Identification of a 14-3-3 binding sequence in the common B 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-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 beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), 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 (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. 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 was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(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.F.C. Stomski, M. Dottore,W. Winnall, M.A. Guthridge, J.Woodcock, C.J. Bagley, D.T. Thomas, R.K. Andrews, M.C. Berndt, and A.F. Lope

Roles of the N and C terminal domains of the interleukin-3 receptor alpha chain in receptor function.

Copyright © 1997 by The American Society of HematologyThe interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 receptor alpha chains are each composed of three extracellular domains, a transmembrane domain and a short intracellular region. Domains 2 and 3 constitute the cytokine receptor module (CRM), typical of the cytokine receptor superfamily; however, the function of the N-terminal domain is not known. We have investigated the functions of the N-terminal and C-terminal domains of the IL-3 receptor (IL-3R) alpha chain. We find that cells transfected with the receptor beta chain (h beta c) and a truncated IL-3R alpha that is devoid of the intracellular region fail to proliferate or to activate STAT5 in response to human IL-3, despite binding the IL-3 with affinity indistinguishable from that of full-length receptor. In addition, IL-3-induced phosphorylation of h beta c was not detected. Thus, the IL-3R alpha intracellular region does not contribute detectably to stabilization of the receptor/ligand complex, but is essential for signal propagation. In contrast, a truncated IL-3R alpha with the N-terminal domain deleted interacts functionally with the beta chain; mouse cells transfected with these receptor chains proliferate in response to human IL-3 and STAT5 transcription factor is activated. High- and low-affinity binding sites are retained, although the affinity for IL-3 is decreased 15-fold, indicating a significant role for the N-terminal domain in IL-3 binding.S.C. Barry, E. Korpelainen, Q. Sun, F.C. Stomski, P.A.B. Moretti, H. Wakao, R.J. D’Andrea, M.A. Vadas, A.F. Lopez, and G.J. Goodal