Consequences of the disease-related L78R mutation for dimerization and activity of STAT3

Abstract

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is centrally involved in diverse processes including haematopoiesis, immunity and cancer progression. In response to cytokine stimulation, STAT3 is activated through phosphorylation of a single tyrosine residue. The phosphorylated STAT3 dimers are stabilized by intermolecular interactions between SH2 domains and phosphotyrosine. These activated dimers accumulate in the nucleus and bind to specific DNA sequences, resulting in target gene expression. We analysed and compared the structural organizations of the unphosphorylated latent and phosphorylated activated STAT3 dimers using Fö rster resonance energy transfer (FRET) in fixed and living cells. The latent dimers are stabilized by homotypic interactions between the N-terminal domains. A somatic mutation (L78R) found in inflammatory hepatocellular adenoma (IHCA), which is located in the N-terminal domain of STAT3 disturbs latent dimer formation. Applying intramolecular FRET, we verify a functional role of the SH2 domain in latent dimer formation suggesting that the protomers in the latent STAT3 dimer are in a parallel orientation, similar to activated STAT3 dimers but different from the antiparallel orientation of the latent dimers of STAT1 and STAT5. Our findings reveal unique structural characteristics of STAT3 within the STAT family and contribute to the understanding of the L78R mutation found in IHCA.

Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity

“…We further showed that the STAT3 NTD mediates cooperativity via the conserved handshake dimer interface (Fig. 8B and C) rather than the Ni 2ϩ interface, consistent with reported mutagenesis data for STAT family members (13,20,54,(56)(57)(58)(59)(60)(61). The data suggest a structural model of two STAT3 dimers on tandem DNA sites "holding hands" by NTD dimerization in a syn geometric arrangement (Fig.…”

“…Indeed, a peptide mimetic of the ␣2 helix in the handshake interface induced apoptosis of breast and prostate cancer cells but not normal cells (7,18,71). A somatic mutation in this interface (Leu 78 Arg) has been found in inflammatory hepatocellular adenoma, where it disrupts homotypic interactions between unphosphorylated STAT3 dimers (20). This suggests that targeting the NTD handshake interface may selectively inhibit the expression of a subset of genes normally regulated by unphosphorylated STAT3.…”

“…Second, the NTD mediates dimerization of unphosphorylated STAT3 (U-STAT3) and is essential for its nuclear accumulation (14)(15)(16), DNA binding (17), chromatin remodeling (17)(18)(19), and regulation of gene expression (18). A point mutation in the NTD (L78R) that disrupts U-STAT3 dimerization has been identified in inflammatory hepatocellular adenoma (IHCA) (20,21). The NTD is also necessary for U-STAT3 to suppress proapoptotic genes, which drives the proliferation and survival of breast cancer cells (22).…”

Molecular Determinants for Unphosphorylated STAT3 Dimerization Determined by Integrative Modeling

“…21 Interestingly, their findings support a direct interaction between the two Nterminal domains, in agreement with previous studies reporting the significance of N-terminal domain for USTATs dimerization. 22 Given the increased interest in the dimerization of USTAT3 and the availability of novel biophysical data, in this study we used an integrative modeling approach to investigate structure and dynamics of 6 this macromolecular assembly.…”

Mutations leading to constitutive active gp130/JAK1/STAT3 pathway

“…Interestingly, substitution of the corresponding Y631 to phenylalanine in STAT2 promotes type I IFN signaling [29,30]. The three other mutations found in IHCA were distributed throughout the protein: the altered leucine-78, which disturbs latent dimer formation [31,32]; glutamate-166, which is part of helix alpha 1 involved in the interaction with gp130 [33]; and aspartate-502, which is located in the alpha-helical ''connector'' domain.…”