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-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.
Stomski
F C
FC
The Cytokine Receptor Laboratory, The Hanson Centre for Cancer Research and Institute of Medical and Veterinary Science, Adelaide, Australia.
Dottore
M
M
Winnall
W
W
Guthridge
M A
MA
Woodcock
J
J
Bagley
C J
CJ
Thomas
D T
DT
Andrews
R K
RK
Berndt
M C
MC
Lopez
A F
AF
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017354:Point Mutation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D016185:Receptors, Interleukin-3 / Q000737:chemistry / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D046911:Macromolecular Substances; D011506:Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D018123:Receptors, Interleukin; D016185:Receptors, Interleukin-3; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D010768:Phosphoserine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D014446:Tyrosine 3-Monooxygenase