Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. IP6Ks convert IP6 to pyrophosphates such as diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. The inositol hexakisphosphate kinase 2 (IP6K2) controls cellular apoptosis. To explore roles for IP6K2 in brain function, we elucidated its protein interactome in mouse brain revealing a robust association of IP6K2 with creatine kinase-B (CK-B), a key enzyme in energy homeostasis. Cerebella of IP6K2-deleted mice (IP6K2-knockout [KO]) produced less phosphocreatine and ATP and generated higher levels of reactive oxygen species and protein oxidative damage. In IP6K2-KO mice, mitochondrial dysfunction was associated with impaired expression of the cytochrome-c1 subunit of complex III of the electron transport chain. We reversed some of these effects by combined treatment with N-acetylcysteine and phosphocreatine. These findings establish a role for IP6K2–CK-B interaction in energy homeostasis associated with neuroprotection.

Inositol pyrophosphates are versatile messenger molecules that mediate a variety of cellular functions, including cell growth, apoptosis, endocytosis, and cell differentiation. The most extensively studied inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), displays a 5′-diphosphate (1, 2). IP7 is generated in mammals by a family of inositol hexakisphosphate kinases (IP6Ks) (3, 4). IP6Ks exists in three isoforms: IP6K1, IP6K2, and IP6K3. Inositol hexakisphosphate kinase-2 (IP6K2) sensitizes cells to apoptosis (5, 6). Mice with targeted deletion of IP6K2 display an increased incidence of aero-digestive tract carcinoma (7). Cell survival associated with heat shock protein 90 also involves IP6K2 (8, 9).We previously reported a major role for IP6K2 in the disposition of cerebellar granule cells as well as Purkinje cell morphology and motor coordination. The influence of IP6K2 upon cerebellar disposition involved protein 4.1N, both of which were highly expressed in cerebellar granule cells (10).To further assess the functions of IP6K2 in the brain, we explored its binding partners using coimmunoprecipitation and tandem liquid chromatography mass spectrometry (LC-MS/MS). Here, we report that IP6K2 robustly interacts with creatine kinase-B (CK-B), which regulates energy homeostasis of cells and exists in two forms, brain type (CK-B) and muscle type (CK-M). CK catalyzes the reversible transfer of the phosphate group of phosphocreatine to ADP to yield ATP (11, 12). A functional interplay between mitochondrial and cytosolic isoforms of CK regulates cellular energy homeostasis. Cytosolic CK rephosphorylates locally produced free ADP and increases creatine globally, while the mitochondrial enzyme catalyzes the conversion of creatine to phosphocreatine utilizing mitochondrial ATP (13–15).Here, we show that IP6K2 loss leads to decreased CK-B expression, reduced ATP levels, and diminished mitochondrial activity associated with increased oxidative stress. About 80 to 90% of ATP is generated in the mitochondria by oxidative phosphorylation, and diminished ATP levels are the immediate effect of mitochondrial dysfunction. Loss of IP6K2 and CK-B reflects the suppression of the mitochondrial cytochrome c1 expression, a component of complex III of the mitochondrial electron transport chain. In the present study, we report a physiologic association of CK-B and IP6K2, whose disruption impacts mitochondrial functions.Dendritic morphogenesis was reduced in IP6K2-deficient neurons and was rescued by restoring normal levels of ATP. These observations reveal an essential role of IP6K2 in the energy production of the brain. Our findings indicate that IP6K2 is a key regulator of mitochondrial homeostasis which promotes neuroprotection.