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

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. STATs1 were originally discovered as latent cytoplasmic transcription factors that mediate cellular responses to diverse cytokines and growth factors (1-6). Following ligand binding and activation of cytokine or growth factor receptors, STATs are recruited to the cytoplasmic portion of receptors where they are tyrosine-phosphorylated. Tyrosine kinases that mediate STAT phosphorylation and activation include growth factor receptors and cytoplasmic tyrosine kinases, particularly Janus kinase and Src kinase families. Once tyrosine-phosphorylated, two STAT monomers form dimers through reciprocal phosphotyrosine-SH2 interactions, translocate to the nucleus, and bind to STAT-specific DNA-response elements of target genes to induce gene expression. To date, there are seven STAT family members identified in mammals, designated Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6. STATs have diverse normal biological functions, which include roles in cell differentiation, proliferation, development, apoptosis, and inflammation (7-18). In addition to their roles in normal cellular processes, some STATs have been observed to participate in signaling events that lead to oncogenic transformation. Constitutively active Stat3 is observed in transformation by...

Stat3 contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration

“…Besides siRNA and Cucurbitacin I, the efficacy of other Stat3 inhibitors could additionally be explored, including other pharmacological agents such as Cucurbitacin Q, which is an analog of Cucurbitacin I and a selective Stat3 inhibitor (Sun et al, 2005), STA-21 , Stat3 decoy oligonucleotide (Leong et al, 2003), or phosphotyrosyl peptides (Turkson et al, 2001), all of which either exhibited antitumor activity, increased apoptosis and inhibited proliferation or suppressed cell transformation of cancer-derived or v-Src-transformed cell lines. These potential cancer therapeutic agents that target the inhibition of Stat3 may also be useful for the prospective clinical treatment of keloid scars.…”

Emerging therapeutic targets in myeloproliferative neoplasms and peripheral T-cell leukemia and lymphomas

“…The first inhibitor of a STAT protein was a peptide molecule [130] and efforts to target STAT signaling for therapeutic purposes are ongoing. To date, inhibition of STAT function has been attempted through several approaches, including N-terminal domain binders [131], oligonucleotides targeting the DNA binding domain [132], and most effectively through use of small molecule compounds that bind the SH 2 domain to block STAT phosphorylation, dimerization, nuclear transport, and target gene expression [133–135].…”

“…The vast majority of medicinal chemistry efforts to target STAT proteins have been conducted to develop specific inhibitors against STAT3 [130–132,136–148], with fewer reports of inhibitory modulators of STAT5A/B. The FDA-approved neuroleptic agent Pimozide was identified in a high-throughput screen as an inhibitor of STAT5 phosphorylation and an inducer of apoptosis in CML cell lines [149].…”

Src SH3/2 Domain-mediated Peripheral Accumulation of Src and Phospho-myosin Is Linked to Deregulation of E-cadherin and the Epithelial-Mesenchymal Transition

“…We used the PI 3-kinase selective inhibitor LY294002, which blocks Akt phosphorylation downstream of PI 3-kinase (Figure 7A, left panels) and the STAT3 inhibitor peptide (Turkson et al, 2001). In neither case was there restoration of E-cadherin-dependent cell-cell contacts ( Figure 7B).…”