Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival.

Bcl-2 interacts with the inositol 1,4,5-trisphosphate receptor (InsP3R) and thus prevents InsP3-induced Ca(2+) elevation that induces apoptosis. Here we report that Bcl-2 binds dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a protein kinase A (PKA)-activated and calcineurin (CaN)-deactivated inhibitor of protein phosphatase 1 (PP1). Bcl-2 docks DARPP-32 and CaN in a complex on the InsP3R, creating a negative feedback loop that prevents exaggerated Ca(2+) release by decreasing PKA-mediated InsP3R phosphorylation. T-cell activation increases PKA activity, phosphorylating both the InsP3R and DARPP-32. Phosphorylated DARPP-32 inhibits PP1, enhancing InsP3R phosphorylation and Ca(2+) release. Elevated Ca(2+) activates CaN, which dephosphorylates DARPP-32 to dampen Ca(2+) release by eliminating PP1 inhibition to enable it to dephosphorylate the InsP3R. Knocking down either Bcl-2 or DARPP-32 abrogates this feedback mechanism, resulting in increased Ca(2+) elevation and apoptosis. This feedback mechanism appears to be exploited by high levels of Bcl-2 in chronic lymphocytic leukemia cells, repressing B-cell receptor-induced Ca(2+) elevation and apoptosis.

Bcl-2 regulation of the inositol 1,4,5-trisphosphate receptor and calcium signaling in normal and malignant lymphocytes: potential new target for cancer treatment.

The anti-apoptotic protein Bcl-2 is a versatile regulator of cell survival. Its interactions with its own pro-apoptotic family members are widely recognized for their role in promoting the survival of cancer cells. These interactions are thus being targeted for cancer treatment. Less widely recognized is the interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (InsP3R), an InsP3-gated Ca(2+) channel located on the endoplasmic reticulum. The nature of this interaction, the mechanism by which it controls Ca(2+) release from the ER, its role in T-cell development and survival, and the possibility of targeting it as a novel cancer treatment strategy are summarized in this review. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors.

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP3R)-interacting protein by mass spectrometry. The PKM2:IP3R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP3R isoforms. Moreover, fluorescence microscopy indicated that both IP3R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP3R at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IP3R isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IP3R interaction and potentiated IP3R-mediated Ca2+ release both in intact cells (TAT-D5SD peptide) and in a unidirectional 45Ca2+ flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP3R-mediated Ca2+ signalling in intact cells without altering the ER Ca2+ content. These data identify PKM2 as an IP3R-interacting protein that inhibits intracellular Ca2+ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.

Trends in Type 1 Diabetic Ketoacidosis During COVID-19 Surges at 7 US Centers: Highest Burden on non-Hispanic Black Patients.

CONTEXT: The impact of the COVID-19 pandemic on individuals with type 1 diabetes remains poorly defined. OBJECTIVE: We examined United States trends in diabetic ketoacidosis (DKA) among individuals with type 1 diabetes (T1D) during the COVID-19 pandemic at 7 large US medical centers and factors associated with these trends. METHODS: We compared DKA events among children and adults with T1D during COVID-19 surge 1 (March-May 2020) and COVID-19 surge 2 (August-October 2020) to the same periods in 2019. Analysis was performed using descriptive statistics and chi-square tests. RESULTS: We found no difference in the absolute number of T1D patients experiencing DKA in 2019 vs 2020. However, a higher proportion of non-Hispanic Black (NHB) individuals experienced DKA in 2019 than non-Hispanic White (NHW) individuals (44.6% vs 16.0%; P < .001), and this disparity persisted during the COVID-19 pandemic (48.6% vs 18.6%; P < .001). DKA was less common among patients on continuous glucose monitor (CGM) or insulin pump in 2020 compared to 2019 (CGM: 13.2% vs 15.0%, P < .001; insulin pump: 8.0% vs 10.6%, P < .001). In contrast to annual DKA totals, a higher proportion of patients had DKA during COVID-19 surges 1 and 2 compared to the same months in 2019 (surge 1: 7.1% vs 5.4%, P < .001; surge 2: 6.6% vs 5.7%, P = .001). CONCLUSION: DKA frequency increased among T1D patients during COVID-19 surges with highest frequency among NHB patients. DKA was less common among patients using CGM or insulin pumps. These findings highlight the urgent need for improved strategies to prevent DKA among patients with T1D-not only under pandemic conditions, but under all conditions-especially among populations most affected by health inequities.

A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2.

Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.