Analysis of tyrosine phosphorylation-dependent interactions between stimulatory effector proteins and the B cell co-receptor CD22.
The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22-derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2.Sos complex was observed to bind exclusively to the fourth phosphotyrosine motif (Y828ENV) from CD22 and does so via a direct interaction based on Far Western and reverse Far Western blotting. Although both PLCgamma and PI3K were observed to bind to multiple phosphopeptides in precipitation experiments, subsequent studies using reverse Far Western blot analysis demonstrated that only the carboxyl-terminal phosphopeptide of CD22 (Y863VTL) binds directly to either one. This finding suggests that PLCgamma and PI3K may be recruited to CD22 either through a direct interaction with Tyr863 or indirectly through an association with one or more intermediate proteins.
Yohannan
J
J
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Wienands
J
J
Coggeshall
K M
KM
Justement
L B
LB
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D015815:Cell Adhesion Molecules; D003593:Cytoplasm / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism*; D037102:Lectins; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2; D015398:Signal Transduction; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; C497868:Cd22 protein, mouse; D015815:Cell Adhesion Molecules; D004792:Enzyme Precursors; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D037102:Lectins; D011506:Proteins; D011947:Receptors, Antigen, B-Cell; D051918:Sialic Acid Binding Ig-like Lectin 2; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C000606898:Syk protein, rat; D010738:Type C Phospholipases; D051966:Phospholipase C gamma