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Regulation of CaMKKβ Dependent Signaling Pathways

dc.contributor.advisor Means, Anthony R Green, Michelle Frances 2012-05-29T16:45:00Z 2012-05-29T16:45:00Z 2011
dc.description.abstract <p>Ca<super>2+</super>/Calmodulin-dependent protein kinase kinase &beta;(CaMKK&beta;) is a serine/threonine directed kinase which is activated following increases in intracellular Ca<super>2+</super>. CaMKK&beta; activates Ca<super>2+</super>/Calmodulin-dependent protein kinase I (CaMKI), Ca<super>2+</super>/Calmodulin-dependent protein kinase IV (CaMKIV), and the AMP-dependent protein kinase (AMPK) in a number of physiological pathways including learning and memory formation, neuronal differentiation, and regulation of energy balance. The purpose of the work presented in this dissertation is to better understand the regulation of CaMKK&beta; activity and specificity in CaMKK&beta;-dependent signaling cascades. First, the CaMKK&beta;-AMPK signaling complex is examined using biochemical assays. In both brain and cell lysates CaMKK&beta; and AMPK form a stable complex which can be examined by co-immunoprecipitation. This complex lacks the AMPK&gamma; subunit and is not allosterically activated by adenosine 5'-monophohphate (AMP) binding. Using a series of CaMKK&beta; and AMPK mutants it was determined that the kinase domains of CaMKK&beta; and AMPK are necessary for their interaction and CaMKK&beta; must be active and bound to adenosine 5'-triphosphate (ATP) to form a complex with AMPK. However, CaMKK&beta; need not be active or bound to ATP to bind CaMKIV. This illustrates that the CaMKK&beta;-AMPK signaling complex differs from the CaMKK&beta;-CaMKIV signaling complex. These observations indicate that the CaMKK&beta;-AMPK signaling complex could be specifically targeted without effecting CaMKK&beta;-CaMKIV signaling.</p><p>Second, the regulation of CaMKK&beta; by multi-site phosphorylation is examined. Three phosphorylation sites in the N-terminus of CaMKK&beta; were identified by mass spectrometry which regulates its Ca<super>2+</super>/CaM-independent autonomous activity. The kinases responsible for these phosphorylations are identified as CDK5 and GSK3. These phosphorylation events are sequential with CDK5 priming for subsequent GSK3 phosphorylation. In addition to regulation of autonomous activity, phosphorylation of CaMKK&beta; regulates its half-life as determined in a radioactive pulse-chase assay. Examination of CaMKK&beta; in a cerebellar granule neuron model system demonstrates that CaMKK&beta; levels correlate with CDK5 activity and are regulated developmentally. In addition, appropriate phosphorylation of CaMKK&beta; is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKK&beta;-dependent signaling cascades. </p><p>Overall the work presented in this dissertation illustrates additional levels of regulation of CaMKK&beta;-dependent signaling pathways. In the future, these novel methods of CaMKK&beta; regulation will need to be considered when studying CaMKK&beta;-dependent signaling pathways.</p>
dc.subject Molecular Biology
dc.subject Pharmacology
dc.subject Biochemistry
dc.subject Calmodulin
dc.subject Cell Signaling
dc.subject Kinases
dc.title Regulation of CaMKK&beta; Dependent Signaling Pathways
dc.type Dissertation
dc.department Pharmacology

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