Mammalian protein-protein interaction trap (MAPPIT) analysis of STAT5, CIS, and SOCS2 interactions with the growth hormone receptor.
Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5. A non-overlapping recruitment pattern is observed for SOCS2 and CIS with positions Y487 and Y595 as major binding sites, ruling out SOCS-mediated inhibition of STAT5 activation by competition for shared binding sites. More detailed analysis revealed that CIS binding to the Y595, but not to the Y487 motif, depends on both its SH2 domain and the C-terminal part of its SOCS box, with a critical role for the CIS Y253 residue. This functional divergence of the two CIS/SOCS2 recruitment sites is also observed upon substitution of the Y+1 residue by leucine, turning the Y487, but not the Y595 motif into a functional STAT5 recruitment site.
Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Ghent University, Faculty of Medicine and Health Sciences, A. Baertsoenkaai 3, B-9000 Ghent, Belgium.
Isabel
I
Uyttendaele
Irma
I
Lemmens
Annick
A
Verhee
Anne-Sophie
AS
De Smet
Joël
J
Vandekerckhove
Delphine
D
Lavens
Frank
F
Peelman
Jan
J
Tavernier
D020816:Amino Acid Motifs; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D008968:Molecular Conformation; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011986:Receptors, Somatotropin / Q000378:metabolism*; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D050826:Suppressor of Cytokine Signaling Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D016207:Cytokines; D017931:DNA Primers; D010455:Peptides; D011986:Receptors, Somatotropin; D050799:STAT5 Transcription Factor; D050826:Suppressor of Cytokine Signaling Proteins