CKB

Summary: Phylogenetic (IBA) inference that B-CK is active in the extracellular region. B-CK is a cytosolic enzyme; it is detected in exosomes/secretome but is not active extracellularly. The "is_active_in extracellular region" call is an over-propagation and is biologically incorrect as a site of activity.

Reason: Creatine kinase catalysis requires cytosolic ATP/ADP and creatine; the enzyme is not known to function in the extracellular region. Detection in secreted vesicles does not make the extracellular region a site of activity, so an "is_active_in extracellular region" assertion is incorrect. Aligned with the parallel CKM review, which REMOVEs the identical IBA annotation; genuine extracellular/exosome detection is retained separately under the extracellular-exosome annotations.

Summary: Creatine kinase activity is the core molecular function of CKB, supported phylogenetically (IBA) and experimentally. Accept.

Reason: This is the defining, experimentally established activity of CKB (EC 2.7.3.2); the IBA inference is consistent with direct assays.

Summary: The biological process corresponding to the forward (ATP -> phosphocreatine) direction of the creatine kinase reaction. Core process for CKB. Accept.

Reason: Directly reflects the physiological left-to-right reaction direction catalyzed by CKB; consistent with the experimentally demonstrated activity.

Summary: Generic root catalytic-activity term assigned by InterPro2GO. Correct but uninformative; subsumed by the specific creatine kinase activity annotation.

Reason: A high-level ancestor of GO:0004111; adds no information beyond the specific molecular function and should not be retained as a meaningful annotation.

Summary: Electronic (IEA) assignment of the core creatine kinase activity, consistent with experimental evidence. Accept (redundant with the experimental annotation but correct).

Reason: Same correct core molecular function; electronic support via RHEA/EC mapping aligns with direct evidence.

Summary: Electronic mitochondrion localization transferred by similarity from the mouse ortholog (Q04447) in the context of thermogenic-fat / futile-creatine-cycle biology. Human CKB is predominantly cytosolic; a mitochondrial pool is not directly demonstrated for human B-CK.

Reason: B-CK is the cytosolic creatine kinase isoform; mitochondrial creatine kinase is a distinct gene product (uMtCK/CKMT1). The mitochondrial call is a by-similarity transfer specific to adipocyte thermogenesis and is not the core localization of human CKB. The UniProt combined-IEA mapping (SL-0173) restates the ISS location. ORGANELLES check (2026-10-25): the earlier MARK_AS_OVER_ANNOTATED is revised to KEEP_AS_NON_CORE. The mouse source Q04447 carries an IDA from PMID:33597756 (now cached, full text) plus MGI HDA rows from mitochondrial proteomics (PMID:14651853). That paper shows endogenous FLAG-tagged CKB protected from protease in purified BAT mitochondria in vivo while the ER marker CANX and the outer-membrane TOM70 were degraded, so the signal is internal to the organelle, not MAM contamination; import depends on an internal MTS-like element, and protease-treated mitochondria carry CKB-dependent creatine kinase activity. A small pool also exists in heart and kidney (about 30-fold less than BAT). The organelle claim is therefore right for the ortholog, and the projection to human is sound in kind, though no human mitochondrial assay exists. It is non-core: the bulk of B-CK is cytosolic and the mitochondrial pool is tissue-restricted (ADO precedent). Stays at the root (rule 4): protease protection places CKB inside the outer membrane but does not resolve intermembrane space from matrix, and there is no TM segment or cleavable presequence.

Summary: Electronic cytosol localization from the UniProt subcellular-location mapping. Correct core localization. Accept.

Reason: B-CK is a cytosolic enzyme; consistent with IDA (HPA) and Reactome TAS annotations to cytosol.

Summary: Electronic plasma-membrane call from UniProt subcellular-location mapping, reflecting peripheral recruitment of B-CK to membrane ATPase microcompartments (e.g. with KCC3). Real but peripheral, not the core compartment.

Reason: CKB is not an integral membrane protein; it is recruited to the membrane to supply ATP to transporters such as KCC3 (PMID:18566107). Retain as a non-core peripheral localization.

Summary: Generic kinase-activity parent from InterPro2GO; subsumed by the specific creatine kinase activity. Uninformative.

Reason: Ancestor of GO:0004111; carries no information beyond the specific molecular function.

Summary: Generic phosphotransferase parent from InterPro2GO; subsumed by the specific creatine kinase activity. Uninformative.

Reason: High-level ancestor of GO:0004111; redundant generic term.

