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The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed Lineage Kinase 3 and Mixed Lineage Kinase 4 Beta

Blessing, Natalya
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430158298

Abstract Details

2015, Doctor of Philosophy, University of Toledo, Biology (Cell-Molecular Biology).
The mixed lineage kinases (MLKs) are serine/threonine mitogen-activated protein kinase kinase kinases (MAP3Ks) that modulate the activities of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 signaling pathways. MLK3 plays a pivotal role in cell invasion, tumorigenesis and metastasis. Wild type MLK4 negatively regulates MAPK signaling by possibly inhibiting MLK3 activation, while mutant MLK4 plays in important role in driving colorectal cancer and glioblastomal tumorigenesis (Martini et al., 2013). The mechanisms by which MLK3 and MLK4 protein levels are regulated in cells are unknown. The carboxyl terminus of HSC-70 interacting protein (CHIP) is a U-box E3 ubiquitin ligase that regulates cytosolic protein degradation in response to stress. We examined the regulation of MLK3 and MLK4 protein levels by stresses. Treatment of SKOV3 ovarian cancer cells with the Hsp90 inhibitor, geldanamycin (GA), promotes the dissociation of endogenous MLK3 from Hsp90, and association of MLK3 with Hsp70. GA treatment induces K-48 linked ubiquitination of MLK3, and reduction of endogenous MLK3 and MLK4ß protein levels. Heat shock and osmotic stress cause a similar reduction of MLK3 and MLK4ß. MLK4ß suppresses heat and osmotic shock-induced activation of ERK, p38, and JNK. We assessed the role of CHIP in modulating MLK3 and MLK4ß protein levels. GA, thermostress, and osmotic shock-induced degradation of MLK3 is abolished in cells with CHIP siRNA knockdown. CHIP overexpression promotes ubiquitination and a proteasome-dependent reduction of MLK3. CHIP interacts with both MLK3 and MLK4ß proteins in vitro and with MLK3 in cells; and CHIP ubiquitinates MLK3 and MLK4ß (with UbcH5 E2s) in vitro. Furthermore, CHIP-dependent regulation of MLK3 is required for invasion of SKOV3 cells. These findings suggest that CHIP regulates MLK3 and MLK4ß protein levels and, in part, cell invasiveness by facilitating ubiquitination and proteasome-dependent degradation of MLK3.
Deborah Chadee (Committee Chair)
Richard Komuniecki (Committee Member)
Malathi Krishnamurthy (Committee Member)
Donald Ronning (Committee Member)
Frank Pizza (Committee Member)
122 p.
Cellular Biology; Molecular Biology

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Citations

  • Blessing, N. (2015). The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed Lineage Kinase 3 and Mixed Lineage Kinase 4 Beta [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430158298

    APA Style (7th edition)

  • Blessing, Natalya. The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed Lineage Kinase 3 and Mixed Lineage Kinase 4 Beta. 2015. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430158298.

    MLA Style (8th edition)

  • Blessing, Natalya. "The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed Lineage Kinase 3 and Mixed Lineage Kinase 4 Beta." Doctoral dissertation, University of Toledo, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1430158298

    Chicago Manual of Style (17th edition)

toledo1430158298
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© 2015, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.