We hypothesize that AKAP abundance and localization within the cell may play a role in cellular differentiation processes through regulation of PKA holoenzyme activity. We will test this hypothesis by determining the changes in expression and distribution of candidate AKAPs and the corresponding changes in the activity and localization of PKA holoenzymes during cell differentiation. As a model system we will use L6 myoblast cell lines, because they are characterized by a well-established pattern of coordinate activation of muscle specific genes and the arrest of cell proliferation.
Specific Aim 1. We will identify AKAPs whose expression changes as myogenic differentiation occurs. To characterize those AKAPs that are potentially involved in the differentiation process, we will examine changes in AKAP expression during L6 myoblast differentiation using cell fractionation, and identify those differentially expressed AKAPs through traditional western blotting methods. To determine whether changes in AKAP localization and distribution are associated with the transition from myoblasts to myotubes, we will use immunolabeling of selected AKAPs and associated PKA, combined with co-localization with subcellular compartments using fluorescence microscopy. We will also examine mRNA expression during differentiation.
Specific Aim 2. We will test the hypothesis that changes in AKAP expression and localization influence the progression of myogenic differentiation. By transfecting myoblasts with the PKA blocking peptide Ht31, the effects of blocking PKA localization on development can be determined. We will then mutate the PKA binding domain on candidate AKAPs using site directed mutagnesis, and transfect these mutants into myoblasts. Alterations in differentiation patterns associated with the changes in PKA localization will be determined.by examining myosin heavy chain expression and formation of multinucleate myotubes. Knowledge of how scaffolding or anchoring proteins contribute to cellular differentiation programs will provide new opportunities to investigate how AKAP anchoring of PKA can influence cell differentiation. The project will also provide new opportunities for an underserved population of undergraduate researchers.
