Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF

The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimul ating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the majo r signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosi ne-phosphorylated residues in the cytoplasmic domain of beta(c). However, t he contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phos phoserine motif in the cytoplasmic domain of beta(c) that interacts with th e adaptor protein 14-3-3 zeta, Coimmunoprecipitation and pull-down experime nts with a glutathione S-transferase (GST):14-3-3 zeta fusion protein showe d that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor a chain. C-terminal truncation mutants of beta(c) further showed that a reg ion between amino acids 544 and 626 in beta(c) was required for its associa tion with 14-3-3 zeta. This region contains the sequence (HSRSLP587)-H-582, which closely resembles the RSXSXP (where S is phosphorylated) consensus 1 4-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (HSRSLP587)-H-582 for EFAAAA complet ely abolished interaction of beta(c) with GST-14-3-3 zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser wa s phosphorylated. Direct binding experiments showed that the peptide contai ning phosphorylated (585)Ser bound 14-3-3 zeta with an affinity of 150 nmol /L, To study the regulation of S-585 phosphorylation in vivo, we raised ant ibodies that specifically recognized (585)Ser-phosphorylated beta(c) Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (5 85)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the S HC-binding site ((577)Tyr) to th, 14-3-3-binding site ((HSRSLP587)-H-582) a nd their conservation between mouse, rat, and human beta(c) but not in othe r cytokine receptors suggest that they form a distinct motif that may subse rve specialized functions associated with the GM-CSF, IL-3, and IL-5 recept ors. (C) 1999 by The American Society of Hematology.