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*
Department of Microbiology and Immunology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan;
Institute for Protein Research, Osaka University, Suita, Japan; and
University of Chicago School of Medicine, Chicago, IL 60637
Using CD45-deficient clones from the immature B cell line,
WEHI-231, we previously demonstrated that CD45 selectively
dephosphorylates the Src-family protein tyrosine kinase Lyn and
inhibits its kinase activity. To further define the mechanisms of CD45
action on Lyn, we metabolically labeled Lyn from CD45-positive and
-negative WEHI-231 cells and analyzed cyanogen bromide fragments by
SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is
tyrosine phosphorylated with little serine or threonine
phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa
were phosphorylated in the absence of B cell Ag receptor (BCR)
ligation. The 8.2-kDa band corresponded to a fragment containing the
positive regulatory site (Tyr397), as assessed by its size
and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band
was phosphorylated by COOH-terminal Src kinase, suggesting that it
contains the COOH-terminal negative regulatory site
(Tyr508). CD45 was also shown to dephosphorylate
autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only
the negative but also the positive regulatory tyrosine residues of Lyn.
Furthermore, coimmunoprecipitations using anti-Ig
Ab
demonstrated that Lyn associated with the resting BCR was
constitutively phosphorylated and activated in CD45-negative cells. In
the parental cells, both regulatory sites were phosphorylated on BCR
ligation. Taken collectively, these results suggest that CD45 keeps
both BCR-associated and total cytoplasmic pools of Lyn in an inactive
state, and a mechanism by which Lyn is activated by relative reduction
of CD45 effect may be operative on BCR ligation.
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