Frank Otto Gombert - Characterization of Phosphopeptide Motifs Specific for the Src Homology 2 Domains of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT3

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  Wiederkehr-Adam,   M.; Ernst, P.; Müller, K.; Bieck, E.; Gombert, F. O.; Ottl, J.;   Graff, P.; Grossmuller, F.; Heim, M. H. Characterization of   Phosphopeptide Motifs Specific for the Src Homology 2 Domains of   Signal Transducer and Activator of Transcription 1 (STAT1) and   STAT3. J. Biol. Chem. 2003,  278, 16117-16128.


  Signal transducers and activators of transcription (STAT) 1 and   STAT3 are activated by overlapping but distinct sets of   cytokines. STATs are recruited to the different cytokine   receptors through their Src homology (SH) 2 domains that make   highly specific interactions with phosphotyrosine-docking sites   on the receptors. We used a degenerate phosphopeptide library   synthesized on 35-{micro}m TentaGel beads and   fluorescence-activated bead sorting to determine the sequence   specificity of the peptide-binding sites of the SH2 domains of   STAT1 and STAT3. The large bead library allowed not only peptide   sequencing of pools of beads but also of single beads. The method   was validated through surface plasmon resonance measurements of   the affinities of different peptides to the STAT SH2 domains.   Furthermore, when selected peptides were attached to a truncated   erythropoietin receptor and stably expressed in DA3 cells,   activation of STAT1 or STAT3 could be achieved by stimulation   with erythropoietin. The combined analysis of pool sequencing,   the individual peptide sequences, and plasmon resonance   measurements allowed the definition of SH2 domain binding motifs.   STAT1 preferentially binds peptides with the motif   phosphotyrosine-(aspartic acid/glutamic   acid)-(proline/arginine)-(arginine/proline/glutamine), whereby a   negatively charged amino acid at +1 excludes a proline at +2 and   vice versa. STAT3 preferentially binds peptides with the motif   phosphotyrosine-(basic or hydrophobic)-(proline or   basic)-glutamine. For both STAT1 and STAT3, specific high   affinity phosphopeptides were identified that can be used for the   design of inhibitory molecules.

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