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Scaffold proteins are important regulatory molecules for signal transduction. Protein scaffolds regulate the spatiotemporal signaling within a cell by providing a framework for the formation of multi-enzyme signaling complexes. These complexes are a key mechanism to sequester a signaling enzyme to a specific subcellular environment, thereby ensuring that the enzyme is near its relevant targets and preventing indiscriminate enzyme activity.
Work in the Carnegie laboratory is directed towards understanding scaffolding complexes mediated by A-Kinase Anchoring Proteins. AKAPs are a diverse family of scaffold proteins that form multi-protein complexes, integrating cAMP-signaling with protein kinases, phosphatases and other effector proteins.
Figure 1. Properties of A-Kinase Anchoring Proteins (AKAPs).
AKAPs nucleate signaling complexes composed of protein kinase A and additional signaling enzymes. AKAPs possess targeting sequences, directing signaling complexes to discrete subcellular locations.
Many AKAPs have been characterized in the heart, where they play a critical role in modulating cardiac function.
Figure 2. AKAPs coordinate signaling complexes in cardiac myocytes that function in cardiac remodeling.
The adult heart responds to injury or stress by activating a variety of intracellular signaling pathways that may affect calcium handling, the cytoskeleton, and sarcomeric and mitochondrial function. Different AKAPs in cardiac myocytes play a critical role in coordinating these signaling events to promote re-expression of an embryonic gene program, myocyte hypertrophy and extracellular matrix remodeling.
Currently, we are predominantly studying AKAP-Lbc (also known as AKAP13) in cardiac cellular function and pathogenesis. AKAP-Lbc cordinates signal transduction through multiple protein kinases and plays a role in pathological cardiac hypertrophy, which is often a major component underlying cardiovascular disease.
Figure 3. AKAP-Lbc coordinates hypertrophic signaling to promote cytoskeletal and gene remodeling.
AKAP-Lbc is present in the cytoplasm, displaying a cytoskeletal and perinuclear localization. This anchoring protein serves as a scaffold for PKA, PKD and its upstream activating kinase PKC. By bringing PKC and PKD into close proximity, AKAP-Lbc facilitates the phosphorylation and subsequent activation of PKD by PKC. Upon activation, PKD translocates to the nucleus promoting hypertrophic gene expression through phosphorylation of a histone deacetylase (HDAC5); leading to HDAC5 nuclear export and de-repression of Mef2-transcription.
AKAP-Lbc is a guanine nucleotide exchange factor (GEF) for Rho. The Rho-GEF activity of AKAP-Lbc is stimulated via the Gα12 family of heterotrimeric G-proteins in response to α1-adrenergic receptor (AR) activation. GEF activity can be inactivated by an AKAP-Lbc anchored PKA-dependent mechanism.
AKAP-Lbc also coordinates a p38a MAPK complex, downstream of Rho, composed of PKNα, MLTK, MKK3 and p38a. Presently, the downstream targets and functional consequences of this AKAP-Lbc-associated signaling cascade are unknown.
Figure 4. Immunocytochemical detection of AKAP-Lbc in rat neonatal ventricular myocytes.
AKAP-Lbc (blue), co-stained with the sarcomeric marker protein a-actinin (green) and a nuclear marker (red).
Using biochemistry, molecular biology, and live cell-imaging techniques, we are dissecting signaling through AKAP-Lbc. Our aim is to determine how the AKAP-Lbc complex coordinates multiple signals that may contribute to disease pathogenesis.