Every cell is faced continuously with the task of transducing a vast number
of external signals into appropriate intracellular responses. In mammalian cells,
membrane bound receptors modulate the production of secondary messenger
molecules, such as cyclic AMP (cAMP). Cell signaling through second
messengers relies on the diffusion of such second messengers from sites of
production to sites of intracellular action to modulate the activities of an
intracellular effector molecule, which in turn modulates a cellular response.
Protein kinase A is the primary sensor of intracellular concentrations of cAMP, and
protein kinase A (PKA)-mediated phosphorylation events are regulated by
fluctuations in cAMP concentrations. This thesis examines the mechanism by
which protein kinase A functions within fish melanophore to regulate intracellular
activities in response to cAMP.
A novel in situ assay to monitor protein kinase A-dependent
phosphorylation is described here. By using a fluorescent substrate, the activities
of protein kinase A are characterized in living cells as a function of
phosphorylation-dependent fluorescence decay. Physiologically manipulated cells
were microinjected with the fluorescent substrate to correlate the activities of
protein kinase A and the subsequent effects on melanosome distribution.
Cytoplasmic compartmentalization of protein kinase A is one means to
regulate the specificity of a multifunctional kinase. The physiologic effects of an
overexpressed PKA anchoring protein, Ht31, demonstrated that protein kinase A
anchoring is required to direct the movements of melanosomes. Additional studies
demonstrated that protein kinase A redistributes with the stimulated movements of
melanosomes and interacts with proteins from a motor-protein complex. These
observations shed light on how a transduction complex, consisting of a group of
proteins, which localize components within the cell and provide motor activity, is
able to integrate signaling information that regulate melanosome movements. I
propose a model and suggest that protein kinase A is a component of the
transduction complex: the proposed PKA complex constitutes a molecular
servomechanism comprised of a sensor, an effector, and a regulator that function
together in feedback loop to direct the motor-protein dependent transport of
melanosomes. / Graduation date: 2001
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32482 |
| Date | 15 November 2000 |
| Creators | Sellers, Drew L. |
| Contributors | McFadden, Philip N. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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