Phosphorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody.
Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcepsilonRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcepsilonRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcepsilonRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivo and in vitro. In FcepsilonRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.
Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA. Lzhang@yoda.nidr.nih.gov
J
J
Zhang
M L
ML
Billingsley
R L
RL
Kincaid
R P
RP
Siraganian
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism* / Q000502:physiology*; D006632:Histamine / Q000378:metabolism; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008407:Mast Cells / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism* / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction; D000072377:Syk Kinase; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D014443:Tyrosine; D006632:Histamine; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat