identifier retained as supplied by GOA; this is not an additional independent localization assay. UniProtKB-SubCell:SL-0191 SUPPORTS TRANSFER UniProt nuclear-location vocabulary mapping; it supplies a mapping to nucleus, not independent experimental evidence. Supporting Evidence: PMID:11418590 The MIDORI protein was localized in the nucleus PMID:36321451 Depth-encoded projections of confocal z-slices confirmed that the majority of nuclear ALPK3 signal was at the nuclear envelope, as opposed to within the nucleus (Figure 6D). PMID:40135575 we did not observe the nuclear localization of endogenous ALPK3. PMID:39196058 In three-dimensional (3D) reconstructions of live ALPK3โtdTomato hPSC CMs, ALPK3 formed striated patterns consistent with sarcomere localization; no nuclear localization was observed (Supplementary Videos 2 and 3). |
| GO:0005634 nucleus | ISS GO_REF:0000024 | ACCEPT | Summary: Retain nuclear association with a resolved nuclear-envelope context. Reason: The ISS donor Q924C5 is mouse ALPK3, not ALPK1. Its nuclear localization is experimentally grounded in the original Midori paper PMID:11418590, using tagged mouse protein expressed in COS cells. Independent human evidence in PMID:36321451 Figure 6 places transient ALPK3-FLAG predominantly at the nuclear envelope within its nuclear-associated pool; the nuclear envelope is part_of nucleus. This supports retention of the broad location without claiming a constitutive nucleoplasmic function. The absence of detected nuclear localization in endogenously tagged human cardiomyocytes in PMID:39196058 and mouse cardiomyocytes in PMID:40135575 limits generalization but does not erase the positive localization in the other model. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q924C5 SUPPORTS TRANSFER Mouse ALPK3 ortholog; its nuclear evidence traces to tagged mouse Midori expressed in COS cells, not endogenous human cardiomyocytes. Independent human nuclear-envelope localization supports the broad target term. Supporting Evidence: PMID:11418590 The MIDORI protein was localized in the nucleus PMID:36321451 Depth-encoded projections of confocal z-slices confirmed that the majority of nuclear ALPK3 signal was at the nuclear envelope, as opposed to within the nucleus (Figure 6D). PMID:40135575 we did not observe the nuclear localization of endogenous ALPK3. PMID:39196058 In three-dimensional (3D) reconstructions of live ALPK3โtdTomato hPSC CMs, ALPK3 formed striated patterns consistent with sarcomere localization; no nuclear localization was observed (Supplementary Videos 2 and 3). |
| GO:0007507 heart development | ISS GO_REF:0000024 | ACCEPT | Summary: ALPK3 contributes to development of a functional heart through cardiomyocyte organization. Reason: The transferred mouse assertion is linked in MGI to the original Midori IMP (PMID:11418590), with altered cardiomyocyte differentiation in sense/antisense P19CL6 lines. Independent human cardiomyocyte studies (PMID:27106955 and PMID:36321451) establish sarcomeric organization and myomesin positioning, providing positive cellular participation rather than inferring a process solely from lethal disease. Retain the broad developmental term without claiming a specific chamber patterning or transcription-factor mechanism. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q924C5 SUPPORTS TRANSFER Source identifier is retained; the target-specific evidence and limits are described in the reason. Supporting Evidence: PMID:11418590 Permanent P19CL6 cell lines overexpressing Midori more efficiently differentiated into cardiomyocytes than did parental cells, whereas those overexpressing the antisense Midori less efficiently differentiated. These results suggest that Midori may promote the differentiation of P19CL6 into cardiomyocytes. PMID:27106955 Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. PMID:36321451 Together, these analyses show that ALPK3, MYOM1, and MYOM2 form a critical protein network in both the nucleus and the sarcomere M-band at various stages in cardiomyocyte development and demonstrate that loss of ALPK3 causes the profound mislocalization and accumulation of myomesin and thick filament proteins. |
| GO:0055007 cardiac muscle cell differentiation | IEA GO_REF:0000107 | ACCEPT | Summary: Retain acquisition of specialized cardiomyocyte structure and function. Reason: The actual mouse donor IMP traces to PMID:11418590: stable sense/antisense Midori lines alter differentiation under DMSO, beyond expression correlation. GO:0055007 includes acquisition of specialized cardiac muscle-cell features, and its developmental sub-process includes maturation of the contractile apparatus. Human ALPK3 loss disrupts sarcomere organization and function. The comparable lineage-marker maturation reported for isogenic human cells in PMID:36321451 means ALPK3 is not established as universally required for initial cardiac fate specification; it does not negate the broader differentiated structural role. