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John Sherman Dissertation Final.pdf (3.56 MB)

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A Metabolic Checkpoint in G2 Regulates Mitotic Entry in Response to Metabolic Stress

Sherman, John William, Jr
http://rave.ohiolink.edu/etdc/view?acc_num=osu1607026548250923

Abstract Details

2020, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Coordination of the G1/S transition and metabolic activity has been the subject of extensive study, while coordination between the G2/M transition and metabolic activity is less well understood. In order to thoroughly investigate the G2 to M transition, we have developed a novel flow cytometry based method to quickly and precisely measure G2 to M phase progression using nocodazole as well as EdU and phospho-histone H3 staining. This method allows us to measure G2 to M phase progression using cell lines and treatments that are incompatible with double thymidine block. Our data shows that a G2 metabolic checkpoint delays mitotic entry in response to a variety of metabolic stresses such as starvation, mitochondrial injury, reactive oxygen species, and endoplasmic reticulum stress. All of these stresses are known to activate autophagy, a protective response which can degrade damaged mitochondria and misfolded proteins to salvage key metabolic intermediates. Autophagy is dispensable for mitotic entry under nutrient replete conditions, but the chemical inhibitor chloroquine or the removal of key autophagy proteins ATG5 or ATG7 further suppresses mitotic entry during starvation. Knockout of AMPK and the overexpression of ATG5 and TFEB all failed to induce any change in the rate of G2/M phase transition. In addition to supporting mitotic entry, autophagy also supports cellular viability over extended starvation. As mitotic entry is predominantly regulated by phosphorylation of the cyclin B-Cdk1 complex, we focused on identifying kinases or phosphatases necessary for checkpoint activation. Kinase and phosphatase inhibitor screening revealed several chemical inhibitors that enhance mitotic entry during starvation and suggest that the G2 metabolic checkpoint is transduced by the JNK and p38 kinase cascades. JNK and p38 are both activated in response to ROS and ER stress, and both phosphorylate and inhibit a key activator of mitotic entry, Cdc25. Complimenting this, siRNA knockdown of several kinases involved in JNK and p38 activation show some ability to rescue mitotic entry. An additional siRNA, which rescues mitotic entry through an unconfirmed off target, knocks down expression of 14-3-3. 14-3-3 binds to Cdc25 in response to phosphorylation caused by p38, and is required for efficient inhibition. In conclusion, our data suggests that starvation in G2 activates both ROS and ER stress, which act as sensors for metabolic stress. ROS and ER stress then transduce this signal through p38 and 14-3-3, as well as JNK to inhibit Cdc25 and cause G2 arrest. We have outlined a potential signaling axis for the G2 metabolic checkpoint, but future studies will be important for confirming the necessity of p38, JNK and 14-3-3 for checkpoint activation, and for investigating the consequences of checkpoint abrogation.
Ruoning Wang (Advisor)
Anne Strohecker (Committee Member)
Harold Fisk (Committee Member)
Matthew Summers (Committee Member)
100 p.
Biology
Cell Cycle; G2 phase; Mitosis; Metabolism; Checkpoint

Recommended Citations

Citations

  • Sherman, Jr, J. W. (2020). A Metabolic Checkpoint in G2 Regulates Mitotic Entry in Response to Metabolic Stress [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1607026548250923

    APA Style (7th edition)

  • Sherman, Jr, John. A Metabolic Checkpoint in G2 Regulates Mitotic Entry in Response to Metabolic Stress. 2020. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1607026548250923.

    MLA Style (8th edition)

  • Sherman, Jr, John. "A Metabolic Checkpoint in G2 Regulates Mitotic Entry in Response to Metabolic Stress." Doctoral dissertation, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1607026548250923

    Chicago Manual of Style (17th edition)

osu1607026548250923
170
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