Identification of serine residues in the connexin43 carboxyl tail important for BCR-mediated spreading of B-lymphocytes.

B-lymphocytes recognize antigen via B-cell antigen receptors (BCRs). This binding induces signaling, leading to B-cell activation, proliferation and differentiation. Early events of BCR signaling include reorganization of actin and membrane spreading, which facilitates increased antigen gathering. We have previously shown that the gap junction protein connexin43 (Cx43; also known as GJA1) is phosphorylated upon BCR signaling, and its carboxyl tail (CT) is important for BCR-mediated spreading. Here, specific serine residues in the Cx43 CT that are phosphorylated following BCR stimulation were identified. A chimeric protein containing the extracellular and transmembrane domains of CD8 fused to the Cx43 CT was sufficient to support cell spreading. Cx43 CT truncations showed that the region between amino acids 246-307 is necessary for B-cell spreading. Site-specific serine-to-alanine mutations (S255A, S262A, S279A and S282A) resulted in differential effects on both BCR signaling and BCR-mediated spreading. These serine residues can serve as potential binding sites for actin remodeling mediators and/or BCR signaling effectors; therefore, our results may reflect unique roles for each of these serines in terms of linking the Cx43 CT to actin remodeling.

The role of the gap junction protein connexin43 in B lymphocyte motility and migration.

The gap junction family of proteins is widely expressed in mammalian cells and form intercellular channels between adjacent cells, as well as hemichannels, for transport of molecules between the cell and the surrounding environment. In addition, gap junction proteins have recently been implicated as important for the regulation of cell adhesion and migration in a variety of cell types. The gap junction protein connexin43 (Cx43) regulates B lymphocyte adhesion, BCR- and LFA-1-mediated activation of the GTPase Rap1, and cytoskeletal rearrangements resulting in changes to cell shape and membrane spreading. We demonstrate here that the actin cytoskeleton is important for the distribution of Cx43 in the B cell plasma membrane and for other cell processes involving the cytoskeleton. Using shRNA knockdown of Cx43 in B lymphoma cells we show that Cx43 is also necessary for chemokine-mediated Rap 1 activation, motility, CXCL12-directed migration, and movement across an endothelial cell monolayer. These results demonstrate that in addition to its role in B cell spreading, Cx43 is an important regulator of B-cell motility and migration, processes essential for normal B-cell development and immune responses.

Mutations of Cx43 that affect B cell spreading in response to BCR signaling.

The gap junction (GJ) protein connexin 43 (Cx43) is both necessary and sufficient for B cell receptor (BCR)-mediated cell spreading. To address how Cx43 mediates this effect, we blocked its function genetically, by expressing mutants of Cx43, and pharmacologically, by using chemical inhibitors. While various point mutations of Cx43 inhibited B cell spreading, treatment with channel blocking drugs did not, suggesting that this response was independent of channel function. The critical region of Cx43 appears to be the cytoplasmic carboxyl-terminal (CT) domain, which has previously been shown to be important for B cell spreading. Consistent with this, mutations of either tyrosine 247 or 265 found in the CT were sufficient to inhibit spreading. Thus Cx43 may influence B cell spreading by mechanisms requiring protein binding to, or modification of, these sites in the CT tail.