Association of tyrosine and serine kinases with the B cell surface antigen CD20. Induction via CD20 of tyrosine phosphorylation and activation of phospholipase C-gamma 1 and PLC phospholipase C-gamma 2.
CD20 is a B cell-specific 35/37 kDa integral membrane protein which modulates proliferation and differentiation of normal resting B cells when stimulated by CD20 antibodies. An increase in c-myc mRNA levels occurs within hours after treatment of resting B cells with CD20 mAb; however earlier events in the CD20 signal transduction pathway have not been described. Here we demonstrate that anti-CD20 mediated induction of c-myc mRNA is inhibited by the tyrosine kinase inhibitor herbimycin A, that CD20 is associated with both tyrosine and serine kinase activity, and that tyrosine phosphorylation of multiple substrates is induced within minutes upon ligation of CD20 with mAb. Association of the tyrosine and serine kinases with CD20 was stable in lysis buffer containing 1% NP40 and 0.25% deoxycholate. Under the same conditions, antibodies against several other B cell surface molecules failed to co-precipitate tyrosine kinase activity, however, a serine kinase was precipitated by the anti-CD19 mAb, B43. Both phospholipase C-gamma 1 and -gamma 2 were phosphorylated on tyrosine after cross-linking of CD20-bound mAb, and this correlated with increases in intracellular calcium that were partially resistant to depletion of extracellular calcium with EGTA. The pattern of tyrosine phosphorylated proteins observed in whole cell lysates after anti-CD20 cross-linking appeared to be a subset of those induced by anti-IgM; however, differences in phosphoproteins induced by anti-IgM and anti-CD20 were detected using a fynSH2-fusion protein.
Deans
J P
JP
Department of Microbiology, University of Washington, Seattle 98195.
Schieven
G L
GL
Shu
G L
GL
Valentine
M A
MA
Gilliland
L A
LA
Aruffo
A
A
Clark
E A
EA
Ledbetter
J A
JA
D015703:Antigens, CD / Q000502:physiology*; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte / Q000502:physiology*; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D016227:Benzoquinones; D002478:Cells, Cultured; D004789:Enzyme Activation; D016259:Genes, myc; D006801:Humans; D007075:Immunoglobulin M / Q000502:physiology; D047029:Lactams, Macrocyclic; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011809:Quinones / Q000494:pharmacology; D011947:Receptors, Antigen, B-Cell / Q000502:physiology; D017828:Rifabutin / Q000031:analogs & derivatives; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D015703:Antigens, CD; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte; D016227:Benzoquinones; D007075:Immunoglobulin M; D047029:Lactams, Macrocyclic; D011809:Quinones; D011947:Receptors, Antigen, B-Cell; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D010738:Type C Phospholipases