No abstract

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in signal transduction. The regulatory role of protein-tyrosine phosphatases (PTPs) in this process was explored by studying the effects of a powerful PTP inhibitor, pervanadate, on the activation of the mitogen-activated protein (MAP) kinase cascade. Treatment of HeLa cells with pervanadate resulted in a marked inhibition of PTP activity, accompanied by a drastic increase in tyrosine phosphorylation of cellular proteins. The increased tyrosine phosphorylation coincided with the activation of the MAP kinase cascade as indicated by enzymatic activity assays of MEK (MAP kinase/ERK-kinase) and MAP kinase and gel mobility shift analyses of Raf-1 and MAP kinase. The activation was sustained but reversible. Upon removal of pervanadate, both tyrosine phosphorylation and MAP kinase activation declined to basal levels. Therefore, inhibition of PTP activity is sufficient per se to initiate a complete MAP kinase activation program.

The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.

The Ras signaling pathway is thought to control the expression of a subset of yet to be defined genes that are crucial for cell growth and differentiation. Ras proteins are among the most important molecular switch molecules that relay mitogenic or differentiation signals from the cell surface to the nucleus where selective gene expression takes place. Oncogenic mutations lock the Ras proteins into a permanent "on" position, leading to unregulated cell proliferation, which is the hallmark of cancer (1, 2). Mutations in ras oncogenes have been detected in over 90% of pancreatic cancers and 50% of colorectal cancer (3). In contrast to the extensive body of knowledge related to the genetics of ras activation (4), relatively little is known of the transcriptional events triggered by Ras. Of a few ras target genes previously identified, including transin/stromelysin-1 (5), glucose transporter (6, 7), Pai-2 (8), heparin binding epidermal growth factor (9), and mob-1/ IP10 (10, 11), nearly all were shown to be inducible in normal cells by serum growth factors, likely through the activation of endogenous ras (12)(13)(14). One long standing puzzle surrounding ras has been that although the effects of Ras activation are very similar to those of serum growth factors, ras-transformed cells may be oncogenic, whereas the parental cells continuously exposed to serum growth factors are not.To search for additional ras target genes, especially those that may be oncogenic ras-specific, optimized differential display technology (15, 16) was employed to systematically search for such genes in comparative studies involving two carefully chosen paradigms. Since the development of the method, it has been realized that differential display, like other competitive methods such as DNA microarray, is mechanism-based, rather than function-based gene screening tool (17). Therefore, much effort should be made to establish the comparative systems based on as direct and simple a mechanism as possible to identify the relevant genes. To this end, alteration in gene expression in Rat-1 embryo fibroblasts upon conditional expression of oncogenic Ha-ras was analyzed to identify the early ras inducible genes. In the other complementary screening, changes in gene expression was followed in the oncogenic Haras transformed Rat-1 cells upon the treatment with inhibitors of either Ras farnesyltransferase (FTI) 1 (18) or MAP kinase kinase (19), which is one of the major Ras downstream kinases required for cell transformation. Here we describe the identification and biochemical characterization of mob-5, the only oncogenic ras-specific gene identified in both paradigms. We demonstrate that mob-5 encodes a cytokine-like secreted protein, which binds specifically to its putative cell surface receptor (Mob-5R) expressed by the ras-transformed cells, but not by their normal parental controls. We propose that the coordinate activation of this novel ligand/receptor loop may play an important role in ras oncogene-mediated neoplasia. MATERIALS AND METHODSCell Line...