Brown, Kalyn A
(2016)
Optical Control of Signal Transduction and Other Cellular Processes.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Nature uses precise spatio-temporal control to maintain proper cellular function. Being able to replicate this control is an important step in investigating proteins. The research presented here uses photo-labile “caging” groups and light to control protein function including signaling cascades, protein localization and dimerization. Cell signaling is an essential process that allows cells to respond to extracellular stimuli. Four kinases, ERK2, p38α, JNK1 and PAK1, were targeted to optochemically control signal transduction. ERK2, p38α, and JNK1 are MAP kinases that are implicated in cell proliferation, apoptosis, motility, and differentiation. PAK1 is a serine/threonine kinase that affects focal adhesion dynamic and action reorganization. Incorporation of a photocaged lysine into the ATP binding pocket of PAK1 allowed for optical control of PAK1 kinase activity and paxillin focal adhesions. Ras GTPases are membrane bound molecular switches involved in various pathways that convert stimuli into a cellular response. Membrane localization of Ras GTPases is determined by the C-terminal CaaX domain. Within the CaaX domain, cysteine residues are modified by the addition of a farnesyl group and two palmitoyl groups. Ultimately, photochemical control of membrane localization by CaaX domain signaling was used to determine the kinetics of CaaX domain processing. Src family tyrosine kinases are involved in cell proliferation, cytoskeletal alterations, differentiation, survival, adhesion, and migration and are localized to the membrane through the modification of a SH4 domain with myristoylation and palmitoylation. Optochemical control of SH4 domain mediated membrane localization was not achieved. Finally, the dimerization of FKBP12 and FRB was photochemically controlled by the development of a photo-cleavable rapamycin dimer. Rapamycin heterodimerizes FKBP12 and FRB and has been exploited as a research tool in a wide array of cellular processes. Photochemical control of protein localization and dimerization allows for precise spatial and temporal control over these processes, which will lend to the development of useful biological tools.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
1 June 2016 |
| Date Type: |
Publication |
| Defense Date: |
5 October 2015 |
| Approval Date: |
1 June 2016 |
| Submission Date: |
14 April 2016 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
151 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Chemical Biology, Photocaging, Kinases, Localization, Conditional Control |
| Date Deposited: |
01 Jun 2016 13:56 |
| Last Modified: |
01 Jun 2018 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/27697 |
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