Abstract
We previously demonstrated that loss of Cdk5 in breast cancer cells promotes ROS-mediated cell death by inducing mitochondrial permeability transition pore (mPTP) opening (Oncogene 37, 1788–1804). However, the molecular mechanism by which Cdk5 loss causes mPTP opening remains to be investigated. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5−/− mouse embryos, we show that absence of Cdk5 causes a significant increase in both mPTP opening and mitochondrial Ca2+ level. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and Cdk5 loss in MAMs causes increased ER-mitochondria tethering, a process required for Ca2+ transfer from the ER to the mitochondria. Loss of Cdk5 also causes increased ATP-mediated mitochondrial Ca2+ uptake from the ER. Inhibition of ER Ca2+ release or mitochondrial Ca2+ uptake in Cdk5−/− MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5−/− MEFs is due to increased Ca2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 in MAMs regulates mitochondrial Ca2+ homeostasis that is disturbed upon Cdk5 loss, which leads to mPTP opening.
Highlights
Cyclin dependent kinase 5 (Cdk5) is a small prolinedirected serine/threonine kinase that serves various cell functions such as in the regulation of oxidative stress [1, 2], mitochondrial functions [3] and apoptosis [4, 5]
To further characterize the molecular and cellular mechanisms that lead to mitochondrial permeability transition pore (mPTP) opening upon Cdk5 loss, we utilized primary mouse embryonic fibroblasts (MEFs) isolated from wt and Cdk5−/− mouse embryos as knockout of the Cdk5 gene in mice is associated with perinatal lethality [39]
Upon treatment with CoCl2, Cdk5−/− MEFs exhibited less calcein fluorescence intensity compared with wt, indicating greater quenching of mitochondrial calcein fluorescence and increased mPTP opening in Cdk5−/− MEFs compared with wt
Summary
Cyclin dependent kinase 5 (Cdk5) is a small prolinedirected serine/threonine kinase that serves various cell functions such as in the regulation of oxidative stress [1, 2], mitochondrial functions [3] and apoptosis [4, 5]. We previously reported that loss of Cdk promotes mitochondrial permeability transition pore (mPTP)-mediated apoptosis in breast cancer cells [6]. We found that Cdk loss induces mPTP opening, which leads to increased cellular levels of reactive oxygen species (ROS) that subsequently cause increased susceptibility of breast cancer cells to apoptosis. The ER is closely tethered to approximately 5–20% of mitochondrial surface within a 10–30 nm space [26, 27]. This distinct structure is crucial for intracellular calcium homeostasis. Disruption of MAM causes deregulation of calcium mobilization and disruption of [Ca2+]mt homeostasis
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