Ca²⁺/Calmodulin-dependent protein kinase kinase β(CaMKKβ) is a serine/threonine directed kinase which is activated following increases in intracellular Ca²⁺. CaMKKβ activates Ca²⁺/Calmodulin-dependent protein kinase I (CaMKI), Ca²⁺/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 CaMKKβ activity and specificity in CaMKKβ-dependent signaling cascades. First, the CaMKKβ-AMPK signaling complex is examined using biochemical assays. In both brain and cell lysates CaMKKβ and AMPK form a stable complex which can be examined by co-immunoprecipitation. This complex lacks the AMPKγ subunit and is not allosterically activated by adenosine 5'-monophohphate (AMP) binding. Using a series of CaMKKβ and AMPK mutants it was determined that the kinase domains of CaMKKβ and AMPK are necessary for their interaction and CaMKKβ must be active and bound to adenosine 5'-triphosphate (ATP) to form a complex with AMPK. However, CaMKKβ need not be active or bound to ATP to bind CaMKIV. This illustrates that the CaMKKβ-AMPK signaling complex differs from the CaMKKβ-CaMKIV signaling complex. These observations indicate that the CaMKKβ-AMPK signaling complex could be specifically targeted without effecting CaMKKβ-CaMKIV signaling.

Second, the regulation of CaMKKβ by multi-site phosphorylation is examined. Three phosphorylation sites in the N-terminus of CaMKKβ were identified by mass spectrometry which regulates its Ca²⁺/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 CaMKKβ regulates its half-life as determined in a radioactive pulse-chase assay. Examination of CaMKKβ in a cerebellar granule neuron model system demonstrates that CaMKKβ levels correlate with CDK5 activity and are regulated developmentally. In addition, appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.

Overall the work presented in this dissertation illustrates additional levels of regulation of CaMKKβ-dependent signaling pathways. In the future, these novel methods of CaMKKβ regulation will need to be considered when studying CaMKKβ-dependent signaling pathways.