Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.
Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation.
Minoguchi
K
K
Laboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
Benhamou
M
M
Swaim
W D
WD
Kawakami
Y
Y
Kawakami
T
T
Siraganian
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology* / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D051381:Rats; D017455:Receptors, IgE / Q000302:isolation & purification / Q000378:metabolism*; D000072377:Syk Kinase; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D000906:Antibodies; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D010750:Phosphoproteins; D017455:Receptors, IgE; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat