Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
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Antibodies, Monoclonal / pharmacology*
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Antigens, CD / drug effects
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Antigens, CD / immunology
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Antigens, CD / physiology*
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Antigens, Differentiation, B-Lymphocyte / drug effects
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Antigens, Differentiation, B-Lymphocyte / immunology
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Antigens, Differentiation, B-Lymphocyte / physiology*
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B-Lymphocytes
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Benzoquinones
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Burkitt Lymphoma
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CD40 Antigens
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Cell Line
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Cell Line, Transformed
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Enzyme Precursors / metabolism*
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Genistein
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Herpesvirus 4, Human / genetics
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Humans
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Intracellular Signaling Peptides and Proteins
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Isoflavones / pharmacology
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Isoquinolines / pharmacology
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Lactams, Macrocyclic
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
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Molecular Weight
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Palatine Tonsil / immunology
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Phosphoproteins / isolation & purification
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Phosphoproteins / metabolism*
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Phosphorylation
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Phosphotyrosine
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Piperazines / pharmacology
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Protein Tyrosine Phosphatases / antagonists & inhibitors
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Protein Tyrosine Phosphatases / metabolism
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Protein-Tyrosine Kinases / antagonists & inhibitors
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Protein-Tyrosine Kinases / metabolism*
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-fyn
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Quinones / pharmacology
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Rifabutin / analogs & derivatives
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Sulfonamides*
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Syk Kinase
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Tumor Cells, Cultured
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Tyrosine / analogs & derivatives
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Tyrosine / analysis
Substances
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Antibodies, Monoclonal
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Antigens, CD
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Antigens, Differentiation, B-Lymphocyte
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Benzoquinones
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CD40 Antigens
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Enzyme Precursors
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Intracellular Signaling Peptides and Proteins
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Isoflavones
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Isoquinolines
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Lactams, Macrocyclic
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Phosphoproteins
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Piperazines
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Proto-Oncogene Proteins
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Quinones
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Sulfonamides
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Rifabutin
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Phosphotyrosine
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Tyrosine
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herbimycin
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1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
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N-(2-guanidinoethyl)-5-isoquinolinesulfonamide
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Genistein
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Protein-Tyrosine Kinases
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FYN protein, human
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
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Proto-Oncogene Proteins c-fyn
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SYK protein, human
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Syk Kinase
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Protein Tyrosine Phosphatases