Abstract View |
PROTEOMIC IDENTIFICATION OF NOVEL PROTEIN
KINASE SUBSTRATES IN RAT BRAIN DURING PAVLOVIAN FEAR CONDITIONING
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M.W. Swank1*; G.E.
Schafe2; J.E. LeDoux2 |
1. Molecular & Human Genetics, Baylor College
of Medicine, Houston, TX, USA |
2. Center for Neural Science, New York University,
New York, NY, USA |
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The lateral nucleus of the amygdala
is thought to be a primary site of plasticity underlying fear
conditioning, a form of associative learning shown to be dependent
on activation of a number of protein kinase cascades, including
PKA, PKC, MAPK, and more recently, PI3-kinase/Akt and CDK5.
However, little is known about the relevant kinase substrates
that may mediate the downstream effects of kinase activation.
Here we demonstrate the utility of proteomic methods which
have been used to identify a number of novel protein kinase
substrates. Using antibodies to specific substrate phospho-motifs,
western blots revealed that a number of Akt, PKA, PKC, and
proline-directed kinase substrates were significantly increased
in the LA of fear conditioned animals, and these showed distinctive
temporal features. Using a combination of ion-exchange chromatography,
2D electrophoresis, and MS/MALDI, we have begun the first
stage of kinase substrate identification from a large pool
of rat brain protein, and have generated a list of substrates
to analyze further. Several of the more interesting substrates
have the same apparent molecular weight as those regulated
in LA, and are targets of proline-directed kinases such as
MAPK and CDK5; these include adenylyl cyclase-associated protein
2 (ACAP2), collapsin-response mediator protein 2 (CRMP-2/DRP-2/TOAD-64),
and valosin-containing protein (VCP/p97). Further experiments
are designed to examine directly the phosphorylation status
of these and other candidate substrates in the LA of fear
conditioned animals.
Supported by: MH38774, MH46516, & MH62519
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