Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation.
Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of ... Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.
Mesh Terms:
3T3 Cells, Alanine, Animals, Baculoviridae, Cell Line, Cell Nucleus, Cell Transformation, Neoplastic, Cytokines, Cytosol, DNA, DNA-Binding Proteins, Dimerization, Dose-Response Relationship, Drug, Drug Design, Gene Expression Regulation, Growth Substances, Insects, Luciferases, Mice, Models, Biological, Mutation, Peptides, Phosphopeptides, Phosphotyrosine, Plasmids, Protein Binding, Protein Structure, Tertiary, STAT3 Transcription Factor, Signal Transduction, Time Factors, Trans-Activators, Transcription, Genetic, Transfection, src Homology Domains
3T3 Cells, Alanine, Animals, Baculoviridae, Cell Line, Cell Nucleus, Cell Transformation, Neoplastic, Cytokines, Cytosol, DNA, DNA-Binding Proteins, Dimerization, Dose-Response Relationship, Drug, Drug Design, Gene Expression Regulation, Growth Substances, Insects, Luciferases, Mice, Models, Biological, Mutation, Peptides, Phosphopeptides, Phosphotyrosine, Plasmids, Protein Binding, Protein Structure, Tertiary, STAT3 Transcription Factor, Signal Transduction, Time Factors, Trans-Activators, Transcription, Genetic, Transfection, src Homology Domains
J. Biol. Chem.
Date: Nov. 30, 2001
PubMed ID: 11579100
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