Calcimembrin (CLMB; formerly C16orf74, also MICT1) is a 76-aa intrinsically disordered microprotein of the DUF4597 family that lacks transmembrane segments and associates with the cytoplasmic face of the plasma membrane through N-myristoylation of Gly2 and reversible S-acylation of Cys7 and Cys14. It carries a composite LxVPxIxIT calcineurin-docking motif (33-LDVPDIIIT-41) whose PxIxIT part, when Thr44 is phosphorylated, binds the catalytic subunit of calcineurin (PPP3CA) with very high affinity, recruiting calcineurin to membranes. CLMB is itself dephosphorylated by calcineurin through its LxVP motif in multimeric complexes, so the CLMB:calcineurin ratio, CLMB phosphorylation and dynamic acylation tune membrane calcineurin signalling. It is highly expressed in brown and beige adipocytes and induced by cold; there it limits calcineurin-mediated dephosphorylation of the PKA regulatory subunit RIIbeta, prolonging beta3-adrenergic PKA signalling and promoting thermogenesis, as shown in mouse brown adipose tissue and human beige adipocytes. CLMB is overexpressed in pancreatic and other carcinomas, where high expression is associated with invasion and poor prognosis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:16189514 Towards a proteome-scale map of the human protein-protein in... | MODIFY | Summary: Binding of CLMB to the calcineurin catalytic subunit PPP3CA (UniProtKB:Q08209), reported here by high-throughput yeast two-hybrid. The interaction is well established and mapped to the PxIxIT (PDIIIT) motif and phospho-Thr44. Reason: The interaction is real and central to CLMB function, confirmed by co-IP of endogenous proteins, PxIxIT deletion/mutation and NMR of the complex. Bare protein binding is uninformative; the specific term protein phosphatase 2B binding (GO:0030346) captures that CLMB is a calcineurin docking protein. Proposed replacements: protein phosphatase 2B binding Supporting Evidence: PMID:41224739 Together, these data show that both the LxVP and PxIxIT components of the composite motif are functional, that CLMB is a calcineurin substrate, and that pThr44 increases CLMB binding to calcineurin. PMID:28881575 IP assays demonstrated that a mutant in which the PDIIIT sequence within C16orf74 is deleted (βPDIIIT) completely abolished the interaction with PPP3CA (Figure 5C) |
| GO:0005515 protein binding | IPI PMID:16189514 Towards a proteome-scale map of the human protein-protein in... | REMOVE | Summary: High-throughput yeast two-hybrid interaction with UNC119 (Q13432), detected in successive human interactome screens (PMID:16189514). Reason: Bare protein binding conveys no function. UNC119 is a lipid-binding chaperone that solubilises N-myristoylated cargo proteins, so this interaction plausibly reflects CLMB acting as N-myristoylated cargo of UNC119 rather than any activity of CLMB itself; no more informative MF for CLMB can be derived from it, and the interaction has not been studied in a low-throughput setting. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MODIFY | Summary: Binding of CLMB to the calcineurin catalytic subunit PPP3CA (UniProtKB:Q08209), reported here by high-throughput yeast two-hybrid. The interaction is well established and mapped to the PxIxIT (PDIIIT) motif and phospho-Thr44. Reason: The interaction is real and central to CLMB function, confirmed by co-IP of endogenous proteins, PxIxIT deletion/mutation and NMR of the complex. Bare protein binding is uninformative; the specific term protein phosphatase 2B binding (GO:0030346) captures that CLMB is a calcineurin docking protein. Proposed replacements: protein phosphatase 2B binding Supporting Evidence: PMID:41224739 Together, these data show that both the LxVP and PxIxIT components of the composite motif are functional, that CLMB is a calcineurin substrate, and that pThr44 increases CLMB binding to calcineurin. PMID:28881575 IP assays demonstrated that a mutant in which the PDIIIT sequence within C16orf74 is deleted (βPDIIIT) completely abolished the interaction with PPP3CA (Figure 5C) |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | REMOVE | Summary: High-throughput yeast two-hybrid interaction with UNC119 (Q13432), detected in successive human interactome screens (PMID:25416956). Reason: Bare protein binding conveys no function. UNC119 is a lipid-binding chaperone that solubilises N-myristoylated cargo proteins, so this interaction plausibly reflects CLMB acting as N-myristoylated cargo of UNC119 rather than any activity of CLMB itself; no more informative MF for CLMB can be derived from it, and the interaction has not been studied in a low-throughput setting. |
