Rho-associated kinase 1 (ROCK1) is a serine/threonine kinase important for the regulation of the cellular cytoskeleton through the induction of actin stress fibres and acto-myosin contractility. The cleavage and subsequent activation of ROCK1 by caspase 3 during apoptosis is believed to cause many morphological phenomena associated with programmed cell death such as dynamic membrane blebbing. I now formally prove the necessity of ROCK1 cleavage for apoptotic blebbing by knocking-in a caspase cleavage resistant mutant of ROCK1 in a genetically modified model. In addition, animals homozygous for non-cleavable ROCK1 demonstrate a phenotype consistent with auto-immune disease suggesting that apoptotic blebbing is important to mediate rapid efferocytosis, which is a rapid phagocytic clearance of the cellular corpse, and thus maintain self-tolerance. Furthermore, apoptotic blebbing is important for the clearance of apoptotic cells and I demonstrate a novel mechanism for ROCK to mediate the release of factors participating in macrophage migration to dying cells. ROCK induced apoptotic blebs and bodies lose membrane integrity prior to secondary necrosis and leak intracellular material. Using quantitative mass spectrometry I identified numerous proteins that were previously unrecognized to be released during apoptosis. The release of protein was found to be impaired following ROCK antagonism with Y27632 which underscores the importance of ROCK activity in apoptotic protein release. One of these proteins, gelsolin, was released following caspase cleavage and encourages macrophage motility towards apoptotic cells. Finally, I now demonstrate that the three nonsynonymous somatic mutations in the ROCK1 gene identified in the Cancer Genome Project lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy-terminus combined with structural modeling provides insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire ‘driver’ mutations that result in kinase activation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:542137 |
| Date | January 2011 |
| Creators | Wickman, Grant Raymond |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/3021/ |
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