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Mixed Lineage Kinase 3 Signaling in Ovarian Cancer and Neurofibromatosis-2

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2011, Doctor of Philosophy, University of Toledo, Biology (Cell-Molecular Biology).
Mixed lineage kinase 3 (MLK3), a 97 kDa Ser/Thr protein kinase, is a MAP kinase kinase kinase (MAP3K) that phosphorylates and activates MAP kinase kinases (MAP2Ks), resulting in the activation of JNK, p38 and ERK MAPK pathways. The Rho GTPases Rac and Cdc42, activate MLK3 via binding to the CRIB domain of MLK3, relieving the SH3 mediated auto-inhibition, and promoting MLK3 auto-phosphorylation and activation. Though the role of MLK3 in tumorigenesis is still under investigation, previous studies have indicated that MLK3 can transform NIH3T3 cells in a MEK dependent manner, and recently, MLK3 expression was found to induce invasion in mammary epithelial cells. In addition, our results indicate higher MLK3 expression and kinase activity in SKOV3, HEY and HEY1B ovarian tumor cells in comparison to immortalized ovarian epithelial T29 and T80 cells. Thus, we hypothesized that MLK3 could promote aberrant MAPK activity and cell invasion in ovarian cancer cells. In our study, we found that silencing mlk3 substantially reduced ERK and JNK signaling, cell invasion, and matrix metalloproteinases (MMP)-1, -2, -9 and -12 expression in SKOV3 cells. Thus we propose that MLK3 may promote invasion by up-regulating specific MMP expression in ovarian cancer cells. The Neurofibromatosis Type 2 (NF2) gene encodes a 595 amino acid tumor suppressor protein, merlin, which has homology to the Ezrin, Radixin and Moesin (ERM) group of cellular proteins that link integral membrane proteins to the actin cytoskeleton. Merlin undergoes intramolecular interactions between the N-terminus and C-terminus, and phosphorylation on Ser518 inhibits this interaction and inactivates merlin. Activated merlin inhibits cell proliferation, cell cycle progression and motility, however, a detailed biochemical mechanism for the growth suppressive function of merlin remains elusive. We previously found that merlin is a MLK3 inhibitor, and we wished to investigate if MLK3 is required for merlin-mediated suppression of cell growth, invasion and MAPK signaling in ovarian and NF2 tumor cells. Comparison of different human cell lines revealed high basal MLK3 activity in HEI193, SKOV3 and NCIH460 cell lines. Interestingly, there was a correlation between high MLK3 activity and reduced merlin expression in the tumor cell lines. The carboxyl-terminus of merlin (340-590) is required for merlin-MLK3 interaction, and is sufficient to inhibit MLK3 kinase acitivity. Induction of merlin protein expression in RT4-NF2.17 cells (rat schwannoma cells with Tet-inducible merlin) inhibited MLK3, B-Raf, ERK and JNK activities. In contrast, depletion of merlin elevated MLK3, B-Raf, ERK and JNK activities. Merlin expression blocked the interaction between MLK3 and the upstream activator, Rho GTPase, Cdc42. In addition, MLK3 was required for proliferation and invasion of cells that lacked functional merlin. Our data suggests that merlin inhibits MLK3 by blocking the cdc42-MLK3 interaction, and that elevated basal MLK3 activity in NF2, ovarian and other tumor cells may be due to the loss of functional merlin in these cells.
Deborah Chadee (Advisor)
Douglas Leaman (Committee Member)
William Taylor (Committee Member)
William Maltese (Committee Member)
Ivana de la Serna (Committee Member)
115 p.

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Citations

  • Zhan, Y. (2011). Mixed Lineage Kinase 3 Signaling in Ovarian Cancer and Neurofibromatosis-2 [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1310127039

    APA Style (7th edition)

  • Zhan, Yu. Mixed Lineage Kinase 3 Signaling in Ovarian Cancer and Neurofibromatosis-2. 2011. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1310127039.

    MLA Style (8th edition)

  • Zhan, Yu. "Mixed Lineage Kinase 3 Signaling in Ovarian Cancer and Neurofibromatosis-2." Doctoral dissertation, University of Toledo, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1310127039

    Chicago Manual of Style (17th edition)