Regulation of the Association of p120cbl with Grb2 in Jurkat T Cells*
- From the ‡ CBMB/NICHD, National Institutes of Health, Bethesda, Maryland, 20892 and the
- § University of Western Australia, Nedlands, Western Australia, 6009, Australia
- ¶ To whom correspondence should be addressed: NIH, Bldg. 18T, Rm. 101, Bethesda, MD 20892. Tel.: 301-496-5216; Fax: 301-402-0078.
Abstract
The c-cbl protooncogene product (p120cbl) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120cbl complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120cbl and other substrates. This dissociation persists in vivo even when p120cbl becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120cbl from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120cbl does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120cbl to map the p120cbl-Grb2 interaction to amino acids 481-528 of p120cbl; this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120cbl complex further supports the idea of a significant role for p120cbl in receptor-mediated signaling pathways.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- TCR
-
T-cell receptor
- PTK
-
protein- tyrosine kinase
- GST
-
glutathione S-transferase
- HA
-
hemagglutinin
- PAGE
-
polyacrylamide gel electrophoresis.
-
- Received June 26, 1996.
- Revision received August 2, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
