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.
Turkson
J
J
Molecular Oncology and Drug Discovery Programs, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, Tampa, Florida 33612, USA. turksonj@moffitt.usf.edu
Ryan
D
D
Kim
J S
JS
Zhang
Y
Y
Chen
Z
Z
Haura
E
E
Laudano
A
A
Sebti
S
S
Hamilton
A D
AD
Jove
R
R
D016475:3T3 Cells; D000409:Alanine / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D016207:Cytokines / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000378:metabolism; D007313:Insecta; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010748:Phosphopeptides / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D018909:src Homology Domains
D016207:Cytokines; D004268:DNA-Binding Proteins; D006133:Growth Substances; D010455:Peptides; D010748:Phosphopeptides; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D019000:Phosphotyrosine; D004247:DNA; D008156:Luciferases; D000409:Alanine