Phosphotyrosyl Peptides Block Stat3-mediated DNA Binding Activity, Gene Regulation, and Cell Transformation

James Turkson, Declan Ryan, Joon S. Kim, Yi Zhang, Zhi Chen, Eric Haura, Andy Laudano, Said Sebti, Andrew Hamilton, Richard Jove

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)45443-45455
Number of pages13
JournalJournal of Biological Chemistry
Volume276
Issue number48
DOIs
StatePublished - Nov 30 2001

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Gene expression
Chemical activation
Transcription
Membranes
Transducers
Peptides
DNA
Genes
Peptidomimetics
Phosphopeptides
Mutagenesis
Phosphotyrosine
Aptitude
src Homology Domains
Drug Design
Oncogene Proteins
Alanine
Dimers
Intercellular Signaling Peptides and Proteins
Monomers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphotyrosyl Peptides Block Stat3-mediated DNA Binding Activity, Gene Regulation, and Cell Transformation. / Turkson, James; Ryan, Declan; Kim, Joon S.; Zhang, Yi; Chen, Zhi; Haura, Eric; Laudano, Andy; Sebti, Said; Hamilton, Andrew; Jove, Richard.

In: Journal of Biological Chemistry, Vol. 276, No. 48, 30.11.2001, p. 45443-45455.

Research output: Contribution to journalArticle

Turkson, J, Ryan, D, Kim, JS, Zhang, Y, Chen, Z, Haura, E, Laudano, A, Sebti, S, Hamilton, A & Jove, R 2001, 'Phosphotyrosyl Peptides Block Stat3-mediated DNA Binding Activity, Gene Regulation, and Cell Transformation', Journal of Biological Chemistry, vol. 276, no. 48, pp. 45443-45455. https://doi.org/10.1074/jbc.M107527200
Turkson, James ; Ryan, Declan ; Kim, Joon S. ; Zhang, Yi ; Chen, Zhi ; Haura, Eric ; Laudano, Andy ; Sebti, Said ; Hamilton, Andrew ; Jove, Richard. / Phosphotyrosyl Peptides Block Stat3-mediated DNA Binding Activity, Gene Regulation, and Cell Transformation. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 48. pp. 45443-45455.
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AU - Turkson, James

AU - Ryan, Declan

AU - Kim, Joon S.

AU - Zhang, Yi

AU - Chen, Zhi

AU - Haura, Eric

AU - Laudano, Andy

AU - Sebti, Said

AU - Hamilton, Andrew

AU - Jove, Richard

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AB - 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.

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