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q924C5 SUPPORTS TRANSFER Source identifier is retained; the target-specific evidence and limits are described in the reason. ensembl:ENSMUSP00000102971 SUPPORTS TRANSFER Source identifier is retained; the target-specific evidence and limits are described in the reason. Supporting Evidence: PMID:11418590 Permanent P19CL6 cell lines overexpressing Midori more efficiently differentiated into cardiomyocytes than did parental cells, whereas those overexpressing the antisense Midori less efficiently differentiated. These results suggest that Midori may promote the differentiation of P19CL6 into cardiomyocytes. PMID:27106955 Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. PMID:36321451 Together, these analyses show that ALPK3, MYOM1, and MYOM2 form a critical protein network in both the nucleus and the sarcomere M-band at various stages in cardiomyocyte development and demonstrate that loss of ALPK3 causes the profound mislocalization and accumulation of myomesin and thick filament proteins. |
| GO:0055013 cardiac muscle cell development | IBA GO_REF:0000033 | ACCEPT | Summary: ALPK3 supports structural and functional development of cardiomyocytes. Reason: MGI links the mouse experimental cardiac-cell-development assertion to J:222361/PMID:21441111, which reports abnormal cardiomyocyte architecture in knockout mice. Independent human ALPK3 studies demonstrate organization of myomesins and the M band, giving the product a structural role in progression to a functional cardiomyocyte. The PAINT ancestral judgment is not a pairwise similarity score and is not weakened by a short donor list; exact PTN002931915 placement remains unreconstructed rather than being assigned a fabricated failure. Propagation Review Root cause: NO FAILURE CORE Sources checked: MGI:MGI:2151224 SUPPORTS TRANSFER Source identifier is retained; the target-specific evidence and limits are described in the reason. PANTHER:PTN002931915 UNRESOLVED Exact ancestral IBD/tree was not recovered; retain source object and biological term on experimental mouse and human evidence. Supporting Evidence: PMID:21441111 light and electron microscopy revealed altered cardiomyocyte architecture, characterized by reduced numbers of abnormal intercalated discs being associated with mild disarray of myofibrils. PMID:27106955 Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. PMID:36321451 Together, these analyses show that ALPK3, MYOM1, and MYOM2 form a critical protein network in both the nucleus and the sarcomere M-band at various stages in cardiomyocyte development and demonstrate that loss of ALPK3 causes the profound mislocalization and accumulation of myomesin and thick filament proteins. PMID:39196058 ALPK3mut CMs displayed disorganization of the sarcomeres and loss of the M-band protein myomesin (MYOM1). |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | UNDECIDED | Summary: The Rhea-mapped serine-specific activity remains unresolved for ALPK3. Reason: RHEA:17989 correctly describes protein-serine phosphorylation, but the human UniProt reaction is inferred from ALPK1/Q96QP1. Mapping a reaction does not independently validate target catalysis. The positive SQSTM1 assay in PMID:39196058 uses a combined T269/S272 phospho readout, while the negative human cellular and mouse biochemical studies dispute intrinsic kinase activity. Neither the broad reaction mapping nor this combined signal settles an autonomous human serine-specific activity. Propagation Review Root cause: UNRESOLVED Sources checked: RHEA:17989 UNRESOLVED Source identifier is retained; the target-specific evidence and limits are described in the reason. Supporting Evidence: PMID:36321451 Our phosphoproteomic studies of ALPK3dIC/dIC hiPSC-CMs, which altered the conformation of the kinase domain, ALPK3sIC/sIC, which targeted the predicted aspartate residue critical for catalysis, and massive overexpression of ALPK3 in HEK293T cells indicated that none of these perturbations significantly affected phosphorylation across the proteome. PMID:38048395 However, our experimental results revealed the absence of detectable kinase activity in ALPK3 under the tested conditions (Figure 1B). PMID:39196058 Before the assay, lysates were treated with an irreversible pan-kinase inhibitor to block endogenous kinase activity32,33. When recombinant ALPK3 kinase was applied to lysates, we identified 500 phosphosites that were significantly elevated compared to controls. |
