The Juxtamembrane Domain: a Central Guardian of the Ron Receptor Tyrosine Kinase Activity

Open Access
Author:
Wang, Xin
Graduate Program:
Cell and Developmental Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 18, 2013
Committee Members:
  • Pamela Hankey Giblin, Dissertation Advisor
  • Pamela Hankey Giblin, Committee Chair
  • Zhi Chun Lai, Committee Member
  • Avery August, Special Member
  • B Tracy Nixon, Committee Member
  • Kumble Sandeep Prabhu, Committee Member
Keywords:
  • Juxtamembrane domain
  • Ron Receptor Tyrosine Kinase
  • Autoinhibition
Abstract:
As a fundamental player in the cell signaling, the Ron receptor tyrosine kinase (RTK) mediates a number of biological events such as cell proliferation, motility, survival and regulation of immune responses. Deregulated expression and/or activation of the Ron receptor has been observed in a variety of pathogenic conditions including cancer, therefore understanding the molecular mechanisms governing Ron receptor activity is of great significance. Recently, the juxtamembrane domain of RTKs has gained increasing attention because of its significant but diverse regulatory role in receptor activation. The studies described herein demonstrate that the juxtamembrane domain of Ron is a crucial regulator of receptor activity. We have identified several critical regions in the juxtamembrane domain which collaboratively mediate Ron receptor autoinhibition. We have characterized the functionally important interplay among different protein segments, and proposed a molecular model of the autoinhibited and active Ron structure. These studies provide a better understanding of the mechanisms governing Ron activation, which will lay the groundwork for the development of novel clinical approaches for the treatment of Ron-related human malignancies. Although the sequence and structure of the juxtamembrane domain vary greatly among RTKs, a central role for tyrosines in the juxtamembrane domain in regulation of receptor activity has been demonstrated in a number of RTKs. However, unlike other RTKs described to date, tyrosines in the juxtamembrane domain of Ron are inconsequential for receptor activation. Rather, we have identified an acidic JM-C region in the juxtamembrane domain which plays a central role in Ron autoinhibition. We propose that the JM-B region where the tyrosines are located modulates Ron activity indirectly by affecting the steric configuration of the JM-C region. The JM-D region at the C terminus of the Ron juxtamembrane domain is relatively conserved between family members, and crystallographic studies show that it forms a helical structure in the vicinity of the αC helix in the kinase N lobe. We have uncovered potential hydrogen bonding linkages between the charged residues in the JM-D and the αC helix. We propose that these interactions inhibit receptor activity by stabilizing the distorted conformation of the C helix in the kinase domain. Interestingly, we have also uncovered an essential role for the JM-D helix in promoting receptor activation. We propose that the structural integrity of the JM-D helix and van der waals contacts between hydrophobic residues in the JM-D and the C helix are indispensable for the kinase activation. As far as we know, this is the first example of a juxtamembrane domain which contributes to both receptor autoinhibition and activation. Interplay between the juxtamembrane domain and the kinase domain of Ron is not limited to direct contacts. Studies of the Met and the murine Ron receptor have demonstrated a critical role for the homologous residues of Y1198 in the Ron kinase C lobe. Here we highlight the necessity of an intact Y1198 for Ron activation and show that release of the autoinhibition mediated by the juxtamembrane domain overcomes the essential role of Y1198 in Ron activation. These data suggest that the primary role of Y1198 in the kinase domain is to alleviate the autoinhibition imposed by the juxtamembrane domain. Based on experimental data and crystallographic studies of Ron, we have developed a novel and comprehensive molecular model for the role of the juxtamembrane domain in mediating Ron activation. In this model, the JM-C region primarily coordinates with protein segments around the active site including the αC helix, P loop and activation loop to promote the stabilization of Ron in the autoinhibited state. The JM-D region, by forming intensive contacts with the αC helix, is a crucial mediator of both the receptor autoinhibition and activation. We propose that phosphorylation of Y1198 facilitates movement of the αC helix and the closely associated JM-D helix, which in turn promotes displacement of the JM-C region and consequent realignment of the active site for kinase catalysis. Taken together, the work presented in this dissertation has unveiled a novel mechanism by which the juxtamembrane domain regulates Ron receptor activity. Our studies also provide a new perspective regarding the regulation of RTK activity by precise utilization of conformational plasticity and the functional cooperation of different protein segments, and may provide a platform for development of the next-generation anti-cancer therapies.