Regulation of the Association of p120 with Grb2 in Jurkat T Cells

Abstract

The c-cbl protooncogene product (p120(cbl)) 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-p120(cbl) 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 p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) 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 p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.

Specific association of phosphatidylinositol 3‐kinase with the protooncogene product Cbl in Fcγ receptor signaling

“…The phosphorylation of most of these proteins was observed within 15 s after stimulation and peaked at 1 min and then gradually decreased up to 16 min. Several lines of evidence have raised the possibility that tyrosine phosphorylation of Cbl might be involved in immune recognition systems in hematopoietic cells [11][12][13][14][15][16][17]. To test the tyrosine phosphorylation of Cbl upon FcvR stimulation, whole cell lysates were immunoprecipitated with anti-phosphotyrosine antibodies (PY20) and analyzed by blotting with anti-Cbl antibodies.…”

“…Although relatively few tyrosine phosphorylated peptides have as yet been identified, a protooncogene product, Cbl, has recently attracted attention among them [5]. Cbl is tyrosine phosphorylated upon stimulation of T-cell receptor, B-cell receptor and some cytokine receptors as well [11][12][13][14][15][16][17]. Some Src family tyrosine kinases are reported to be associated with Cbl in hematopoietic cells [5,12,15].…”

Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl

“…Cbl lacks a de®ned catalytic domain but is rapidly phosphorylated on tyrosine residues following the stimulation of a wide range of cell surface receptors which include growth factor receptors, immunoglobulin receptors, antigen receptors and integrin receptors (Donovan et al, 1994;Tanaka et al, 1995;Galisteo et al, 1995;Bowtell and Langdon, 1995;Odai et al, 1995;Marcilla et al, 1995;Wang et al, 1996;Cory et al, 1995;Kontani et al, 1996;Ojaniemi et al, 1997). Indeed in many cell types Cbl appears to be one of the most prominent and rapidly phosphorylated substrates of protein tyrosine kinases (Andoniou et al, 1994;Donovan et al, 1994;. This suggests a pivotal role in early events following signal transduction across the cell membrane into the cytoplasm.…”

“…This suggests a pivotal role in early events following signal transduction across the cell membrane into the cytoplasm. Furthermore, Cbl has been found to associate constitutively or inductively with many signalling proteins such as the Grb2, Crk and Nck adaptor proteins, members of the Src, Abl and Syk tyrosine kinase families, the p85 regulatory subunit of PI 3-kinase and 14-3-3 proteins (Donovan et al, 1994;de Jong et al, 1995;Buday et al, 1996;RiveroLezcano et al, 1994;Ribon et al, 1996;Andoniou et al, 1994Andoniou et al, , 1996Smit et al, 1996;Tanaka et al, 1996;Tsygankov et al, 1996;Fournel et al, 1996;Kim et al, 1995;Meisner et al, 1995;Solto and Cantley, 1996;Liu et al, 1996). To date however a de®nite function for Cbl has not emerged from these studies.…”

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2

“…Recently, c-Cbl has been reported to be highly phosphorylated upon stimulation through T cell receptor (TCR) (Donovan et al, 1994), B cell receptor (BCR) , Fcg receptor , granulocyte macrophage-colony stimulating factor receptor (GM-CSFR) and erythroprotein receptor (EPOR) (Odai et al, 1995). These data strongly suggest that c-Cbl is likely to play a major role in the events occurring downstream of hematopoietic receptors coupled to c-Src-like kinases.…”