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. 2007 Mar 6;104(10):4060-4.
doi: 10.1073/pnas.0611665104. Epub 2007 Feb 21.

Identification of STAT3 as a substrate of receptor protein tyrosine phosphatase T

Xiaodong Zhang  1 , Ailan Guo, Jianshi Yu, Anthony Possemato, Yueting Chen, Weiping Zheng, Roberto D Polakiewicz, Kenneth W Kinzler, Bert Vogelstein, Victor E Velculescu, Zhenghe John Wang
Affiliations

Identification of STAT3 as a substrate of receptor protein tyrosine phosphatase T

Xiaodong Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Protein tyrosine phosphatase (PTP) receptor T (PTPRT) is the most frequently mutated PTP in human cancers. However, the cell signaling pathways regulated by PTPRT have not yet been elucidated. Here, we report identification of signal transducer and activator of transcription 3 (STAT3) as a substrate of PTPRT. Phosphorylation of a tyrosine at amino acid Y705 is essential for the function of STAT3, and PTPRT specifically dephosphorylated STAT3 at this position. Accordingly, overexpression of normal PTPRT in colorectal cancer cells reduced the expression of STAT3 target genes. These studies illuminate a mechanism regulating the STAT3 pathway and suggest that this signaling pathway plays an important role in colorectal tumorigenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
STAT3 is a substrate of PTPRT. (A) Cell lysates from inducible cell lines expressing either the intracellular (Intra) or extracellular fragment (Extra) of PTPRT were probed with antibodies specific for pY705 of STAT3. The same membrane was stripped and probed with anti-STAT3 antibodies or anti-FLAG tag antibodies to detect PTPRT protein fragments. I and U indicate cells with and without induction of PTPRT, respectively. (B) HEK293T cells were infected with adenoviruses expressing either full-length PTPRT or GFP (control) for 24 h. Cell lysates were probed with antibodies to pSTAT3 or STAT3. (C) MCF-7 cells were transfected with siRNA against PTPRT or control siRNA. Expression of PTPRT GAPDH was determined by RT-PCR. STAT3 and pSTAT3 protein levels were determined by Western blots. (D) HCT116 cells were infected with adenoviruses expressing PTPRT or GFP and starved for 24 h and then stimulated with IL-6 for the indicated time. (E) Western blots were performed with the same cell lysates using antibodies to pSTAT5 or STAT5. (F–I) The indicated CRC cell lines were starved for 24 h then stimulated with IL-6 for various time periods. Western blot was performed with pSTAT3 and STAT3 antibodies.
Fig. 2.
Fig. 2.
STAT3 is a direct substrate of PTPRT. (A) Coomassie-stained gels of GST fusion proteins eluted from the indicated beads before their incubation with cell lysates. The arrow indicates the fusion proteins. (B) SW480 cell lysates were incubated with beads bound to the indicated GST-fusion proteins. Proteins bound to the beads were resolved by 10% SDS/PAGE, and Western blots were performed with the indicated antibodies. (C) Lysates from the indicated lines were incubated with beads as in B. (D) pSTAT3 proteins were immunoprecipitated from lysate of HEK293 cells pretreated with 50 μM pervanadate. The immunocomplexes were incubated with 1 μg of the indicated recombinant proteins with or without Na3VO4. Western blots were performed to quantitate pSTAT3.
Fig. 3.
Fig. 3.
Regulation of STAT3 activity by PTPRT. (A) HCT116 cells were infected with adenoviruses expressing PTPRT or GFP, starved for 24 h, and then stimulated with IL-6. Lysates were used in Western blots to assess the levels of the indicated proteins. (B) Schematic diagram of PTPRT deletion constructs. (C and D) HCT116 cells were infected with the indicated adenoviruses, starved for 24 h, and then stimulated with IL-6. Western blots were performed to detect the indicated proteins.
Fig. 4.
Fig. 4.
PTPRT regulates STAT3 cellular localization. HCT116 cells were infected with adenoviruses expressing GFP or the indicated forms of PTPRT. Virus-infected cells were starved for 24 h and then stimulated with or without IL-6 for 30 min. Immunofluorescent staining was performed to detect STAT3 protein. DAPI was used to stain nuclei, and GFP served as a marker of adenovirus-infected cells.
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