Hormone action heavily utilizes multifunctional serine/threonine protein kinases and phosphoprotein phosphatases. These enzymes regulate cellular events by altering the phosphorylation state of metabolic enzymes, structural proteins and ion-channels. Recently, it has been proposed that subcellular targeting of kinases and phosphatases at sites close to preferred substrates influences the specificity of these seemingly multifunctional enzymes. In support of this concept we have shown that targeting of kinases and phosphatases occurs through association with a family of """"""""anchoring proteins"""""""". During the past funding period we cloned and characterized a neuronal A-Kinase Anchoring Protein, called AKAP79, which targets protein kinase A (PKA) to the postsynaptic density (PSD), a cytoskeletal-like structure attached to the internal surface of excitatory synapses. Several lines of evidence suggest that AKAP79 is, in fact, a multifunctional anchoring protein which is capable of associating with three signaling enzymes, PKA, protein kinase C (PKC) and the phosphatase 2B, calcineurin (CaN). Each enzyme binds to distinct regions of AKAP79 and is inhibited when anchored. Therefore, we propose that AKAP79 targets these enzymes to sites just below the postsynaptic membrane where they are optimally positioned to become activated in response to the second messengers Ca2+ and cAMP and to phosphorylate neuronal substrates such as the AMPA/kainate or NMDA ion-channels. In order to test this hypothesis this proposal has two phases.
Aim 's 1-3 will conduct structure/function analysis of the AKAP79 signaling complex to define the protein-protein interactions that occur between the anchoring protein and calcineurin (aim-1), PLC (aim-2) and map the AKAP79-subcellular targeting domain (aim- 3). The functional significance of enzyme targeting via the anchoring protein will be tested in cultured hippocampal neurons in aim-4. This will be achieved by measuring changes in AMPA/kainate or NMDA evoked currents under conditions where AKAP79 derived peptides are used as reagents to inhibit CaN activity, inhibit PKC activity and disrupt targeting of the AKAP signaling complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048231-08
Application #
2900793
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1992-08-01
Project End
2000-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
8
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Murphy, Jonathan G; Sanderson, Jennifer L; Gorski, Jessica A et al. (2014) AKAP-anchored PKA maintains neuronal L-type calcium channel activity and NFAT transcriptional signaling. Cell Rep 7:1577-1588
Mercado, Jose; Baylie, Rachael; Navedo, Manuel F et al. (2014) Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle. J Gen Physiol 143:559-75
Gold, Matthew G; Fowler, Douglas M; Means, Christopher K et al. (2013) Engineering A-kinase anchoring protein (AKAP)-selective regulatory subunits of protein kinase A (PKA) through structure-based phage selection. J Biol Chem 288:17111-21
Smith, F Donelson; Scott, John D (2013) Scaffolding proteins: not such innocent bystanders. Curr Biol 23:R515-7
Scott, John D; Dessauer, Carmen W; Tasken, Kjetil (2013) Creating order from chaos: cellular regulation by kinase anchoring. Annu Rev Pharmacol Toxicol 53:187-210
Smith, F Donelson; Reichow, Steve L; Esseltine, Jessica L et al. (2013) Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation. Elife 2:e01319
Havekes, Robbert; Canton, David A; Park, Alan J et al. (2012) Gravin orchestrates protein kinase A and ?2-adrenergic receptor signaling critical for synaptic plasticity and memory. J Neurosci 32:18137-49
Kosenko, Anastasia; Kang, Seungwoo; Smith, Ida M et al. (2012) Coordinated signal integration at the M-type potassium channel upon muscarinic stimulation. EMBO J 31:3147-56
Altier, Christophe; Dubel, Stefan J; Barrere, Christian et al. (2012) AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit. Channels (Austin) 6:157-65
Gold, Matthew G; Reichow, Steve L; O'Neill, Susan E et al. (2012) AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency. EMBO Mol Med 4:15-26

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