Identification of a 14-3-3 binding sequence in the common β 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 β chain (β(c)) of the granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major 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 tyrosine-phosphorylated residues in the cytoplasmic domain of β(c). However, the contribution of serine phosphorylation in β(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of β(c) that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with β(c) but not the GM-CSF receptor α chain. C-terminal truncation mutants of β(c) further showed that a region between amino acids 544 and 626 in β(c) was required for its association with 14-3- 3ζ. This region contains the sequence ⁵⁸²HSRSLP⁵⁸⁷, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of ⁵⁸²HSRSLP⁵⁸⁷ for EFAAAA completely abolished interaction of β(c) with GST-14-3-3ζ. Furthermore, the interaction of β(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 ⁵⁸⁵Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated ⁵⁸⁵Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of ⁵⁸⁵S phosphorylation in vivo, we raised antibodies that specifically recognized ⁵⁸⁵Ser-phosphorylated β(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated ⁵⁸⁵Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC- binding site (⁵⁷⁷Tyr) to the 14-3-3-binding site (⁵⁸²HSRSLP⁵⁸⁷) and their conservation between mouse, rat, and human β(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.