THESIS
2013
xix, 160 pages : illustrations (some color) ; 30 cm
Abstract
Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase,
plays important roles in various neuronal functions including neuronal migration, neurite
outgrowth, synapse development, and plasticity. The dysfunction of Cdk5 activity is
associated with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s
disease. Therefore, the precise regulation of Cdk5 activity is important for proper
neuronal development and functioning. Unlike other cyclin-dependent kinase family
members, which are regulated by cyclins, Cdk5 is activated through its association with
the neuronal specific activators, p35 and p39. However, whether and how Cdk5 activity
is regulated in neurons by other molecular mechanisms remain unclear. This study
revealed Cdk5 and its activa...[
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Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase,
plays important roles in various neuronal functions including neuronal migration, neurite
outgrowth, synapse development, and plasticity. The dysfunction of Cdk5 activity is
associated with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s
disease. Therefore, the precise regulation of Cdk5 activity is important for proper
neuronal development and functioning. Unlike other cyclin-dependent kinase family
members, which are regulated by cyclins, Cdk5 is activated through its association with
the neuronal specific activators, p35 and p39. However, whether and how Cdk5 activity
is regulated in neurons by other molecular mechanisms remain unclear. This study
revealed Cdk5 and its activator p35 can undergo post-translational modification by
S-nitrosylation, which involves a reaction in which a nitrogen monoxide group is
attached to the thiol side chain of the cysteine residue(s) of a protein. Cdk5
S-nitrosylation occurs at Cys83, which is one of the critical amino acids within the
ATP-binding pocket of the kinase. Upon S-nitrosylation, Cdk5 exhibits reduced kinase
activity, whereas the mutation of Cys83 to alanine on Cdk5 renders the kinase refractory
to such inhibition. Meanwhile, p35 S-nitrosylation occurs at Cys92; this modification promotes its proteasome-dependent degradation, consequently resulting in reduced Cdk5
activity. Importantly, S-nitrosylated Cdk5 and p35 can be detected in vivo. While the
S-nitrosylation of Cdk5 and p35 was detected in wild-type mouse brain lysate, this
specific S-nitrosylation was absent in neuronal nitric oxide synthase-knockout (nNOS)
mice. More importantly, Cdk5/p35 S-nitrosylation negatively regulates the activity of
Cdk5 in vivo as revealed by enhanced Cdk5 activity in nNOS-knockout mouse brains.
Furthermore, Cdk5/p35 S-nitrosylation is implicated in the regulation of neuronal
morphogenesis and synaptic plasticity. Blockade of Cdk5 S-nitrosylation in cultured
hippocampal neurons enhanced dendritic growth and branching, indicating that Cdk5
S-nitrosylation is a novel mechanism in the regulation of dendritic growth during
development. Moreover, Cdk5/p35 S-nitrosylation was found to be involved in the
regulation of synaptic function. Cdk5 hyperactivation contributed to synaptic failure upon
NO production blockade. Notably, Cdk5/p35 overexpression and silencing phenocopied
and reversed these synaptic deficits, respectively, upon pharmacological inhibition or
genetic depletion of nNOS. More importantly, blocking p35 S-nitrosylation reduced
dendritic spine density and the percentage of mature spines in cultured hippocampal
neurons as well as the surface expression of GluA1 in 293T cells. This suggests that p35
S-nitrosylation is critical for the regulation of synaptic function. The present rules
collectively demonstrate a novel mechanism in the regulation of the multi-faceted kinase
Cdk5 in many neuronal functions including dendritic morphogenesis and synaptic
plasticity.
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