| GO:0005515 protein binding | IPI PMID:28881575 Overexpression of C16orf74 is involved in aggressive pancrea... | MODIFY | Summary: Binding of CLMB to the calcineurin catalytic subunit PPP3CA (UniProtKB:Q08209), reported here by co-immunoprecipitation. The interaction is well established and mapped to the PxIxIT (PDIIIT) motif and phospho-Thr44. Reason: The interaction is real and central to CLMB function, confirmed by co-IP of endogenous proteins, PxIxIT deletion/mutation and NMR of the complex. Bare protein binding is uninformative; the specific term protein phosphatase 2B binding (GO:0030346) captures that CLMB is a calcineurin docking protein. Proposed replacements: protein phosphatase 2B binding Supporting Evidence: PMID:28881575 Endogenous PPP3CA interacted with the phosphorylated form of endogenous C16orf74 (arrow). PMID:28881575 IP assays demonstrated that a mutant in which the PDIIIT sequence within C16orf74 is deleted (βPDIIIT) completely abolished the interaction with PPP3CA (Figure 5C) PMID:41224739 Together, these data show that both the LxVP and PxIxIT components of the composite motif are functional, that CLMB is a calcineurin substrate, and that pThr44 increases CLMB binding to calcineurin. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MODIFY | Summary: Binding of CLMB to the calcineurin catalytic subunit PPP3CA (UniProtKB:Q08209-2), reported here by high-throughput yeast two-hybrid. The interaction is well established and mapped to the PxIxIT (PDIIIT) motif and phospho-Thr44. Reason: The interaction is real and central to CLMB function, confirmed by co-IP of endogenous proteins, PxIxIT deletion/mutation and NMR of the complex. Bare protein binding is uninformative; the specific term protein phosphatase 2B binding (GO:0030346) captures that CLMB is a calcineurin docking protein. Proposed replacements: protein phosphatase 2B binding Supporting Evidence: PMID:41224739 Together, these data show that both the LxVP and PxIxIT components of the composite motif are functional, that CLMB is a calcineurin substrate, and that pThr44 increases CLMB binding to calcineurin. PMID:28881575 IP assays demonstrated that a mutant in which the PDIIIT sequence within C16orf74 is deleted (βPDIIIT) completely abolished the interaction with PPP3CA (Figure 5C) |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: High-throughput yeast two-hybrid interaction with UNC119 (Q13432), detected in successive human interactome screens (PMID:32296183). Reason: Bare protein binding conveys no function. UNC119 is a lipid-binding chaperone that solubilises N-myristoylated cargo proteins, so this interaction plausibly reflects CLMB acting as N-myristoylated cargo of UNC119 rather than any activity of CLMB itself; no more informative MF for CLMB can be derived from it, and the interaction has not been studied in a low-throughput setting. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that CLMB family members are active at the plasma membrane, seeded by mouse Mict1 and human CLMB. Reason: Consistent with direct evidence: CLMB is N-myristoylated and S-acylated and recruits calcineurin to the plasma membrane; mouse MICT1 acts at the plasma membrane in brown adipocytes. Human CLMB appears in its own WITH/FROM because it carries experimental evidence. Supporting Evidence: PMID:41224739 We name C16orf74 calcimembrin (CLMB), because it associates with membranes via lipidation (N-myristoylation and S-acylation) and recruits calcineurin to these sites. PMID:40355556 Overall, we conclude that MICT1 is a microprotein that is mainly found in the plasma membrane and can interact with PP2B. |
