Role of cyclin-dependent kinase 5 (Cdk5) in mitochondrial permeability transition pore (mPTP) opening and intracellular Ca2+ dynamics

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dc.contributor.advisorLee, Ki-Young
dc.contributor.authorNavaneetha Krishnan, Saranya
dc.contributor.committeememberRiabowol, Karl T.
dc.contributor.committeememberLees-Miller, Susan P.
dc.contributor.committeememberShutt, Timothy
dc.contributor.committeememberBraun, Andrew P.
dc.contributor.committeememberEitzen, Gary A.
dc.dateFall Convocation
dc.date.accessioned2022-11-15T17:42:57Z
dc.date.embargolift2022-06-25
dc.date.issued2020-06-25
dc.description.abstractCyclin-dependent kinase 5 (Cdk5), which plays a role in the development and progression of many human cancers, localizes in the mitochondria, a key determinant of apoptotic cell death. Cdk5 is upregulated in breast cancer cells and Cdk5 loss increases cancer cell sensitivity to chemotherapeutic drugs. However, the molecular mechanism by which Cdk5 loss promotes cell death remains unclear. I hypothesized that Cdk5 loss activates the intrinsic apoptotic pathway in breast cancer cells. I demonstrated that Cdk5-deficient breast cancer cells exhibit increased mitochondrial depolarization, mitochondrial reactive oxygen species (mtROS) levels, and mitochondrial fragmentation that is associated with an increase in both intracellular Ca2+ level and calcineurin activity, and dynamin related protein 1 (DRP1) Ser637 dephosphorylation. To define mitochondria-mediated apoptotic pathway, I utilized various inhibitors of mitochondrial function. Apoptosis is completely prevented by mitochondrial permeability transition pore (mPTP) inhibition, almost fully inhibited by blocking ROS and unaffected by inhibition of mitochondrial fission, suggesting that apoptosis in breast cancer cells due to Cdk5 loss occurs via a novel mPTP-dependent mechanism that acts primarily through ROS increase. Since calcium is the major regulator of mPTP opening, I hypothesized that Cdk5 regulates intracellular calcium homeostasis. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5?/? mouse embryos, I showed that loss of Cdk5 increases inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from internal stores. Cdk5 associates with and phosphorylates the IP3R1 Ca2+ channel at Ser421 and 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. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum membrane (MAM) and Cdk5 loss causes increased ER-mitochondria tethering, a process required for Ca2+ transfer from the ER to the mitochondria. 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 regulates IP3R1-mediated calcium release and mitochondrial Ca2+ homeostasis that are disturbed upon Cdk5 loss, which lead to mPTP opening.
dc.identifier.citationNavaneetha Krishnan, S. (2020). Role of cyclin-dependent kinase 5 (Cdk5) in mitochondrial permeability transition pore (mPTP) opening and intracellular Ca2+ dynamics (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.urihttp://hdl.handle.net/1880/115468
dc.identifier.urihttps://dx.doi.org/10.11575/PRISM/40435
dc.language.isoenen
dc.language.isoEnglish
dc.publisher.facultyGraduate Studiesen
dc.publisher.facultyCumming School of Medicine
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en
dc.subjectBreast cancer
dc.subjectCyclin-dependent kinase 5
dc.subjectMitochondria
dc.subjectCalcium,Reactive oxygen species
dc.subjectApoptosis
dc.subject.classificationHealth Sciences--Oncology
dc.subject.classificationBiology--Cell
dc.subject.classificationBiology--Molecular
dc.titleRole of cyclin-dependent kinase 5 (Cdk5) in mitochondrial permeability transition pore (mPTP) opening and intracellular Ca2+ dynamics
dc.typedoctoral thesis
thesis.degree.disciplineMedicine – Medical Sciences
thesis.degree.grantorUniversity of Calgaryen
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
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