CKB (P12277) — Creatine kinase B-type — review notes

Identity

• UniProt P12277, KCRB_HUMAN, gene CKB (HGNC:1991), synonym CKBB. 381 aa, EC 2.7.3.2.
• Brain-type / B-type cytosolic creatine kinase. Forms CK-BB homodimers and CK-MB heterodimers (with CKM, muscle-type).
• Member of the ATP:guanido phosphotransferase (phosphagen kinase) family.

Catalytic function and reaction direction

• Reversibly catalyzes transfer of the high-energy phosphoryl between ATP and creatine:
  creatine + ATP = N-phosphocreatine + ADP + H(+) (Rhea:RHEA:17157, EC 2.7.3.2).
• UniProt FUNCTION: "Reversibly catalyzes the transfer of phosphate between ATP and various phosphogens (e.g. creatine phosphate) (PubMed:8186255). Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa."
• Catalytic residues established by site-directed mutagenesis: Cys-283 and Arg-292 essential for catalysis PMID:8186255; Cys283->Ser/Tyr abolishes activity without affecting dimerization; Asp340->Glu had no effect. This paper grounds GO:0004111 (creatine kinase activity, IDA) and GO:0046314 (phosphocreatine biosynthetic process, IDA).
• Crystal structure of human B-CK with ADP-Mg2+-NO3--creatine transition-state analogue, 2.0 Å (PMID:18977227; PDB 3B6R, 3DRB, 3DRE), confirms active site and dimer.

Dimer composition / subunit

• "Dimer of identical or non-identical chains, which can be either B (brain type) or M (muscle type). With MM being the major form in skeletal muscle and myocardium, MB existing in myocardium, and BB existing in many tissues, especially brain." (UniProt SUBUNIT).
• CK-MB (CKB:CKM heterodimer) — IntAct interaction with CKM (P06732) recorded; clinically the CK-MB heterodimer is a cardiac biomarker.
• PMID:8186255 also shows heterodimerization and dominant-negative inhibition of the normal subunit by the mutant subunit in BB and MB dimers.

Cytosolic localization coupled to ATPases / phosphocreatine shuttle

• Cytosolic (cytoplasm, cytosol) is the canonical localization. GOA: GO:0005829 cytosol IDA (HPA), TAS (Reactome).
• Phosphocreatine/creatine kinase (PCr/CK) shuttle: mitochondrial CK (a separate gene product, uMtCK/CKMT1) regenerates phosphocreatine in mitochondria; PCr diffuses to the cytosol where cytosolic CK (B-CK) coupled to ATPases regenerates ATP locally [Wikipedia/Wallimann review: "PCr generated by mtCK in mitochondria is shuttled to cytosolic CK that is coupled to ATP-dependent processes... CK is associated with the ATPases, forming a coupled microcompartment"].
• Wallimann review (PMID:21448658): CK/PCr functions as (a) temporal energy buffer, (b) spatial energy buffer/shuttle, (c) metabolic regulator.
• CKB is the cytosolic ATP-regenerating limb; it is NOT itself a mitochondrial creatine kinase. The GOA mitochondrion annotations (ISS/IEA from Q04447, the mouse "futile creatine cycle" / thermogenic-fat protein) are by-similarity transfers from the futile-creatine-cycle work and should be treated cautiously — human CKB is overwhelmingly cytosolic. Note Q04447 is itself the brain CK ortholog (mouse Ckb), and the UniProt "futile creatine cycle / thermogenic fat mitochondrion" annotations derive from adipocyte thermogenesis studies (Kazak et al.). For HUMAN CKB the mitochondrial/futile-cycle annotations are by-similarity, not directly demonstrated.

Tissue expression

• BB-CK expressed in brain and in most non-muscle tissues, and in smooth muscle. HPA: "Tissue enhanced (brain, intestine)".
• Brain: cytosolic B-CK is the dominant cytosolic CK isoform; PCr/CK system supports neuronal/astrocytic energy metabolism.

