STAT3 is constitutively phosphorylated by RTK^Metp+1loop with the 2B mutation. (A) Whole extracts prepared from NIH 3T3 cells transfected with wild-type RET or RET-2B (Met⁹¹⁸→Thr) were incubated with the antibody arrays comprising 300 antibodies immobilized on nitrocellulose membrane. After extensive washes, the antibody arrays were blotted with pY20-HRP, followed by ECL analysis. Positive signals detected only in the RET-2B samples include: STAT3 (L6), HDAC2 (N3), SHP-1 (N11), RET (J10), FAF (C7), and EGFR (F2). (B) Whole extracts, were prepared from 293T cells in 6-well dishes transiently transfected with 1 μg of cDNA of wild-type RET or RET-2B for 48 h. STAT3 immunoprecipitates were blotted with anti-pY⁷⁰⁵-STAT3 or anti-STAT3 antibody; SHP-1 immunoprecipitates were blotted with pY20 or anti-SHP-1; and phospho-ERKs were detected with anti-pERK1/2 antibody. (C) 293T cells were transiently transfected with wild-type MET, MET-2B (Met¹²⁶⁸→Thr), wild-type RON, and RON-2B (Met¹²⁵⁴→Thr). Whole-cell extracts prepared from these transfectants were subjected to Western blotting analysis with different antibodies as indicated. (D) Whole extracts of 293T cells transiently transfected with wild-type KIT or KIT-2B (Met⁸³⁶→Thr) were subjected to Western blotting analysis with the antibodies as indicated. (E) Whole extracts of 293T cells transiently cotransfected with cMyc-STAT3 and wild-type EGFR, EGFR-2B (Met⁸⁸¹→Thr), or C terminus (from Tyr974) truncated EGFR were analyzed with anti-pY⁷⁰⁵-STAT3, anti-STAT3 and anti-EGFR antibodies, respectively. Also included were whole extracts of A431 cells treated with or without EGF (100 ng/ml) for 30 min.

Thr^p+1loop residue is critical for tyrosine autophosphorylation. (A) 293T cells were transiently transfected with different forms of RET as indicated. Whole extracts prepared from these 293T transfectants were subjected to Western blotting analysis with anti-pTyr and anti-RET antibodies. (B) 293T cells were transiently transfected with different forms of EGFR. Whole extracts prepared from these 293T transfectants were subjected to Western blotting analysis with anti-pTyr (pY20) or anti-EGFR antibody. (C) 293T cells were transiently transfected with wild-type EphA1, EphA3, EphB3, EphB2, EphB2^T793M, EphA5, and EphA5^T845M. Whole extracts prepared from these transfectants were subjected to Western blotting analysis with pY20 or anti-EphA5 antibody. (D) In 293T cells, wild-type EGFR and EGFR-2B were transiently transfected, followed by treatment with or without EGF as indicated. Whole-cell extracts prepared from these cells were Western blotted with anti-pERK1/2, anti-ERK1/2, anti-pAKT (catalog no. 9271S; Cell Signaling) antibodies (left panel). Same Western blotting analysis was performed with 293T cells transfected with empty vector, wild-type EphB2, EphB2^T793M, wild-type EphA5, and EphA5^T845M as indicated (right panel).

RTK-^Thrp+1loop is highly active in STAT3-dependent transcriptional activation. (A) In 293T cells, different RTKs (1 μg) as indicated were cotransfected with Luc reporter construct (0.5 μg) containing 2× STAT3 binding SIE fragment of the promoter region of mouse IRF1 gene. At 48 h after transfection, cell extracts were prepared and subjected to luciferase activity assay. The averages and standard deviations (error bars) of the values of luciferase activities from triplicates of a representative experiment are shown. Luciferase activities in the cell extracts were measured by using a luminometer to estimate transcriptional activity. (B) In 293T cells, the Luc reporter construct (0.5 μg) described above was cotransfected with 1 μg of cDNA of wild-type EGFR or EGFR-2B. As indicated, in some cases, wild-type STAT3 or STAT3^Y705F was included. Luciferase activity was measured with the whole-cell lysates prepared from these transfectants. (C) In 293T cells, wild-type RET and the 2B-mutant RET of different doses (0.25 μg and 0.5 μg) were cotransfected with the above Luc reporter construct (0.5 μg) and pcDNA3 (empty vector). Luciferase activity was assayed 48 h after transfection.

STAT3 regulates CXCR4 and multiple mucin genes. (A) STAT3-binding SIE motifs present in 5′ upstream promoter regions of CXCR4, MUC1, MUC4, and MUC5B genes. (B) Nuclear extracts prepared from EGF-treated A431 cells were incubated with p32-labeled probes with the SIE sequences of CXCR4, MUC1, MUC4, and MUC5B promoters and separated in 5% polyacrylamide gel. For supershift assays, anti-STAT3 antibody was included in the binding reactions. (C) A431 cells were transfected with the indicated CXCR4-Luc reporter constructs or the control vector pcDNA3. After EGF treatment for 6 h, luciferase activity was measured with the whole lysates as described above. (D) From different cells as indicated, DNA fragments coimmunoprecipitated with anti-STAT3 antibody were amplified with the primers for CXCR4, MUC1, MUC4, and MUC5B gene promoters. (E) MCF-7, MB-468, and T47D cells were transiently delivered with STAT3 siRNA. After incubation for 48 h, whole-cell extracts were prepared and subjected to Western blotting for STAT3, MUC1, MUC4, MUC5B, and β-actin. (F) Medullary thyroid carcinoma metastasized to liver within the blood vessel (left) and liver tissue (right) in MEN-2B patients: anti-CXCR4 immunoperoxidase staining (magnification, ×400).

