P120cbl Is a Major Substrate of Tyrosine Phosphorylation upon B Cell Antigen Receptor Stimulation and Interacts in Vivo with Fyn and Syk Tyrosine Kinases, Grb2 and Shc Adaptors, and the p85 Subunit of Phosphatidylinositol 3-Kinase

Govindaswamy Panchamoorthy,Toru Fukazawa,Sachiko Miyake,Stephen Soltoff,Kris Reedquist,Brian Druker,Steve Shoelson,Lewis Cantley,Hamid Band

doi:10.1074/jbc.271.6.3187

Abstract

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

Highlights

We and others have demonstrated that the c-cbl protooncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation
Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling
The potential signal transduction role of the c-cbl protooncogene product was revealed by its cloning as an NCK SH3domain-binding protein [35], a prominent tyrosine phosphorylation of Cbl polypeptide upon triggering through the TCR [26], and identification of the Fyn/Lck SH3 domain-binding T cell phosphoprotein p120 as Cbl [23,24,25]

Summary

Introduction

We and others have demonstrated that the c-cbl protooncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. B cell activation induced a prominent association of Cbl with Syk tyrosine kinase. Grb and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. The CD3/␨ chains of the TCR and the CD4/8 co-receptors associate with the Src-family PTKs Fyn and Lck, respectively [3,4,5,6]. Upon tyrosine phosphorylation by the Src family kinases, the I-TAMs recruit the Syk/ZAP-70 PTKs in an activation-dependent manner (10 –14). Many of the identified effectors that function downstream of the PTKs in T and B cells are shared; these include phospholipase C-␥1, phosphatidylinositol (PI) 3-kinase, adaptor proteins Shc and Grb that couple receptors to Ras pathway, and the proto-oncogene product Vav [1, 2]. C-cbl resides on human chromosome 11-q23 near a translocation breakpoint found in about 10% of leukemias; a direct involvement of Cbl in the pathogenesis of these leukemias remains to be shown, it is interesting to note that a large proportion of these are of B or mixed cell lineage [31]

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Conclusion