Phosphorylation of Cbl following stimulation with interleukin-3 and its association with Grb2, Fyn, and phosphatidylinositol 3-kinase.
We have demonstrated that a 120-kDa protein, identified as Cbl, becomes rapidly phosphorylated on tyrosine residues following stimulation of factor-dependent cells with interleukin-3 (IL-3). Little or no phosphorylation of Cbl was observed in the absence of IL-3 stimulation and phosphorylation is maximal by 20-30 min after IL-3 stimulation. Association of Cbl with Grb2 was noted in unstimulated cells, and the amount of Cbl associated with Grb2 increased following IL-3 stimulation. The p85 subunit of phosphatidylinositol 3-kinase was constitutively associated with Cbl. Approximately 10% of the PI kinase activity present in anti-phosphotyrosine immunoprecipitates was present in anti-Cbl immunoprecipitates of IL-3-stimulated cells. The constitutive association of Cbl with Fyn was also observed. Cbl was observed to bind to bacterial fusion proteins encoding the unique, SH3, and SH2 domains of Fyn, Hck, and Lyn. The SH2 domain of Fyn alone was able to bind Cbl to nearly the same extent as did the fusion protein encoding the unique, SH3, and SH2 domains. This was not the case for the SH2 domain of Hck, however, as binding of the Hck fusion protein to Cbl appeared to require multiple domains. The binding of the fusion proteins to Cbl occurred regardless of whether Cbl was tyrosine-phosphorylated or not, and the binding could not be disrupted by the addition of 30 mM free phosphotyrosine. These data suggest the unexpected conclusion that the Fyn SH2 domain may bind to Cbl in a phosphotyrosine-independent manner.
Anderson
S M
SM
University of Colorado Health Sciences Center, Department of Pathology, Denver 80262, USA. steve.anderson@uchsc.edu
Burton
E A
EA
Koch
B L
BL
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D051572:Proto-Oncogene Proteins c-hck; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007377:Interleukin-3; D011506:Proteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; C496632:Hck protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases