Binding of src-like kinases to the beta-subunit of the interleukin-3 receptor

We have previously shown that stimulation of 32D cl3 cells with interleukin (IL)-3 results in the activation of three src-like tyrosine kinases, fyn, hck, and lyn. The beta subunit of the IL-3 receptor co-immunoprecipitated with hck in lysates of both unstimulated and IL-3-stimulated cells; however, the beta subunit did not precipitate with either fyn or lyn. The association of these three kinases with the beta subunit of the IL-3 receptor was further investigated using bacterial fusion proteins encoding the unique, SH3, and SH2 domains of these three kinases. Fusion proteins of both hck and fyn bound to a 150-kDa tyrosine-phosphorylated protein present in lysates of IL-3-stimulated cells. This protein was identified as the beta subunit of the IL-3 receptor by immunoblotting with an anti-beta antibody. Glutathione S-transferase (GST) fusion proteins containing the SH2 domain of hck bound to the beta subunit although the amount of beta subunit that bound to the SH2 domain alone was only 30% of that which bound to the fusion protein containing the unique, SH3, and SH2 domains. This indicates that the SH2 domain is one of the motifs involved in binding hck to the beta subunit. A GST fusion protein encoding a 236-amino acid region of the cytoplasmic tail of the beta subunit, which contained four tyrosine residues, bound to hck and fyn. Binding to both proteins was dramatically increased when the GST-beta fusion protein was tyrosine-phosphorylated. Far Western blot analysis was used to demonstrate the binding of the unique, SH3, and SH2 domains of hck to this 236-amino acid region of the beta subunit; tyrosine phosphorylation of this protein increased the binding of both the unique region and the SH2 domain probes. These data indicate that binding of hck to the beta subunit is mediated by both phosphotyrosine-dependent and -independent mechanisms.