CD45 negatively regulates lyn activity by dephosphorylating both positive and negative regulatory tyrosine residues in immature B cells.
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-Igalpha 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.
Katagiri
T
T
Department of Microbiology and Immunology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan.
Ogimoto
M
M
Hasegawa
K
K
Arimura
Y
Y
Mitomo
K
K
Okada
M
M
Clark
M R
MR
Mizuno
K
K
Yakura
H
H
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D002454:Cell Differentiation / Q000276:immunology; D002999:Clone Cells; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology*; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000502:physiology*; D046911:Macromolecular Substances; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D046911:Macromolecular Substances; D011947:Receptors, Antigen, B-Cell; D019000:Phosphotyrosine; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D017493:Leukocyte Common Antigens