Manipulation of B cell antigen receptor tyrosine phosphorylation using aluminum fluoride and sodium orthovanadate

The B cell antigen receptor complex (BCR) is composed of a membrane-spanning immunoglobulin molecule (mIg) non-covalently associated with heterodimers of the transmembrane proteins Ig-α and Ig-β. The cytoplasmic domains of Ig-α and Ig-β do not contain kinase domains but are phosphorylated on tyrosine residues immediately upon receptor ligation. The mechanism and kinase responsible for initial Ig-α and Ig-β phosphorylation following receptor ligation is unknown. In an attempt to better understand this process, Ig-α and Ig-β phosphorylation was examined in response to treatment of permeabilized B cells with the pharmacologic agents, aluminum fluoride (AIF_x) and sodium orthovanadate (Na₃VO₄). AIF_x is known to stimulate GTP-binding proteins while Na₃VO₄ inhibits protein tyrosine phosphatases (PTPs), both of which are involved in the BCR signalling cascade. In these studies, AIF_x and Na₃VO₄ stimulated rapid tyrosine phosphorylation of Ig-α, Ig-β, and additional cellular proteins, including the protein tyrosine kinase (PTK) Lyn. The tyrosine phosphorylation does not appear to be mediated through GTP-binding proteins, since GTPγS did not stimulate tyrosine phosphorylation. As expected, however, PTPs modulate the phosphorylation state of these proteins since another PTP inhibitor, phenylarsine oxide (PAO), increased phosphorylation of Ig-α, Ig-β and other proteins in this system. Interestingly, the extent and kinetics of the mIg-associated Lyn and Ig-α Ig-β phosphorylation was correlated, suggesting that Lyn may mediate receptor phosphorylation. Alternatively, Lyn, may be a downstream effector of phosphorylated Ig-α and Ig-β as suggested by the reported ability of biphosphorylated Ig-α to activate Fyn PTK in vitro. Finally, all components necessary for Na₃VO₄, but not AIF_x, stimulation of phosphorylation are membrane associated. The data are consistent with modulation of phosphorylation of Ig-α and Ig-β through both PTP inhibition and AIF_x treatment, and a common intermediary in or effector of these phosphorylation pathways appears to be the Lyn kinase.