Stimulation of the high-affinity IgE receptor results in the tyrosine phosphorylation of a 60 kD protein which is associated with the protein-tyrosine kinase, Csk.
The protein tyrosine kinase Csk downregulates the activity of the Src family of kinases and has a negative effect on signal transduction through several Src kinase-associated receptors. Because the Src-family kinase Lyn plays a pivotal role in FcepsilonRI-mediated cellular activation, we examined whether Csk is involved in FcepsilonRI signaling events. Using anti-Csk antibodies and recombinant fusion proteins we detected a single tyrosine-phosphorylated protein of 60 kD (herein referred to as 'p60') that associates with the SH2 domain of Csk after stimulation of the FcepsilonRI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggregated; disaggregation of the receptors resulted in rapid dephosphorylation of p60. The phosphorylation of p60 was only detected after activation by IgE and antigen and not by stimulation with PMA and/or ionomycin. Phosphorylated p60 was associated entirely with the membrane fraction of the cells. A considerable fraction of Csk was associated with the membrane in both unstimulated and stimulated cells, this fraction did not change upon activation. p60 coprecipitated with Csk from both unstimulated and FcepsilonRI stimulated cells and was phosphorylated by the immunocomplex. Total kinase activity of Csk immunoprecipitates increased upon FcepsilonRI stimulation. p60 did not react with antibodies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62dok. Our data demonstrate that Csk associates with a membrane-anchored protein complex that is directly involved in FcepsilonRI signal transduction.
Rafnar
T
T
Division of Clinical Immunology, Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA. rafnar@krabb.is
Peebles
R S
RS
Brummet
M E
ME
Catipović
B
B
Imani
F
F
MacGlashan
D W
DW
Marsh
D G
DG
D000818:Animals; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D004268:DNA-Binding Proteins; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000276:immunology; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017455:Receptors, IgE / Q000502:physiology*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000737:chemistry; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000502:physiology; D019061:src-Family Kinases / Q000032:analysis / Q000276:immunology / Q000502:physiology*
D004268:DNA-Binding Proteins; C074561:GAP-associated protein p62; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D015688:Oncogene Protein pp60(v-src); D019061:src-Family Kinases