Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells.
Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells.
Takagi
S
S
Department of Pharmacology, University of Massachusetts Medical School, Worcester 01655.
Daibata
M
M
Last
T J
TJ
Humphreys
R E
RE
Parker
D C
DC
Sairenji
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D002460:Cell Line; D003432:Cross-Linking Reagents; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D019833:Genistein; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007529:Isoflavones / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007529:Isoflavones; D011947:Receptors, Antigen, B-Cell; D019000:Phosphotyrosine; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases