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-sp anning immuno-globulin molecule (mIg) non-covalently associated with h eterodimers of the transmembrane proteins Ig-alpha and Ig-beta. The cy toplasmic domains of Ig-alpha and Ig-beta do not contain kinase domain s but are phosphorylated on tyrosine residues immediately upon recepto r ligation. The mechanism and kinase responsible for initial Ig-alpha and Ig-beta phosphorylation following receptor ligation is unknown. In an attempt to better understand this process, Ig-alpha and Ig-beta ph osphorylation was examined in response to treatment of permeabilized B cells with the pharmacologic agents, aluminum fluoride (AlFx) and sod ium orthovanadate (Na3VO4). AlFx is known to stimulate GTP-binding pro teins while Na3VO4 inhibits protein tyrosine phosphatases (PTPs), both of which are involved in the BCR signalling cascade. In these studies , AlFx and Na3VO4 stimulated rapid tyrosine phosphorylation of Ig-alph a, Ig-beta, and additional cellular proteins, including the protein ty rosine kinase (PTK) Lyn. The tyrosine phosphorylation does not appear to be mediated through GTP-binding proteins, since GTP gamma S did not stimulate tyrosine phosphorylation. As expected, however, PTPs modula te the phosphorylation state of these proteins since another PTP inhib itor, phenylarsine oxide (PAO), increased phosphorylation of Ig-alpha, Ig-beta and other proteins in this system. Interestingly, the extent and kinetics of the mIg-associated Lyn and Ig-alpha/Ig-beta phosphoryl ation was correlated, suggesting that Lyn may mediate receptor phospho rylation. Alternatively, Lyn, may be a downstream effector of phosphor ylated Ig-alpha and Ig-beta as suggested by the reported ability of bi phosphorylated Ig-alpha to activate Fyn PTK in vitro. Finally, all com ponents necessary for Na3VO4, but not AIF(x), stimulation of phosphory lation are membrane associated. The data are consistent with modulatio n of phosphorylation of Ig-alpha and Ig-beta through both PTP inhibiti on and AlFx treatment, and a common intermediary in or effector of the se phosphorylation pathways appears to be the Lyn kinase.