Known interactions / moonlighting roles (most are GO:0005515 protein binding, IPI)

• SLC12A6 / KCC3 (K-Cl cotransporter 3): CK-B binds the C-terminal domain of KCC3; the interaction (and CK-B kinase activity) is required for KCC3 cotransport activity PMID:18566107. CK-B is also a partner of the neuronal KCC2 (PMID:16336223, not in GOA). This is the "ATP-regenerating enzyme coupled to a membrane ATP-consuming transporter" microcompartment paradigm. UniProt records plasma-membrane localization (EXP, PMID:18566107) and the SLC12A6 interaction.
• ASB9 (Q96DX5): substrate-recognition subunit of an ECS(ASB9)/Cullin-RING E3 ligase; CKB is ubiquitinated by ECS(ASB9), leading to proteasomal degradation [UniProt PTM, PMID:33268465]. EM structure of CKB in complex with ASB9-ELOB-ELOC (PMID:32513959, PDB 6V9H). Many IntAct "protein binding" rows (Q96DX5) and the GO:0031625 "ubiquitin protein ligase binding" (IPI, PMID:19725078, with O60260 = Parkin) reflect this E3-ligase/degradation axis. CUL3 (Q13618), CUL5 also recorded.
• HCV NS4A: CKB interacts with hepatitis C virus NS4A and is recruited to the viral replication complex, upregulating NS3-4A helicase/replicase activity — a host-factor/moonlighting role PMID:19264780. (Q03463 xeno interaction.)
• TCF4 (P15884), various high-throughput interactome maps (PMID:16189514, 19060904, 25416956, 28514442, 32296183, 33961781, 35271311, 37207277, 40205054) — generic Y2H/AP-MS "protein binding" hits, low individual functional informativeness.

PTMs

• Phosphorylated (Ser-4, Thr-35, Ser-199, etc.); ubiquitinated (Lys-45, 101, 107, 381) by ECS(ASB9); nitrated (Tyr-269). Mostly large-scale proteomics.

Annotation review judgments (summary)

• CORE: creatine kinase activity (GO:0004111) — multiple lines (IDA PMID:8186255, IBA, ISS, TAS, IEA). ACCEPT the experimental/IBA; the redundant IEA/ISS/TAS duplicates kept as non-core or accepted.
• CORE: phosphocreatine biosynthetic process (GO:0046314) — the BP for the reaction (left-to-right physiological direction). ACCEPT.
• CORE location: cytosol (GO:0005829) — IDA (HPA) ACCEPT.
• Generic parent MF terms from InterPro IEA (GO:0003824 catalytic activity, GO:0016301 kinase activity, GO:0016772 transferase transferring P, GO:0016775 phosphotransferase N acceptor) — these are correct but uninformative parents of creatine kinase activity → MARK_AS_OVER_ANNOTATED (redundant generic ancestors).
• protein binding (GO:0005515) IPI rows — uninformative bare "protein binding"; KEEP_AS_NON_CORE (real interactions, but term is uninformative; the informative ones, KCC3 and ASB9/E3 ligase, are captured elsewhere). Avoid as core.
• ubiquitin protein ligase binding (GO:0031625) IPI — real (ASB9/Parkin E3 axis); KEEP_AS_NON_CORE (CKB is the substrate, not function of CKB per se, but it does bind the ligase).
• mitochondrion (GO:0005739) ISS/IEA, is_active_in — by-similarity from mouse Q04447 thermogenic-fat work; human CKB is cytosolic. MARK_AS_OVER_ANNOTATED / KEEP_AS_NON_CORE (cannot fully exclude small mito pool, but not the core cytosolic function).
• futile creatine cycle (GO:0140651) IEA (from Q04447) — by-similarity, adipocyte thermogenesis; KEEP_AS_NON_CORE for human (not demonstrated in human CKB).
• plasma membrane (GO:0005886) EXP PMID:18566107 — CK-B co-localizes with KCC3 at membrane microcompartment; KEEP_AS_NON_CORE (peripheral/recruited, not a transmembrane protein).
• extracellular exosome (GO:0070062) HDA, extracellular region (GO:0005576/IBA/ISS), extracellular space — CKB detected in exosomes/secretome (proteomic). KEEP_AS_NON_CORE (real proteomic detection but not the functional compartment); the IBA extracellular_region is questionable for a cytosolic enzyme → MARK_AS_OVER_ANNOTATED for the IBA/ISS extracellular_region (likely over-propagation), KEEP exosome HDA as non-core.
• substantia nigra development (GO:0021762) HEP PMID:22926577 — differential expression in a substantia nigra proteomic disease study; HEP from an abstract about differential protein expression in neurodegenerative disease does not establish a developmental role. MARK_AS_OVER_ANNOTATED.