CD40 signaling pathway: anti-CD40 monoclonal antibody induces rapid dephosphorylation and phosphorylation of tyrosine-phosphorylated proteins including protein tyrosine kinase Lyn, Fyn, and Syk and the appearance of a 28-kD tyrosine phosphorylated protein.
CD40 plays an important role in B cell activation, proliferation, and Ig class switching. The signal transduction pathway mediated by CD40 was studied using monoclonal antibody (mAb) 626.1 to CD40. Burkitt's lymphoma and Epstein-Barr virus-transformed B cell lines and tonsilar B lymphocytes were treated with the anti-CD40 mAb for various lengths of time. The early events triggered by CD40 were examined by monitoring the changes in tyrosine phosphorylation of cellular proteins with anti-phosphotyrosine mAb. Dephosphorylation of specific proteins ranging between 50-110 kD and the appearance of a 28-kD tyrosine phosphorylated protein were seen within 30 s in human B cell lines. The dephosphorylation was reversed and the 28-kD protein was dephosphorylated in cells stimulated for 1 min. In resting B cells, the appearance of the 28-kD phosphoprotein was observed in 30 s after the addition of the anti-CD40 mAb. The tyrosine phosphorylation of this protein persisted. The patterns of protein tyrosine phosphorylation differed from those induced by an anti-immunoglobulin M mAb. The changes in the state of tyrosine phosphorylation induced by the anti-CD40 mAb were obviated by mAb to CD45, a protein tyrosine phosphatase (PTP) or by the addition of sodium orthovanadate, a broad PTP inhibitor. They were also blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, and PKC and protein serine/threonine kinase inhibitors, H7 and HA1004. In addition, the alteration in the tyrosine phosphorylation of PTKs Lyn, Fyn, and Syk was directly demonstrated. Engagement of CD40 for 30 s induced a transient decrease in tyrosine phosphorylation of these PTKs. These results indicate that the early events in CD40 signaling involve the complex interaction between PTP and protein kinases.
Faris
M
M
Department of Internal Medicine, University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville 22908.
Gaskin
F
F
Parsons
J T
JT
Fu
S M
SM
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015703:Antigens, CD / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D001402:B-Lymphocytes; D016227:Benzoquinones; D002051:Burkitt Lymphoma; D019013:CD40 Antigens; D002460:Cell Line; D002461:Cell Line, Transformed; D004792:Enzyme Precursors / Q000378:metabolism*; D019833:Genistein; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008970:Molecular Weight; D014066:Palatine Tonsil / Q000276:immunology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D010879:Piperazines / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D013449:Sulfonamides; D000072377:Syk Kinase; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D016227:Benzoquinones; D019013:CD40 Antigens; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007529:Isoflavones; D007546:Isoquinolines; D047029:Lactams, Macrocyclic; D010750:Phosphoproteins; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D011809:Quinones; D013449:Sulfonamides; D017828:Rifabutin; D019000:Phosphotyrosine; D014443:Tyrosine; C020754:herbimycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; C000606896:SYK protein, human; D000072377:Syk Kinase; D017027:Protein Tyrosine Phosphatases