| GO:0106310 protein serine kinase activity | ISS GO_REF:0000024 | UNDECIDED | Summary: ALPK1-derived serine-kinase transfer is not resolved for divergent ALPK3. Reason: The supplied donor Q96QP1 is human ALPK1, a different alpha-kinase-family member; its identity is preserved, not mislabeled as a mouse ALPK3 ortholog. Curated similarity transfer can cross paralogs when activity is conserved, but target-specific ALPK3 experiments conflict. Positive recombinant-domain phosphorylation is weighed against human cellular and mouse negative studies, and no definitive ALPK3 serine-residue specificity is established from the combined SQSTM1 T269/S272 readout. This is an unresolved target-activity question, not evidence that the ALPK1 experiment was wrong. Access to all three primary studies does not resolve their opposing results. Paralogy alone is not evidence of a failed transfer, and the modeled pocket comparison in the negative biochemical study is against TRPM7, not a demonstrated loss of ALPK1-like specificity in ALPK3. Accordingly this source-specific similarity inference remains unresolved; it is not accepted as measured target catalysis and is not labeled over-annotated solely from paper titles. Propagation Review Root cause: UNRESOLVED Sources checked: UniProtKB:Q96QP1 UNRESOLVED Q96QP1 is human ALPK1, the actual paralog donor for UniProt similarity transfer. Its activity does not establish ALPK3 serine specificity. Conflicting target-specific experiments leave conservation of this activity unresolved rather than demonstrating either a sound transfer or a definite paralog-transfer failure. Supporting Evidence: PMID:36321451 Our phosphoproteomic studies of ALPK3dIC/dIC hiPSC-CMs, which altered the conformation of the kinase domain, ALPK3sIC/sIC, which targeted the predicted aspartate residue critical for catalysis, and massive overexpression of ALPK3 in HEK293T cells indicated that none of these perturbations significantly affected phosphorylation across the proteome. PMID:38048395 However, our experimental results revealed the absence of detectable kinase activity in ALPK3 under the tested conditions (Figure 1B). PMID:39196058 Before the assay, lysates were treated with an irreversible pan-kinase inhibitor to block endogenous kinase activity32,33. When recombinant ALPK3 kinase was applied to lysates, we identified 500 phosphosites that were significantly elevated compared to controls. |
| GO:0031430 M band | IDA PMID:39196058 Alpha kinase 3 signaling at the M-band maintains sarcomere i... | NEW | Summary: Human ALPK3 localizes to the sarcomeric M band. Reason: PMID:39196058 directly images two independently tagged endogenous human ALPK3 reporter lines in hPSC-derived cardiomyocytes. The signal lies between Z discs, colocalizes with obscurin and sits between the two MYBPC3-marked A bands; this directly matches the M-band location rather than inferring it from a cardiomyopathy phenotype. Endogenous C-terminal FLAG-HA knock-in in mouse neonatal and adult cardiomyocytes independently corroborates M-band localization in PMID:40135575. The IDA proposal is grounded in the human experiment; the mouse work is corroboration, not mislabeled human evidence. No existing source annotation is an ancestor or descendant of this location, and no additional generic sarcomere parent is proposed. Supporting Evidence: PMID:39196058 To determine the subcellular localization of ALPK3, we generated a series of ALPK3 reporter hPSC lines in which either the tdTomato fluorescent protein or a streptavidin-binding peptide 3 (SBP3)รFLAG tag (SBP3รFLAG) was fused to the C terminus of endogenous ALPK3 (Extended Data Fig. 2aโc). PMID:39196058 Critically, both the ALPK3โtdTomato and ALPK3โSBP3รFLAG fusion proteins localized to the M-band of the sarcomere (Fig. 1d,e, Extended Data Fig. 2d and Supplementary Video 1) as demonstrated by interdigitation with Z-disc ฮฑ-actinin-2 (ACTN2), colocalization with OBSCN and its position between the two A-bands of the sarcomere (MYBPC3). PMID:40135575 Our results showed that ALPK3 colocalized with the M band marker myomesinโ1 (Figureย 3G and 3H) but not zโdisk marker ฮฑโactininโ2 in both neonatal and adult cardiomyocytes (Figureย 3I and 3J). |
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Download this section (compressed HTML)Q: Does native full-length human ALPK3 bind ATP and catalyze phosphotransfer, or do its alpha-kinase fold and partner interactions primarily support a noncatalytic organizing role?
Q: Which ALPK3 interactions form a simultaneous functional bridge or stable scaffold, rather than independent associations with MYOM2, obscurin, SQSTM1 and MuRF proteins?
Q: How does nuclear-envelope localization vary between endogenous and tagged ALPK3, developmental stages and species?
Experiment: Compare purified full-length human ALPK3 and its isolated domain with matched mouse proteins, multiple independent purification steps, inactive-site variants and active alpha-kinase controls; measure direct ATP binding and site-resolved phosphotransfer onto purified SQSTM1 separately from lysate or immunoprecipitate assays.
Experiment: Use endogenous knock-in tags and orthogonal antibodies in staged human cardiomyocytes to quantify nuclear-envelope and M-band pools, and reconstitute defined ALPK3-partner combinations to distinguish structural assembly from indirect proteostasis changes.
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