| GO:0005886 plasma membrane | IDA PMID:40355556 The microprotein C16orf74/MICT1 promotes thermogenesis in br... | ACCEPT | Summary: Human CLMB/MICT1 detected in the plasma membrane fraction of transfected HEK293 cells and colocalising with PP2B at the plasma membrane (is_active_in). Reason: Direct evidence with the human protein (fractionation and immunofluorescence); membrane localisation requires the myristoylated Gly2 and is required for the thermogenic activity, which supports the is_active_in qualifier as well as located_in. Supporting Evidence: PMID:40355556 When we overexpressed the human ortholog, which corresponds to the mouse 76 aa MICT1 in HEK293 cells, human MICT1 was also found in the plasma membrane fraction (Fig. PMID:40355556 Overall, we conclude that MICT1 is a microprotein that is mainly found in the plasma membrane and can interact with PP2B. PMID:40355556 Thus, myristoylation of MICT1 and thus plasma membrane localization is required for its promotion of thermogenesis. |
| GO:0005886 plasma membrane | IDA PMID:40355556 The microprotein C16orf74/MICT1 promotes thermogenesis in br... | ACCEPT | Summary: Human CLMB/MICT1 detected in the plasma membrane fraction of transfected HEK293 cells and colocalising with PP2B at the plasma membrane (located_in). Reason: Direct evidence with the human protein (fractionation and immunofluorescence); membrane localisation requires the myristoylated Gly2 and is required for the thermogenic activity, which supports the is_active_in qualifier as well as located_in. Supporting Evidence: PMID:40355556 When we overexpressed the human ortholog, which corresponds to the mouse 76 aa MICT1 in HEK293 cells, human MICT1 was also found in the plasma membrane fraction (Fig. PMID:40355556 Overall, we conclude that MICT1 is a microprotein that is mainly found in the plasma membrane and can interact with PP2B. PMID:40355556 Thus, myristoylation of MICT1 and thus plasma membrane localization is required for its promotion of thermogenesis. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Cell membrane, SL-0039) to plasma membrane. Reason: Mapping reflects the experimentally supported UniProt cell membrane annotation from PMID:40355556. Supporting Evidence: PMID:40355556 When we overexpressed the human ortholog, which corresponds to the mouse 76 aa MICT1 in HEK293 cells, human MICT1 was also found in the plasma membrane fraction (Fig. |
| GO:0016020 membrane | IDA PMID:41224739 A composite motif in calcimembrin/C16orf74 dictates multimer... | ACCEPT | Summary: CLMB associates with membranes via N-myristoylation and dynamic S-acylation (membrane fractionation and imaging of lipidation mutants). Reason: Correct, directly shown. Less specific than plasma membrane (which the same paper also shows by colocalisation) but not wrong. Supporting Evidence: PMID:41224739 Together, these analyses indicate that N-myristoylation, in combination with dynamic dual S-acylation, promotes the stable association of CLMB with membranes. PMID:41224739 Arrows indicate some examples of CLMB and CNA colocalization to the plasma membrane. |
| GO:0016020 membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Membrane, SL-0162) to membrane. Reason: Correct but general; redundant with plasma membrane annotations. Retained as a valid parent-level location. Supporting Evidence: PMID:41224739 Together, these analyses indicate that N-myristoylation, in combination with dynamic dual S-acylation, promotes the stable association of CLMB with membranes. |
| GO:1990845 adaptive thermogenesis | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of a role in adaptive thermogenesis for the CLMB family, seeded by mouse Mict1 and human CLMB. Reason: Consistent with the mouse BAT-specific knockout and human beige adipocyte data. The family is vertebrate-specific and highly conserved in the PxIxIT and lipidation motifs, so inheritance from the mouse/human experimental node is reasonable. Supporting Evidence: PMID:40355556 Moreover, BAT-specific MICT1 ablation in mice suppresses thermogenic capacity to increase adiposity and insulin resistance. PMID:40355556 Together, these data show that MICT1 promotes thermogenic gene expression and can improve insulin sensitivity in human beige adipocytes. |
