The regulation of signaling and cytoskeletal rearrangements in B-lymphocytes

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Interactions among signaling pathways that link antigen receptors to remodeling of the actin cytoskeleton are not completely understood. The initiation of signaling involves receptors that nucleate the formation of "signalosomes" that contain adaptor proteins, kinases, phosphatases, and their targets. Previous work described and analyzed the role of the B-cell antigen receptor (BCR) as the key signaling receptor that regulates B-lymphocyte (B cell) activation, development, and antigen gathering, responses requiring actin rearrangements. We discovered that the gap junction protein connexin43 (Cx43) that forms channels between cells and the environment, also has key non-channel functions, due to its unique carboxyl-tail (CT). These roles include regulating the actin cytoskeleton, through mechanisms that are unclear. Our goal is to understand how receptor signaling is coupled to scaffolding/adaptor proteins that regulate actin rearrangements, using B cells as a model. We will test the hypothesis that BCR signaling is influenced by Cx43 expression and that they both work collaboratively with interacting proteins to regulate cytoskeletal responses. Aim 1. To determine how Cx43 effects BCR signaling. Aim 2. To determine how the BCR and Cx43 are organized at the plasma membrane. Aim 3. To determine the effects of the cytoplasmic tail (CT) of Cx43 on rearrangements of the cytoskeleton in B cells. We propose that Cx43 acts as a scaffolding or adaptor protein to regulate either receptor organization in the membrane, or in the process of successive breakdown and rearrangement of actin to promote changes in the cytoskeleton. We have used a genetic mutational approach to identify important amino acids in the Cx43 CT and made a series of deletions and point mutations, many normally phosphorylated.  We have defined the region of the Cx43 CT between amino acids 246 to 307 that is necessary for BCR-mediated cell spreading in our model, as well as identified critical tyrosines and serines in the CT that when mutated inhibit cytoskeletal rearrangements. We will focus on a subset of the mutants. We predict that these targeted residues in the CT are sites of critical protein interactions that are nucleated by Cx43 and link receptors to the machinery that controls cytoskeletal architecture. Since cytoskeletal rearrangements are common to all cell types, we propose that we will gain an overall understanding of the complex interplay between similar families of proteins and the actin cytoskeletal network. In addition to B cells, Cx43 has been shown to be important for neuronal cell migration, cardiac and artery development, and invasiveness of cancer cells during tumor progression. The common features of Cx43's influence in different cell and organ systems suggests that it is involved in fundamental mechanisms that underlie cytoskeletal functions, and our studies will reveal interactions that are important for these basic effects.