Date of Graduation
5-2015
Document Type
Dissertation (PhD)
Program Affiliation
Genes and Development
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Andrew Gladden, Ph.D.
Committee Member
Russell Broaddus, M.D., Ph.D.
Committee Member
Michael Galko, Ph.D.
Committee Member
Gary Gallick, Ph.D.
Committee Member
Pierre McCrea, Ph.D.
Abstract
Cell-cell adhesion is critical for the control of tissue organization and homeostasis. A family of proteins that regulate cell-cell adhesions is the FERM (4.1 protein, Ezrin, Radixin, Moesin) domain-containing proteins.One FERM domain protein, the non-receptor tyrosine phosphatase PTPN14, is mutated or deleted in several human cancers suggesting that it may be involved in tumor development and/or progression. Additionally, the loss of the FERM domain protein Merlin is associated with tumor development and metastasis.Both PTPN14 and Merlin have been shown to localize and possibly regulate adherens junction (AJ) functions. This work sought to determine if PTPN14 is required for the establishment of epithelial adhesion and polarity. Using mammalian epithelial cells as a model to test these hypotheses, we down-regulated the tyrosine phosphatase, PTPN14. Decreased levels of PTPN14 increased E-cadherin membrane movement at cell contacts, disrupting cell contacts and abolishing E-cadherin localization during primordial lumen formation. In cells with established AJs and TJs, decreased levels of PTPN14 led to an increase in β-catenin Y654 phosphorylation, promoting increased membrane mobility of E-cadherin and subsequent destabilization of cell contacts. This was dependent on PTPN14 phosphatase activity at b-cateninY654 opposing Src-mediated disruption of AJs. In cells that lacked PTPN14, for an extended time, I observed alterations in the morphology of the epithelial cells to a spindle morphologycoupled with increased cell migration,transcriptional repression of E-cadherin and activation of Slug. Similar to the regulation of the AJ by PTPN14, we examined another FERM domain protein, Merlin, and found that specific post-translational modifications in its unique N-terminal region regulate distinct AJ-mediated activities. Mutation of a single serine residue in the N-terminal region of Merlin blocked Merlin loading onto the AJ during early cell-cell adhesion formation. Disruption of Merlin AJ loading leads to actin disorganization and loss of contact inhibition of proliferation. Additionally, I found that phosphorylation of a second site in the N-terminal region of Merlin affects the release of the apical proteins, atypical protein kinase C (aPKC) and partitioning defective 3 (Par3), to the apical membrane for the development of functional TJs. Blocking this specific phosphorylation site disrupts the formation of functional polarized epithelia. From this, we see that distinct post-translational modifications of Merlin can alter its interaction with polarity proteins and thus Merlin functionin regulating epithelial cell adhesion and polarity. Overall, these results indicate that mutation or post-translation regulation of FERM proteins could promote increased migration and invasion in cells altering normal epithelial development possibly leading to loss of tissue function and disease.
Keywords
PTPN14, Merlin, FERM, epithelial adhesion, polarity, adherens and tight junctions, aPKC
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