To address whether B cells expressing a disease-associated autospecificity are regulated in normal mice, we have established a rheumatoid factor (RF) transgenic model of autoimmunity, using V genes derived from an IgA anti-IgG2a RF isolated from an autoimmune MRL/lpr mouse. As we wished to study induction of tolerance during B cell development, we cloned the VH gene into an IgM expression vector. The RF we chose binds only IgG2a of the 'a' allotype (IgG2a) but not IgG2ab allowing us to produce transgenic animals on IgHa and IgHb backgrounds, which either express or lack the self-antigen. Two transgenic lines were studied. Using mice which lack the self-antigen, we show by fluorescence activated cell sorting and hybridoma analysis that the H and L transgenes are expressed to the exclusion of endogenous genes in most splenic B cells. In spite of good allelic exclusion, transgenic mice which are genetically capable of expressing IgG2aa have reduced but significant (approximately 50 micrograms/ml) serum levels. Nonetheless, the frequency and numbers of transgene-expressing B cells in peripheral lymphoid organs of such mice which have the self-antigen are similar to those which lack it (IgHb mice). Thus, B cells expressing an anti-self IgG2a surface receptor can develop in this system. Whether such B cells are anergic or otherwise regulated in autoantigen-expressing mice is discussed.

The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono-or non-phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.

When cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.z 2000 Federation of European Biochemical Societies.