The SH3 Domain-binding T Cell Tyrosyl Phosphoprotein p120

Abstract

Previously, we have identified p120 as a Fyn/Lck SH3 and SH2 domain-binding protein that is tyrosine phosphorylated rapidly after T cell receptor triggering. Here, we used direct protein purification, amino acid sequence analysis, reactivity with antibodies, and two-dimensional gel analyses to identify p120 as the human c-cbl protooncogene product. We demonstrate in vivo complexes of p120cbl with Fyn tyrosine kinase, the adaptor protein Grb2, and the p85 subunit of phosphatidylinositol (PI) 3-kinase. The association of p120cbl with Fyn and the p85 subunit of PI 3-kinase (together with PI 3-kinase activity) was markedly increased by T cell activation, consistent with in vitro binding of p120cbl to their SH2 as well as SH3 domains. In contrast, a large fraction of p120cbl was associated with Grb2 prior to activation, and this association did not change upon T cell activation. In vitro, p120cbl interacted with Grb2 exclusively through its SH3 domains. These results demonstrate a novel Grb2-p120cbl signaling complex in T cells, distinct from the previously analyzed Grb2-Sos complex. The association of p120cbl with ubiquitous signaling proteins strongly suggests a general signal transducing function for this enigmatic protooncogene with established leukemogenic potential but unknown physiological function.

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

“…A possibility could not be excluded, however, for other signaling roles of Cbl, which is capable of binding to SH3 domains of intracellular proteins via its proline-rich motifs [36,37]. In fact, Grb2/Ash coprecipitated with Cbl from lysates of stimulated or unstimulated THP-1 cells (unpublished observation), in accordance with previous reports on T-cell lines [12,15]. Such adaptor proteins might mediate Ras-MAP kinase cascades via Sos, an activator of small GTP-binding proteins.…”

“…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].…”

Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase

“…GST constructs of Lyn have been found to associate with and activate PI 3-kinase ( Pleiman et al, 1994). Lyn and other Srcrelated PTK have been found to associate with Cbl (Cory et al, 1995;Fukazawa et al, 1995). A role for the su ciency of an SH3 ± Cbl interaction has been reported in studies with a GST fusion protein of Nck's SH3 (Rivero-Lezcano et al, 1995).…”

“…The adaptor protein Cbl, when expressed as an oncoprotein, results in B cell and myeloid leukemias (Langdon et al, 1989). Because Cbl has been found to co-precipitate with p85, cytosolic and receptor-encoded protein tyrosine kinases (Cory et al, 1995;Fukazawa et al, 1995;Hartley et al, 1995;Kim et al, 1995;Solto and Cantley, 1996), it is possible that dysregulation of PI 3-kinase may be oncogenic.…”