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.colorectal cancer ͉ tyrosine phosphorylation ͉ signaling T yrosine phosphorylation is coordinately controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) and is a central feature of many signaling pathways involved in tumor development (1). While activating mutations in protein tyrosine kinases have been shown to play vital roles in tumorigenesis, the role of PTPs is less well defined. We recently identified PTP receptor T (PTPRT), also known as PTP , as the most frequently mutated PTP gene in colorectal cancers (CRCs) (2). PTPRT was also mutationally altered in lung and gastric cancers (2). The spectrum of mutations, which included nonsense mutations and frameshift changes, suggested that these alterations were inactivating. Biochemical analyses demonstrated that missense mutations in the catalytic domains of PTPRT diminished its phosphatase activity and overexpression of PTPRT inhibited CRC cell growth (2). Taken together, these studies strongly support the notion that PTPRT normally acts as a tumor suppressor gene. This conclusion was buttressed by a transposon-based somatic mutagenesis screen in mice, wherein PTPRT was isolated as a target gene from two different mouse transgenic sarcomas (3). In light of these data, it is important to understand the mechanisms through which PTPRT is involved in the neoplastic process. Identifying substrates of PTPRT is an important step to elucidating the signal transduction pathways regulated by this phosphatase. Here, we report identification of signal transducer and activator of transcription 3 (STAT3) as a substrate of PTPRT.STAT3 has been shown to play an important role in leukemias, and persistent STAT3 activation has been detected in a variety of hematopoietic malignancies and solid tumors (4-6), including CRCs (7,8). In general, latent cytoplasmic STAT3 becomes activated through phosphorylation of amino acid residue Y705 by cytokine receptor-associated kinase (Jak) or growth factor receptor-associated tyrosine kinase (Src) (6). Phosphorylated STAT3 dimerizes through reciprocal Src homology 2-phosphotyrosine interaction and accumulates in the nucleus (6). STAT3 then activates the transcription of a wide array of genes, including Bcl-XL and SOCS3 (4). In the current study, we demonstrate that PTPRT specifically dephos...
We developed a strategy to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated 'knock-in'. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation-microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.For ease in constructing directed knock-in of Flag-epitope tags, we developed a universal knock-in vector. The vector contains two multiple cloning sites, sequences that encode a triple Flag epitope tag (3×Flag), a neomycin gene flanked by loxP sites and two inverted terminal repeats (Fig. 1). For targeted knock-in of 3×Flag, we inserted sequences homologous to 5′ and 3′ regions flanking the target locus into the two respective cloning sites, and packaged the resulting vector into recombinant adeno-associated virus (rAAV). Then we infected cells with targeting virus and selected neomycin-resistant clones. We identified correctly targeted clones by genomic PCR and then excised the neomycin gene by infection with adenovirus expressing Cre recombinase. As detailed in Supplementary Methods online, we successfully used this strategy to Flag-tag the C terminus of the following five proteins in three different colorectal cancer cell (CRC) lines: STAT3 (signal transducer and activator of transcription 3; Fig. 1c Table 1). The resultant epitope tagged proteins were readily detectable by western blot, immunoprecipitation and immunofluorescence, using commercially available anti-Flag ( Fig. 2 and Supplementary Fig. 1). Moreover, Flag-STAT3 retained the ability to activate the expression of a target gene 1 , and Flag-MRE11 remained associated with known interacting proteins 2 , suggesting that the presence of the 3×Flag tag is not likely to interfere with targeted protein function ( Supplementary Fig. 2 online). These data indicate that the rAAV-mediated targeting method is feasible for multiple loci across several different CRC cell lines and that 3×Flag can serve as a universal epitope for several antibody-based applications. NIH Public AccessTo test whether the tagging method can be used for global chromatin immunoprecipitation (ChIP) analyses, which require antibodies with very high specificity, we performed ChIP-chip analyses on homozygously tagged STAT3 and hemizygously tagged CHD7 loci. We performed ChIP using antibodies either to the Flag tag, or to native STAT3 or CHD7 proteins. Hybridizations were carried out on tiled microarrays that span the Encyclopedia of DNA Elements (ENCODE) regions 3 . A histogram displaying the intensity ratios of oligonucleotide probes after hybridization showed a skewing of the data in the positive direction, consistent with enrichment of DNA fragments captured by ChIP with Flag antibody (Fig. 3a and data not shown). We also plotted intensity ratios by thei...
Novel 1,7-dioxa-4,10-diazacyclododecane artificial receptors with two pendant aminoethyl (3) or guanidinoethyl (4) side arms have been synthesized. Spectroscopy, including fluorescence and CD spectroscopy, of the interactions of 3, 4, and their copper(II) complexes with calf thymus DNA indicated that the DNA binding affinity of these compounds follows the order Cu(2+)-4>Cu(2+)-3>4>3, and the binding constants of Cu(2+)-3 are Cu(2+)-4 are 7.2x10(4) and 8.7x10(4) M(-1), respectively. Assessment by agarose gel electrophoresis of the plasmid pUC 19 DNA cleavage activity in the presence of the receptors showed that the complexes Cu(2+)-3 and Cu(2+)-4 exhibit powerful supercoiled DNA cleavage efficiency. Kinetic data of DNA cleavage promoted by Cu(2+)-3 and Cu(2+)-4 under physiological conditions fit to a saturation kinetic profile with kmax values of 0.865 and 0.596 h(-1), respectively, which give about 10(8)-fold rate acceleration over uncatalyzed supercoiled DNA. This acceleration is due to efficient cooperative catalysis of the copper(II) center and the functional (diamino or bisguanidinium) groups. In-vitro cytotoxic activities toward murine melanoma B16 cells and human leukemia HL-60 cells were also examined: Cu(2+)-4 shows the highest activity with IC(50) values of 1.62x10(-4) and 1.19x10(-5) M, respectively.
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