Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal transduction.
We have shown previously that a 52-kDa phosphoprotein (p52) co-precipitated with Ig receptor (IgR)-associated MB-1 protein was inducibly phosphorylated by the stimulation with 12-O-tetradecanoyl-phorbol-13-acetate. By immunizing the 52-kDa protein co-precipitated with MB-1, we prepared a mAb (19-14), which can immunoprecipitate the p52 and the associated kinase molecule. Immune complex kinase assay with the 19-14 mAb showed that the p52 is associated with a novel kinase molecule and is involved in IgR-mediated signal transduction. Here we isolated a cDNA clone (alpha 4) from a murine bone marrow cDNA library in lambda gt-11 vector by the 19-14 mAb. The alpha 4 cDNA encodes a novel protein of 340 amino acids with multiple potential phosphorylation sites. The 1.4-kb alpha 4 mRNA is expressed in various cells including cell lines of B lineage, T lineage, monocytic, fibroblast, and normal organs such as liver, spleen, thymus, and brain. To study the molecule encoded by the alpha 4 cDNA, we produced a chimeric protein of the glutathione S transferase (GST)-alpha 4 in pGEX-3X expression vector. The 19-14 mAb binds to the GST-alpha 4 fusion protein. Rabbit anti-alpha 4 Ab prepared by immunizing the GST-alpha 4 fusion protein immunoprecipitated a 52-kDa phosphoprotein from WEHI-231 cells. The 52-kDa phosphoprotein immunoprecipitated by the anti-alpha 4 Ab is tightly associated with kinase activity that is similarly observed with the p52 protein reported previously. Moreover, the 52-kDa phosphoprotein immunoprecipitated with the anti-alpha 4 Ab is identical to the p52 by the protein comparison on non-equilibrium pH gradient gel electrophoresis/SDS-PAGE, isoelectric focusing/SDS-PAGE, and V8 protease mapping. The 52-kDa phosphoprotein is associated with functional components that are inducibly phosphorylated by anti-IgM stimulation. These results suggested that the alpha 4 gene-encoded molecule is functionally involved in the IgR-mediated signal transduction in B cells.
Inui
S
S
Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Japan.
Kuwahara
K
K
Mizutani
J
J
Maeda
K
K
Kawai
T
T
Nakayasu
H
H
Sakaguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics* / Q000276:immunology; D011233:Precipitin Tests / Q000379:methods; D011817:Rabbits; D011961:Receptors, Fc / Q000276:immunology*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D010750:Phosphoproteins; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C035311:immunoglobulin M receptor