FcgammaRII tyrosine phosphorylation differs between FcgammaRII cross-linking and platelet-activating anti-platelet monoclonal antibodies.
Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.
Qi
R
R
Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, Japan.
Ozaki
Y
Y
Asazuma
N
N
Satoh
K
K
Yatomi
Y
Y
Law
C L
CL
Hato
T
T
Nomura
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology*; D001667:Binding, Competitive; D001792:Blood Platelets / Q000276:immunology*; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015539:Platelet Activation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D017452:Receptors, IgG / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000072377:Syk Kinase; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D004792:Enzyme Precursors; C080568:Fc gamma receptor IIA; D047908:Intracellular Signaling Peptides and Proteins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase