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Cdk5 regulates IP3R1-mediated Ca²⁺ dynamics and Ca²⁺-mediated cell proliferation.

ABSTRACT: Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER-mitochondria tethering and ER Ca²⁺ transfer to the mitochondria, subsequently increasing mitochondrial Ca²⁺ concentration ([Ca²⁺]_mt). This suggests a role for Cdk5 in regulating intracellular Ca²⁺ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5^-/- mouse embryos, we show here that loss of Cdk5 causes an increase in cytosolic Ca²⁺concentration ([Ca²⁺]_cyt), which is not due to reduced internal Ca²⁺ store capacity or increased Ca²⁺ influx from the extracellular milieu. Instead, by stimulation with ATP that mediates release of Ca²⁺ from internal stores, we determined that the rise in [Ca²⁺]_cyt in Cdk5^-/- MEFs is due to increased inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca²⁺ release from internal stores. Cdk5 interacts with the IP3R1 Ca²⁺ channel and phosphorylates it at Ser₄₂₁. Such phosphorylation controls IP3R1-mediated Ca²⁺ release as loss of Cdk5, and thus, loss of IP3R1 Ser₄₂₁ phosphorylation triggers an increase in IP3R1-mediated Ca²⁺ release in Cdk5^-/- MEFs, resulting in elevated [Ca²⁺]_cyt. Elevated [Ca²⁺]_cyt in these cells further induces the production of reactive oxygen species (ROS), which upregulates the levels of Nrf2 and its targets, Prx1 and Prx2. Cdk5^-/- MEFs, which have elevated [Ca²⁺]_cyt, proliferate at a faster rate compared to wt, and Cdk5^-/- embryos have increased body weight and size compared to their wt littermates. Taken together, we show that altered IP3R1-mediated Ca²⁺ dynamics due to Cdk5 loss correspond to accelerated cell proliferation that correlates with increased body weight and size in Cdk5^-/- embryos.

SUBMITTER: NavaneethaKrishnan S

PROVIDER: S-EPMC9402492 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Cdk5 regulates IP3R1-mediated Ca2+ dynamics and Ca2+-mediated cell proliferation.

NavaneethaKrishnan Saranya S Law Vincent V Lee Jungkwon J Rosales Jesusa L JL Lee Ki-Young KY

Cellular and molecular life sciences : CMLS 20220824 9

Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER-mitochondria tethering and ER Ca2+ transfer to the mitochondria, subsequently increasing mitochondrial Ca2+ concentration ([Ca2+]mt). This suggests a role for Cdk5 in regulating intracellular Ca2+ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibrob ...[more]

PMID: 36001172

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Project description:AimsPhospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling.Methods and resultsFluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10 nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ± 0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ± 0.02. At 50 nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice.ConclusionPLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.

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Cdk5 regulates IP3R1-mediated Ca²⁺ dynamics and Ca²⁺-mediated cell proliferation.

Publications

Cdk5 regulates IP3R1-mediated Ca<sup>2+</sup> dynamics and Ca<sup>2+</sup>-mediated cell proliferation.

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