Fyn Associates with Cbl and Phosphorylates Tyrosine 731 in Cbl, A Binding Site for Phosphatidylinositol 3-Kinase

Seija Hunter,Elizabeth A Burton,Steven C Wu,Steven M Anderson

doi:10.1074/jbc.274.4.2097

Abstract

We have investigated the interaction between Cbl and the Src-related tyrosine kinase Fyn. Fyn was observed to be constitutively associated with Cbl in lysates of several different cell types including the interleukin-3-dependent murine myeloid cell line 32Dcl3, and the prolactin-dependent rat thymoma cell line Nb2. Binding studies indicated that Cbl could bind to glutathione S-transferase (GST) fusion proteins encoding the unique, Src homology domain 3 (SH3), and SH2 domains of Fyn, Hck, or Lyn. Fusion proteins encoding either the SH3 or SH2 domains of Fyn bound to Cbl as effectively as the fusion protein encoding the unique, SH3, and SH2 domains of Fyn. The Fyn SH2 domain bound to both tyrosine-phosphorylated and nonphosphorylated Cbl, implying that this interaction might be phosphotyrosine-independent. Binding of the Fyn SH2 domain to Cbl was not disrupted by the addition of phosphotyrosine, phosphoserine, or phosphothreonine. A GST fusion protein encoding the proline-rich region of Cbl bound to Fyn present in a total cell lysate. Far Western blot analysis also indicated that the SH3 domain of Fyn bound preferentially to the proline-rich region of Cbl. The addition of [gamma-32P]ATP to either anti-Cbl immunoprecipitates or anti-Fyn immunoprecipitates resulted in the phosphorylation of both Cbl and Fyn as demonstrated by immunoprecipitation of the phosphorylated proteins with specific antisera. Fyn directly phosphorylated a GST fusion protein containing the C-terminal region of Cbl (GST-CBL-LZIP). In contrast, immunoprecipitated JAK2 was not able to phosphorylate this same region of Cbl. The GST-CBL-LZIP fusion protein contains a binding site for the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase, which mapped to Tyr731, which is present in the sequence YEAM. Mutation of Tyr731 in GST-CBL-LZIP eliminated binding of the p85 subunit of phosphatidylinositol 3-kinase and substantially reduced the phosphorylation of this fusion protein by Fyn, despite the presence of four other tyrosine residues in this fusion protein. These data are consistent with the hypothesis that Cbl represents a substrate for Src-like kinases that are activated in response to the engagement of cell surface receptors, and that Src-like kinases are responsible for the phosphorylation of a tyrosine residue in Cbl that may regulate activation of phosphatidylinositol 3-kinase.

Highlights

Adapter proteins play a critical role in the regulation and activation of signaling molecules that lie downstream of various cell surface receptors
In this study we show that Fyn is constitutively associated with Cbl in PRLresponsive cells, and that Fyn, but not JAK2, can phosphorylate a fusion protein that contains the region of Cbl to which the p85 subunit of PI 3-kinase appears to bind
One of the critical issues regarding signal transduction by cytokine receptors is the role of different tyrosine kinases in mitogenic signaling

Summary

Introduction

Adapter proteins play a critical role in the regulation and activation of signaling molecules that lie downstream of various cell surface receptors. The constitutive association of the p85 subunit of PI 3-kinase with Cbl was observed in the cell lines used in these studies [18, 19], a cytokine-induced increase in Cbl-associated PI 3-kinase activity was observed following stimulation of cells with either IL-3 or PRL [18, 19] This suggested that Cbl may play a critical role in cytokine receptor signaling events since, as noted above, PI 3-kinase is thought to be important in mitogenesis and/or suppression of apoptosis. In this study we show that Fyn is constitutively associated with Cbl in PRLresponsive cells, and that Fyn, but not JAK2, can phosphorylate a fusion protein that contains the region of Cbl to which the p85 subunit of PI 3-kinase appears to bind This suggests that Src-like kinases phosphorylate Cbl and thereby regulate the activation of PI 3-kinase

Methods

Results

Conclusion