Mitogenic stimulation of cells induces rapid and transient activation of MAP kinases. Previously we have identified PAC1 as an immediate early, mitogen- inducible, dual specificity phosphatase that dephosphorylates and inactivates the MAP kinase ERK2 in vitro and in vivo. PAC1 RNA and protein are short-lived, and it appears that controlling the transcription of PAC1 is a major form of its regulation. Thus, the induction of an early response gene, PAC1, results in the feed back attenuation of a primary signalling pathway. PAC 1 is part of a larger family of related MAP kinase phosphatases. In order to reveal possible unique functions of these phosphatases in the context of development and the immune response, we have developed transgenic mouse lines which constitutively express the phosphatases in T cells starting at a stage of development preceeding antigenic selection. The development of T cells and selection of TCR-transgenic populations in the constitutive presence of the various MAP kinase phosphatases has been investigated. The most profound effect has been seen with transgenic expression of the M3/6 phosphatase which inactivates JNK and p38 MAP kinase. M3/6- expressing mice displayed a decrease in TCR-driven negative selection, and no obvious effect on positive selection. This phenotype was observed as an increase in the number of double-positive thymocytes which survive in male mice expressing a TCR specific for the H-Y antigen. The developmental history of such surviving cells is currently being investigated. In order to define the role of ERK in tumorigenesis and metastasis, we have developed a system of tetracycline-inducible PAC1 expression in 3T3 cells. Such cells were transformed with various constitutively-active oncogenes that have been hypothesized to transform cells as a result of ERK activation. MAP kinase activation of raf and MEK but not ras -transformed cells was required for morphological transformation and experimental metastasis in vivo. Effector mutants of Ras have been used in conjunction with PAC1 inducibility to define the signaling pathways emanating from ras which are required for tumorigenesis and invasion. Significantly, the pathway mediated by the Ras(G37) effector mutant appears to play an important stimulatory role in the development of infiltrating metastasis in vivo and the development of matrix degrading activity in vitro. The Ras(G37) pathway synergizes with the ERK pathway to produce experimental metastasis. The Ras(G37) and ERK pathways regulate pericellular proteolysis. Ras(G37) stimulates invasiveness in human epithelial cells.
