Molecular targets of CD45 in B cell antigen receptor signal transduction.
Expression of the phosphotyrosine phosphatase CD45 is essential for B cell Ag receptor (BCR)-mediated p21ras activation and calcium mobilization. To examine the molecular basis of this requirement, we analyzed signaling events following BCR ligation in CD45-deficient (CD45-) and CD45-reconstituted (CD45+) variants of J558Lmicrom3 cells. Ag stimulation resulted in tyrosine phosphorylation of cellular proteins in both cells. However, the spectrum of proteins phosphorylated in the CD45+ cells was qualitatively and/or quantitatively distinct from that in the CD45- cells. Among the protein tyrosine kinases examined, the Src family kinases Fyn and Blk were inducibly tyrosine phosphorylated and activated by receptor ligation only in CD45+ cells. While Ag-induced Btk tyrosine phosphorylation occurred in both cells, its activation was greatly diminished in the CD45- cells. Analysis of specific effector molecules revealed that tyrosine phosphorylation of Shc, but not rasGAP or Vav, correlated with the unique ability of BCR ligation to trigger p21ras activation in CD45+ cells. BCR-mediated Shc phosphorylation and recruitment of Grb2 depended on CD45 expression. Thus, Shc tyrosine phosphorylation may be the primary CD45-dependent mechanism by which Ag receptors are coupled to the p21ras pathway in J558Lmicrom3. In addition, phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 were tyrosine phosphorylated upon Ag stimulation in CD45- cells, despite much reduced inositol trisphosphate production and lack of calcium mobilization. These findings suggest that CD45 may modulate events other than PLCgamma phosphorylation, which regulate phosphoinositide hydrolysis and the calcium mobilization response following BCR ligation.
Pao
L I
LI
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206, USA.
Bedzyk
W D
WD
Persin
C
C
Cambier
J C
JC
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000077329:Agammaglobulinaemia Tyrosine Kinase; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D051380:GRB2 Adaptor Protein; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010954:Plasmacytoma; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D010738:Type C Phospholipases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018797:Cell Cycle Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C496618:VAV1 protein, human; D019000:Phosphotyrosine; D015544:Inositol 1,4,5-Trisphosphate; C082599:protein-tyrosine kinase p55(blk); D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D017493:Leukocyte Common Antigens; D010738:Type C Phospholipases