Identification and functional characterization of paxillin as a target of protein tyrosine phosphatase receptor T

Abstract

Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis.

Fig. 1.

Paxillin is a direct substrate of PTPRT. (A) Paxillin is pulled down by substrate-trapping mutants of PTPRT. Colon cancer cell lysates were incubated with beads bound to the indicated GST-fusion proteins. Western blots were performed with an anti-paxillin antibody. (B) PTPRT dephosphorylates paxillin at residue Y88. Phospho-paxillin proteins were immunoprecipitated from lysate of HEK 293 cells. The immunocomplexes were incubated with the indicated recombinant proteins with or without Na₃VO₄. (C) DLD1 cells were infected with adenoviruses expressing PTPRT or GFP and starved and then stimulated with PDGF-AA for the indicated times. The intensity of pY88 signals were quantified with NIH image and normalized against total paxillin levels. Fold change of pY88 paxillin for each time point over unstimulated PTPRT-infected cells was calculated.

Fig. 2.

Paxillin Y88F mutant CRC cells are less tumorigenic. (A) Anchorage-independent growth. CRC cells of the indicated clones were mixed in 0.4% soft agar and plated in six-well plates in triplicates. Cells were grown for 30 days, and colony foci were counted (*, P < 0.001; **, P < 0.05). (B) Athymic nude mice were injected s.c. and bilaterally with cells of the indicated genotypes and killed 35 days after cell injection. Mice that formed xenografts of the indicated genotypes were counted. (C) Tumor sizes of the indicated clones were measured weekly for 5 weeks, and the average volume at each time point was plotted.

Fig. 3.

Paxillin Y88F mutant affect cell signaling through altering phosphorylation of AKT, p130CAS, and SHP2. Parental (WT) and paxillin Y88F homozygous KI CRC cells were serum-starved for 16 h and stimulated with PDGF-AA for the indicated times. Western blot analyses were performed with the indicated antibodies.

Fig. 4.

Increased tyrosine phosphorylation of paxilin correlates with azoxymethane (AOM) induces colon tumors in PTPRT knockout mice. (A) PTPRT regulates pY88 paxillin in vivo. Colon lysates were made from PTPRT^+/+ (WT) and PTPRT^−/− mouse littermates. Western blots were performed with the indicated antibodies. (B) Numbers of colon tumors developed in each of the PTPRT^+/+ and ^−/− C57BL/6J mice (n = 20 in each group). Each circle or square represents one mouse. (C) Gross morphology of two tumors (arrowheads) in a PTPRT^−/− mouse (Right) compared with a colon of a PTPRT^+/+ (WT) mouse. (D and E) Representative images of hematoxylin and eosin staining (D) and Ki67 IHC (E) of AOM-induced tumors.

Fig. 5.

Paxillin Y88 phosphorylation is up-regulated in human colon cancers. (A) Representative images of IHC staining of human colon carcinomas and matched normal colon tissues with the indicated antibodies. (Scale bar: 100 μm.) (B) Quantitative IHC staining of 10 pairs of tumors and matched normal colon tissues with anti-pY88 paxillin antibody (*, P < 0.001).