Abstract View
PROTEOMIC IDENTIFICATION OF NOVEL PROTEIN KINASE SUBSTRATES IN RAT BRAIN DURING PAVLOVIAN FEAR CONDITIONING
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
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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