| GO:1990845 adaptive thermogenesis | IDA PMID:40355556 The microprotein C16orf74/MICT1 promotes thermogenesis in br... | ACCEPT | Summary: Overexpression of human MICT1 in human beige adipocytes increased thermogenic gene expression, PKA activity and glucose uptake; knockdown reduced responses. Reason: The paper contains human-protein, human-cell data supporting a role in adaptive thermogenesis, backed by extensive mouse loss- and gain-of-function work. CLMB acts within the beta3-adrenergic/PKA thermogenic signalling route by restraining PP2B dephosphorylation of RIIbeta; this is a modulatory signalling role, and a regulation term may ultimately be more precise (see suggested questions), but the curated process is supported. Supporting Evidence: PMID:40355556 Together, these data show that MICT1 promotes thermogenic gene expression and can improve insulin sensitivity in human beige adipocytes. PMID:40355556 MICT1 interacts with protein phosphatase 2B (PP2B, calcineurin) through the docking motif PNIIIT, thereby interfering with dephosphorylation of the regulatory subunit of protein kinase A (PKA), RIIΞ², and potentiating PKA activity in brown adipocytes. PMID:40355556 Moreover, BAT-specific MICT1 ablation in mice suppresses thermogenic capacity to increase adiposity and insulin resistance. |
| GO:1990845 adaptive thermogenesis | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfer from mouse Mict1 (Q8K1L6). Reason: Mouse evidence is strong (BAT-specific ablation, overexpression) and the human ortholog has independent human-cell support. Supporting Evidence: PMID:40355556 Moreover, BAT-specific MICT1 ablation in mice suppresses thermogenic capacity to increase adiposity and insulin resistance. |
| GO:0043495 protein-membrane adaptor activity | IDA PMID:41224739 A composite motif in calcimembrin/C16orf74 dictates multimer... | NEW | Summary: Lipid-anchored CLMB tethers calcineurin to membranes: calcineurin colocalises with wild-type CLMB at membranes but becomes cytosolic with a PxIxIT-mutant CLMB. Reason: CLMB itself performs the anchoring step: its own lipid modifications attach it to membranes and its own PxIxIT motif captures calcineurin, so the activity belongs to CLMB rather than to its partner. Comparable calcineurin-anchoring proteins (e.g. AKAP5) are characterised as membrane scaffolds. This is more informative than protein phosphatase 2B binding alone and is the activity named by the gene symbol. Supporting Evidence: PMID:41224739 We name C16orf74 calcimembrin (CLMB), because it associates with membranes via lipidation (N-myristoylation and S-acylation) and recruits calcineurin to these sites. PMID:41224739 Thus, CLMB recruits calcineurin to membranes primarily via PxIxT-mediated binding. PMID:41224739 In contrast, when co-expressed with CLMBIxITMut, this colocalization was greatly reduced, and calcineurin was predominantly cytosolic. PMID:41224739 Together, these analyses indicate that N-myristoylation, in combination with dynamic dual S-acylation, promotes the stable association of CLMB with membranes. |
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Download this section (compressed HTML)Q: Is CLMB net-inhibitory to calcineurin (sequestering it from RIIbeta, as proposed in brown adipocytes, PMID:40355556) or a membrane-targeting scaffold that promotes dephosphorylation of LxVP-only membrane substrates (PMID:41224739)? The answer decides between protein phosphatase inhibitor activity and an adaptor/regulator MF.
Q: Should the thermogenesis annotations use positive regulation of cold-induced thermogenesis (GO:0120162) rather than adaptive thermogenesis, given that CLMB modulates beta3-adrenergic PKA signalling rather than producing heat itself?
Q: Is the UNC119 interaction (repeated Y2H hits) a physiological route for transporting myristoylated CLMB between membranes, analogous to other UNC119 cargo?
Experiment: In human brown/beige adipocytes, replace endogenous CLMB with wild-type, G2A (non-myristoylated), IxIT-mutant or T44A CLMB and measure membrane calcineurin, RIIbeta phospho-Ser, PKA activity and UCP1-dependent respiration after beta3-agonist stimulation.
Hypothesis: CLMB-dependent membrane recruitment of calcineurin alters RIIbeta phosphorylation in human brown adipocytes.
Type: genetic rescue with structure-function mutants
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