The spatial and temporal organization of signal transduction pathways influences the precision and fidelity of intracellular events. We have discovered a family of non-catalytic regulatory elements called A-Kinase Anchoring Proteins (AKAPs) that bring together different combinations of calcium- and cAMP-responsive protein kinases and phosphatases to customize the regulation of effector proteins. This application focuses on the molecular pathophysiology of a multivalent anchoring protein known as AKAP79/150 (AKAP79 is the human form, AKAP150 is the murine ortholog). We discovered this protein and defined binding sites for protein kinase A (PKA), the phosphatase PP2B and conventional isoforms of protein kinase C (PKC). Physiological studies have demonstrated that AKAP79/150-tethered enzymes actively participate in the control of glucose homeostasis and coordinate certain extra pancreatic aspects of insulin action. This application is developed around exciting data pertaining to the role of anchored PKC in signaling events that govern arterial constriction and hypertension. Molecular events underlying this pathological state include aberrant calcium influx through ion channels that are controlled, in part, by AKAP79/150-associated PKC. The hypothesis to be tested is that manipulation of anchored PKC affords a measure of vascular benefit to alleviate diabetes-induced hypertension.
Two specific aims are proposed:
Aim 1 : How are individual AKAP79 complexes configured? We will harness three cutting-edge approaches: a) single-molecule pull-down photobleaching (SiMPull) to calculate the range of enzyme combinations on individual anchoring proteins, b) single-particle electron microscopy (EM) to obtain near-atomic structures of higher-order AKAP79 assemblies and c) super resolution imaging of native AKAP-enzyme complexes in vascular smooth muscle.
Aim 2 : Does PKC-anchoring govern diabetes-related hypertension? Sixty-five percent of diabetics develop hypertension. Our AKAP150-/- and AKAP150?PKC knockin mice exhibit lower basal blood pressure suggesting that AKAP-associated PKC participates in the control of vascular tone.
Aim 2 will test if selective disruption of this protein-protein interactin reduces vascular tone in mouse models of diabetic hypertension.

Public Health Relevance

Sixty five percent of diabetics develop hypertension, an increase in vascular tone and contractility of smooth muscle. A-Kinase Anchoring Protein (AKAP) associated protein kinase C has been implicated in the increase vascular tone in arteries. Manipulation of this anchored kinase may provide a measure of vascular benefit to alleviate diabetes-induced hypertension. The ultimate goal of this proposal is to develop molecular strategies to combat this diabetic complication and relieve hypertension.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK105542-03
Application #
9211325
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Silva, Corinne M
Project Start
2015-02-10
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
3
Fiscal Year
2017
Total Cost
$305,633
Indirect Cost
$103,133
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Smith, F Donelson; Esseltine, Jessica L; Nygren, Patrick J et al. (2017) Local protein kinase A action proceeds through intact holoenzymes. Science 356:1288-1293
Nystoriak, Matthew A; Nieves-CintrĂ³n, Madeline; Patriarchi, Tommaso et al. (2017) Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes. Sci Signal 10:
Li, Lei; Li, Jing; Drum, Benjamin M et al. (2017) Loss of AKAP150 promotes pathological remodelling and heart failure propensity by disrupting calcium cycling and contractile reserve. Cardiovasc Res 113:147-159
Nygren, Patrick J; Mehta, Sohum; Schweppe, Devin K et al. (2017) Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity. Elife 6:
Aggarwal-Howarth, Stacey; Scott, John D (2017) Pseudoscaffolds and anchoring proteins: the difference is in the details. Biochem Soc Trans 45:371-379
Nieves-CintrĂ³n, Madeline; Hirenallur-Shanthappa, Dinesh; Nygren, Patrick J et al. (2016) AKAP150 participates in calcineurin/NFAT activation during the down-regulation of voltage-gated K(+) currents in ventricular myocytes following myocardial infarction. Cell Signal 28:733-40
Kountz, Timothy S; Lee, Kyung-Soon; Aggarwal-Howarth, Stacey et al. (2016) Endogenous N-terminal Domain Cleavage Modulates ?1D-Adrenergic Receptor Pharmacodynamics. J Biol Chem 291:18210-21
Riggle, Kevin M; Riehle, Kimberly J; Kenerson, Heidi L et al. (2016) Enhanced cAMP-stimulated protein kinase A activity in human fibrolamellar hepatocellular carcinoma. Pediatr Res 80:110-8
Whiting, Jennifer L; Ogier, Leah; Forbush, Katherine A et al. (2016) AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption. Proc Natl Acad Sci U S A 113:E4328-37
Cherry, Allison E; Haas, Brian R; Naydenov, Alipi V et al. (2016) ST-11: A New Brain-Penetrant Microtubule-Destabilizing Agent with Therapeutic Potential for Glioblastoma Multiforme. Mol Cancer Ther 15:2018-29

Showing the most recent 10 out of 22 publications