Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

Spring 3-10-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Gregory D. Longmore

Abstract

The highly conserved Hippo kinase cascade is critical for both regulation of organ size in development and tumor suppression. Several mechanisms, such as cell-cell contact in vitro and organ size in vivo, have been implicated in the activation of the Hippo pathway, leading to inactivation of the YAP/TAZ transcriptional coactivators and growth arrest. Previous work from our lab has identified the Ajuba LIM proteins as novel negative regulators of the Hippo pathway in both Drosophila and mammals. However, the mechanism by which the Ajuba LIM proteins prevent Hippo pathway activity is not known. Using contact inhibition of proliferation as a functional assay of Hippo pathway activity, we find that the Ajuba LIM proteins bind to the Ndr-kinase Lats1/2 and inhibit Hippo pathway mediated inactivation of YAP only in low density, proliferating cells, suggesting that the Ajuba LIM proteins function to keep the Hippo pathway off in contexts where cell proliferation is needed, but are unable to prevent activation of the pathway by signals such as cell density. We demonstrate that the Ajuba LIM proteins enhance association of the core kinase complex, and, using a panel of LIM domain mutants of the Ajuba LIM protein, LIMD1, identify the domains required for sequestering active Lats2 in the core kinase complex. Using Drosophila wing size as an in vivo read-out of Hippo activity, we find that only the domain mutants that increase complex association suppress Hippo-mediated YAP inactivation. We also determine the role of subcellular localization in Ajuba LIM-mediated inhibition of the Hippo pathway. It has previously been shown that targeting the complex to the plasma membrane, using a membrane targeted Mob1, can lead to its activation; interestingly, we find that LIMD1 is unable to associate with or enhance the association of this membrane-targeted complex. Conversely, targeting LIMD1 to the plasma membrane also abrogates its ability to bind to, and enhance the association of, the cytoplasmic core kinase complex. Given that LIMD1 and the Hippo complex are both recruited to the plasma membrane in high density, growth-arrested cells, these results provide a model for Ajuba LIM-mediated inhibition of Hippo pathway signaling, in which the Ajuba LIM proteins physically interact with the core kinase complex in the cytoplasm of low-density, proliferating cells, thereby preventing inactivation of YAP and premature growth arrest.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K7959FK5

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