Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.
Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.
Ota
Y
Y
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-5430, USA.
Beitz
L O
LO
Scharenberg
A M
AM
Donovan
J A
JA
Kinet
J P
JP
Samelson
L E
LE
D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011518:Proto-Oncogene Proteins; D017455:Receptors, IgE; D011994:Recombinant Proteins; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C000606898:Syk protein, rat; D019061:src-Family Kinases; C067419:CBL protein, human; C493849:Cbl protein, mouse