Summary: The immediate parent class of creatine kinase activity (phosphotransfer to a nitrogenous/guanidino acceptor). Correct but redundant with the specific term.

Reason: Direct ancestor of GO:0004111; the specific creatine kinase activity term is preferred and makes this generic term redundant.

Summary: Electronic assignment of the core phosphocreatine biosynthetic process, consistent with experimental evidence. Accept.

Reason: Correct core process for CKB; redundant with the IDA/IBA annotations but not erroneous.

Summary: Generic 'protein binding' from a high-throughput interactome screen. Uninformative as a molecular function; the biologically meaningful interactions are captured by more specific annotations.

Reason: Bare protein binding does not describe a function; retained as non-core evidence of interaction only.

Summary: Generic 'protein binding' from a high-throughput binary interactome. Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Interaction with hepatitis C virus NS4A, recruiting CKB to the viral replication complex. A genuine moonlighting interaction but represented only as generic 'protein binding'.

Reason: Biologically real host-factor interaction (PMID:19264780) but a non-core, condition-specific role; the bare term is uninformative as a core function.

Summary: Generic 'protein binding' from a cullin-RING ligase network proteomic study (CUL3 association). Uninformative as a function.

Reason: Bare protein binding; relates to CKB's role as a CRL substrate, retained as non-core.

Summary: Generic 'protein binding' from a high-throughput interactome map. Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Generic 'protein binding' from a BioPlex interactome (includes CKM and ASB9 partners). Uninformative as a standalone function.

Reason: Bare protein binding; the meaningful partners (CKM dimerization, ASB9 E3 ligase) are better captured specifically. Retained as non-core.

Summary: Generic 'protein binding' from the HuRI binary interactome (ASB9). Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Generic 'protein binding' from a dual proteome-scale interactome (CKM, ASB9). Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Generic 'protein binding' from the OpenCell endogenous-tagging map. Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Generic 'protein binding' from a brain cell-type interactome (TCF4, CUL3). Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Generic 'protein binding' from a multimodal cell-map interactome (ASB9). Uninformative molecular function.

Reason: Bare protein binding is not a core function; retained as non-core.

Summary: Electronic transfer (from mouse Q04447) of involvement in the futile creatine cycle (adipocyte thermogenesis). This role is characterized for the thermogenic-fat mitochondrial creatine cycle and has not been demonstrated for human B-CK; retain as a non-core, by-similarity possibility.

Reason: The futile creatine cycle is a specialized thermogenic-fat process inferred by orthology; it is plausible but not the core energy-buffering function of cytosolic B-CK in humans.

Summary: Direct immunofluorescence (HPA) localization to the cytosol. This is the core, experimentally supported localization of B-CK. Accept.

Reason: B-CK is a cytosolic enzyme; IDA from HPA directly supports the core localization.

Summary: ISS mitochondrion localization transferred from mouse Q04447 (thermogenic-fat creatine cycle). Not the core compartment of human cytosolic B-CK.

Reason: By-similarity mitochondrial localization tied to adipocyte thermogenesis; human B-CK is the cytosolic isoform and a human mitochondrial pool is not directly established. ORGANELLES check (2026-10-25): the earlier MARK_AS_OVER_ANNOTATED is revised to KEEP_AS_NON_CORE. The mouse source Q04447 carries an IDA from PMID:33597756 (now cached, full text) plus MGI HDA rows from mitochondrial proteomics (PMID:14651853). That paper shows endogenous FLAG-tagged CKB protected from protease in purified BAT mitochondria in vivo while the ER marker CANX and the outer-membrane TOM70 were degraded, so the signal is internal to the organelle, not MAM contamination; import depends on an internal MTS-like element, and protease-treated mitochondria carry CKB-dependent creatine kinase activity. A small pool also exists in heart and kidney (about 30-fold less than BAT). The organelle claim is therefore right for the ortholog, and the projection to human is sound in kind, though no human mitochondrial assay exists. It is non-core: the bulk of B-CK is cytosolic and the mitochondrial pool is tissue-restricted (ADO precedent). Stays at the root (rule 4): protease protection places CKB inside the outer membrane but does not resolve intermembrane space from matrix, and there is no TM segment or cleavable presequence.

Summary: Experimental co-localization of CK-B with KCC3 at the plasma membrane, consistent with recruitment of B-CK to membrane ATPase microcompartments. Real but peripheral, not the core cytosolic compartment.

Reason: CKB associates with the membrane via interaction with the KCC3 transporter to supply ATP locally; it is not an integral membrane protein. Retain as a non-core peripheral localization.

Summary: Direct experimental evidence (site-directed mutagenesis of human B-CK) for creatine kinase activity, including identification of essential catalytic residues. The strongest support for the core molecular function. Accept.

Reason: Direct assay of human CKB enzymatic activity; defines the core molecular function (EC 2.7.3.2).

Summary: Direct evidence for the phosphocreatine-forming (forward) direction of the creatine kinase reaction. Core process for CKB. Accept.

Reason: Reflects the physiological left-to-right reaction catalyzed by CKB, directly demonstrated for the human enzyme.

Summary: ISS support (from ortholog) for the core creatine kinase activity. Consistent with direct evidence. Accept.

Reason: Correct core molecular function; redundant with the experimental annotation but not erroneous.

Summary: ISS 'is_active_in mitochondrion' transferred from mouse Q04447. Human B-CK is the cytosolic isoform; an active mitochondrial pool is not directly established.

Reason: By-similarity mitochondrial activity tied to thermogenic-fat biology; not the core localization or site of activity of human cytosolic B-CK. ORGANELLES check (2026-10-25): the earlier MARK_AS_OVER_ANNOTATED is revised to KEEP_AS_NON_CORE. The mouse source Q04447 carries an IDA from PMID:33597756 (now cached, full text) plus MGI HDA rows from mitochondrial proteomics (PMID:14651853). That paper shows endogenous FLAG-tagged CKB protected from protease in purified BAT mitochondria in vivo while the ER marker CANX and the outer-membrane TOM70 were degraded, so the signal is internal to the organelle, not MAM contamination; import depends on an internal MTS-like element, and protease-treated mitochondria carry CKB-dependent creatine kinase activity. A small pool also exists in heart and kidney (about 30-fold less than BAT). The organelle claim is therefore right for the ortholog, and the projection to human is sound in kind, though no human mitochondrial assay exists. It is non-core: the bulk of B-CK is cytosolic and the mitochondrial pool is tissue-restricted (ADO precedent). Stays at the root (rule 4): protease protection places CKB inside the outer membrane but does not resolve intermembrane space from matrix, and there is no TM segment or cleavable presequence. Qualifier note (is_active_in): the source paper does show the activity in the organelle for mouse, as creatine kinase activity in protease-treated brown adipocyte mitochondria that is absent in Ckb-null mitochondria and restored by wild-type but not iMTS-L-deleted CKB, so is_active_in is justified for the ortholog; not shown for human.

Summary: High-throughput proteomic detection of CKB in exosomes. A real observation but reflects passive secretion/vesicle loading rather than a functional compartment.

Reason: Exosome detection is common for abundant cytosolic enzymes; retain as a non-core localization, not a site of creatine kinase function.

Summary: ISS extracellular-region localization transferred from an ortholog. B-CK is a cytosolic enzyme; extracellular detection (exosomes/secretome) does not establish a functional extracellular localization.

Reason: Over-propagated localization for a cytosolic enzyme; not a site of activity. Exosomal detection is better captured by the exosome annotation.

Summary: CKB binds an E3 ubiquitin ligase component (Parkin/E3 interactant proteomics), consistent with CKB being a substrate of the ECS(ASB9) Cullin-RING E3 ligase that targets it for degradation. A real binding activity but reflecting CKB's role as a degradation substrate rather than a core enzyme function.

Reason: Informative interaction (E3 ligase/degradation axis) but not part of CKB's core energy-metabolism function; retain as non-core.

Summary: High-throughput proteomic detection of CKB in prostatic-secretion exosomes. Same interpretation as other exosome detections.

Reason: Proteomic exosome detection; non-core localization, not a functional compartment for catalysis.

Summary: HEP annotation derived from a disease-state differential-abundance proteomic survey of human substantia nigra. Differential expression in neurodegenerative disease does not establish a role in substantia nigra development.

Reason: The supporting study compares protein abundance in disease vs control substantia nigra; it does not provide evidence that CKB is involved in the developmental process. Over-interpretation of an expression observation.

Summary: High-throughput proteomic detection of CKB in urinary exosomes. Same interpretation as other exosome detections.

Reason: Proteomic exosome detection; non-core localization.

Summary: Traceable author statement (Reactome) placing the creatine kinase reaction in the cytosol. Consistent with the core cytosolic localization. Accept.

Reason: Correct core localization; the creatine kinase reaction catalyzed by CKB occurs in the cytosol.

Summary: Traceable author statement (original CKB cDNA cloning paper) for creatine kinase activity. Core molecular function. Accept.

Reason: Correct core molecular function, supported by the foundational cloning/characterization reference.