BINDING OF SRC-LIKE KINASES TO THE BETA-SUBUNIT OF THE INTERLEUKIN-3 RECEPTOR

We have previously shown that stimulation of 32D c13 cells with interl eukin (IL)-3 results in the activation of three src-like tyrosine kina se's, fyn, hck, and lyn. The beta subunit of the IL-3 receptor co-immu noprecipitated with hck in lysates of both unstimulated and IL-3-stimu lated cells; however, the beta subunit did not precipitate with either fyn or lyn, The association of these three kinases with the beta subu nit of the IL-3 receptor was further investigated using bacterial fusi on proteins encoding the unique, SH3, and SH2 domains of these three k inases. Fusion proteins of both hck and fyn bound to a 150-kDa tyrosin e-phosphorylated protein present in lysates of IL-3-stimulated cells. This protein was identified as the beta subunit of the IL-3 receptor b y 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 c ontaining the unique, SH3, and SH2 domains. This indicates that the SH 2 domain is one of the motifs involved in binding hck. to the beta sub unit, A GST fusion protein encoding a 236-amino acid region of the cyt oplasmic tail of the beta subunit, which contained four tyrosine resid ues, bound to hck and fyn, Binding to both proteins was dramatically i ncreased 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 t he beta subunit; tyrosine phosphorylation of this protein increased th e binding of both the unique region and the SH2 domain probes, These d ata indicate that binding of hck to the beta subunit is mediated by bo th phosphotyrosine-dependent and -independent mechanisms.