Wild-type but not Thr/Tyr^p+1loop→Met substituted EPH or JAK constitutively phosphorylates STAT3. (A) According to the p+1 loop motif, RTKs are divided into two groups: RTK^Metp+1loop and RTK^Thrp+1loop. Secondary structural data were obtained by analyzing the tyrosine kinases with a three-dimensional structural program (http://www.sbg.bio.ic.ac.uk/∼3dpssm). Yellow shading indicates β-sheets, and blue shading indicates α-helix. The amino acids (in red) are the core of the p+1 loop motif, and the autophorphorylated tyrosine residues in activation loop are blue. (B) Wild-type EphA1, EphA2, EphB3, EphB4, EphA5, and EphB2, as well as the EphA5^T845M and EphB2^T793M mutants, were cotransfected with c-Myc-STAT3 in 293T cells. Whole-cell extracts prepared from these transfectants were subjected to Western blotting analysis with anti-pY⁷⁰⁵-STAT3 or anti-STAT3 antibody as indicated. (C) 293T cells were transfected with empty vector, EphB2, or EphB2 and ephrinB1. Cell lysates were analyzed in Western blotting with anti-pY705-STAT3, anti-EphB2, and anti-ephrinB2 antibodies, respectively. (D) In 293T cells cMyc-STAT3 was cotransfected with empty vector, wild-type JAK1, JAK1^Y1033S, JAK1^{Y1033 M}, or JAK1^Y1033T, followed by alpha interferon (2,000 U/ml) treatment for 30 min. Whole lysates prepared from these transfectants were immunoblotted with anti-pY⁷⁰⁵-STAT3, anti-STAT3, and anti-JAK1 antibodies.

The Thr^p+1loop residue is required for RTK to recruit STAT3 constitutively. (A) Different forms of RET were cotransfected with cMyc-STAT3 in 293T cells. Anti-RET immunoprecipitates were subjected to Western blotting analysis with anti-c-Myc or anti-RET antibody as indicated. (B) Different forms of EGFR were cotransfected with c-Myc-STAT3 in 293T cells. Anti-EGFR immunoprecipitates were subjected to Western blotting with anti-c-Myc or anti-EGFR antibody. (C) Wild-type EphA5 or EphA5^2B and wild-type EphB2 or Eph^2B were cotransfected with c-Myc-STAT3 in 293T cells. Anti-EphA5 or anti-STAT3 immunoprecipitates were analyzed by Western blotting with anti-c-Myc and anti-EphA5 or anti-c-Myc and anti-EphB2 as indicated.

STAT3, constitutively activated by RTK^Metp+1loop with 2B mutation or wild-type EPH, is enriched in cancer cell nuclei. (A) Medullary thyroid C-cell carcinoma tumor samples from thyroid gland, lymph nodes, and liver from of a 14-year-old male MEN-2B patient were stained with H&E, anti-calcitonin antibody, or anti-STAT3 antibody as indicated. (B) In the left panel, whole extracts prepared from A431 cells treated with or without EGF were analyzed by Western blotting with anti-pY⁷⁰⁵-STAT3, anti-STAT3, and anti-EGFR antibodies. In the right panel, whole extracts of T47D, MCF-7, and MB-468 cells were analyzed in a Western blot with antibodies to pY⁷⁰⁵-STAT3, STAT3, and different EPH family members as indicated. (C) Whole extracts of EGF treated MCF-7, MD-468, and T47D cells were blotted with anti-pERK1/2 and anti-ERK1/2 antibodies in Western blotting analysis. (D) Confluent MB-468 and T47D cells in six-well dishes received treatment of genistein (30 μM), AG-490 (10 μM), PP1 (10 μM), PD153035 (20 μM), or a combination of these drugs for 2 h prior to harvest. Whole-cell lysates were analyzed by Western blotting with anti-pY⁷⁰⁵-STAT3 or anti-STAT3 antibody.

STAT3 constitutive activation leads to increased invasion. (A) MCF-7, MB-468, and T47D cells were regulated with siRNA (+) or scrambled siRNA (Ctl) against STAT3. Equal numbers of cells (2 × 10⁵) was seeded in the upper chamber. The cells that invaded the low chamber 10 h later were fixed, stained with H&E, and counted. (B) Stable NIH 3T3 (empty vector), NIH 3T3-RET^WT, or NIH 3T3-RET^2B cell lines were transformed with c-Myc-tagged STAT3^Y705F or wild-type STAT3. Similarly, these two forms of STAT3 were introduced into NIH 3T3 cells expressing EphB2 and EphB2^T793M. Equal numbers of cells (2 × 10⁵) were seeded in the upper chamber, and the cells that invaded the lower chamber 10 h later were fixed, stained with H&E, and counted. (C) Cells that migrated through Matrigel and the other side of the membrane in the chambers in panel B were stained with H&E and examined by using light microscopy.