| dc.description.abstract |
<p>Ca<super>2+</super>/Calmodulin-dependent protein kinase kinase β(CaMKKβ)
is a serine/threonine directed kinase which is activated following increases in intracellular
Ca<super>2+</super>. CaMKKβ 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 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.</p><p>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 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 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. </p><p>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.</p>
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