Activation and serine phosphorylation of the p56lck protein tyrosine kinase in response to antigen receptor cross-linking in B lymphocytes.
We show that cross-linking the B cell AgR with anti-Ig Abs activates p56lck (Lck) in both the immature B cell line WEHI-231 and mature resting B cells from mouse spleen. Anti-Ig-stimulated Lck activity peaked after 1 to 2 min, but remained elevated for at least 15 min. Consistent with the proposed role for src family tyrosine kinases in AgR signaling, we found that Lck could phosphorylate the cytoplasmic tails of the Ig-alpha and Ig-beta components of the B cell AgR in vitro. Lck phosphorylated both of the tyrosines in the Ig-beta AgR homology motif and one of the two tyrosines in the Ig-alpha AgR homology motif. Finally, we show that AgR ligation in B cells caused a significant portion of the Lck to migrate with an apparent molecular mass of 60 kDa on SDS-PAGE gels. Conversion of p56lck to p60lck was maximal at 5 to 15 min, at which times Lck activity in the cells was decreasing. This Lck "band shift" has been observed previously in activated T cells and correlates with phosphorylation of Lck at serine 59. We show that the 60-kDa form of Lck induced by AgR cross-linking in B cells is also phosphorylated at serine 59. Phosphorylation of Lck at this site in vitro decreases its activity. Thus, in B cells, AgR cross-linking activates Lck and subsequently activates a kinase that phosphorylates Lck at serine 59, a potential negative regulatory site.
Gold
M R
MR
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Chiu
R
R
Ingham
R J
RJ
Saxton
T M
TM
van Oostveen
I
I
Watts
J D
JD
Affolter
M
M
Aebersold
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000276:immunology; D015151:Immunoblotting; D007136:Immunoglobulins / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013154:Spleen / Q000166:cytology
D007136:Immunoglobulins; D011947:Receptors, Antigen, B-Cell; D012694:Serine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)