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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.

We have isolated and sequenced several cDNAs derived from the sea squirt Ciona intestinalis that encode vitamin K-dependent proteins. Four of these encode γ-carboxyglutamic acid (Gla) domain-containing proteins, which we have named Ci-Gla1 through Ci-Gla4. Two additional cDNAs encode the apparent orthologs of γ-glutamyl carboxylase and vitamin K epoxide reductase. Ci-Gla1 undergoes γ-glutamyl carboxylation when expressed in CHO cells and is homologous to Gla-RTK, a putative receptor tyrosine kinase previously identified in a related ascidian. The remaining three Gla domain proteins are similar to proteins that participate in fundamental developmental processes, complement regulation, and blood coagulation. These proteins are generally expressed at low levels throughout development and exhibit either relatively constant expression (Ci-Gla1, γ-glutamyl carboxylase, and vitamin K epoxide reductase) or spatiotemporal regulation (Ci-Gla2, -3, and -4). These results demonstrate the evolutionary emergence of the vitamin K-dependent Gla domain before the divergence of vertebrates and urochordates and suggest novel functions for Gla domain proteins distinct from their roles in vertebrate hemostasis. In addition, these findings highlight the usefulness of C. intestinalis as a model organism for investigating vitamin K-dependent physiological phenomena, which may be conserved among the chordate subphyla.

The Pebpb2 gene encodes a non-DNA binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF), and is rearranged in inversion of chromosome 16 associated with human acute myeloid leukemia. To investigate its physiological function, Pebpb2 was mutated by a targeting strategy to generate a null mutant. The homozygous mutation in mice proved lethal in embryos around embryonic day 12.5, apparently due to massive hemorrhaging in the central nervous system. In addition, definitive hematopoiesis in the liver was severely impaired. The observed phenotype was indistinguishable from that reported for homozygous disruption of AML1 , which encodes a DNA binding subunit of PEBP2/CBF. Thus, the results indicate that the two subunits function together as a heterodimeric PEBP2/CBF in vivo and that PEBP2/CBF plays an essential role in the development of definitive hematopoiesis.

Polyomavirus (Py) DNA replication is regulated by its enhancer, which contains an AP1 site, c-Jun and c-Fos, the products of nuclear protooncogenes c-jun and c-fos, form the heterodimeric transcriptional activating factor AP1. Overexpression of c-fos and c-jun genes strongly stimulated Py DNA replication from the Py origin of replication as well as transcription from the Py early promoter through the AP1 binding site. The cAMP response element (CRE)-binding protein CREB stimulated only transcription, not DNA replication, through the CRE under similar conditions. The results indicate that AP1 functions as a regulator of DNA replication and that the mechanism of activation of Py DNA replication by AP1 is distinct from that of activation of transcription from the Py early promoter.

The activated c-Ha-ras oncogene induced AP1-site DNA-binding activity in F9 cells. This induction appeared to be due, at least in part, to the induction of c-jun transcription. Both activated c-Ha-ras and c-jun induced the differentiation of F9 cells to endoderm-like cells. Thus, AP1 appears to play a key role in the initial stage of F9 cell differentiation.