The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.

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Constitutive activation of the MAP kinase kinase MEK1 induces oncogenic transformation in intestinal epithelial cells. Loss of cellcell adhesion followed by the dissociation of epithelial structures is a prerequisite for increased cell motility and tumor invasion. This phenotypic switch is designated epithelial-to-mesenchymal transition (EMT). EMT also plays an important role in determining the dissemination of tumors. However, the role of MEK1 in intestinal EMT, tumor invasion and metastasis has not been elucidated. To determine the functions of activated MEK1 in intestinal tumorigenesis, we established intestinal epithelial cell lines that overexpress wild-type MEK1 (wtMEK) or activated MEK1 (caMEK). Our results indicate that expression of caMEK is sufficient to induce EMT as confirmed with the induction of N-cadherin, vimentin, Snail1 and Snail2, whereas a reduction in E-cadherin, occludin, ZO-1 and cortical F-actin was noted. The Snail1 and Snail2 promoter analyses revealed that Egr-1 and Fra-1, an AP-1 protein, are responsible for MEK1-induced Snail1 and Snail2 expression, respectively. Cells expressing activated MEK1 clearly acquired an invasive capacity when compared to wtMEK-expressing cells. Zymography studies confirmed elevated levels of MMP2 and MMP9 activities in media of caMEK-expressing cells. Importantly, cells expressing activated MEK1 induced tumors with short latency in correlation with their ability to induce experimental metastasis in vivo and to express factors known to promote colorectal cancer cell metastasis. In conclusion, our results demonstrate, for the first time, that constitutive activation of MEK1 in intestinal epithelial cells is sufficient to induce an EMT associated with tumor invasion and metastasis. ' UICCKey words: MEK; ERK; intestinal epithelium; epithelialmesenchymal transition; snail; metastasis Ras proteins act as molecular switches that cycle between active GTP-bound and inactive GDP-bound forms and function as essential components of signal transduction pathways regulating cell growth. Activating mutations of the Ras family members are among the most common genetic events in human tumorigenesis. 1 For example, K-ras is mutated in nearly 50% of colorectal tumors at a relatively early stage of the carcinogenic process 2 and despite extensive research, the primary reason for this high frequency remains unclear.The most studied downstream effector pathway of K-ras is the mitogenic serine/threonine kinase cascade called Raf/MEK/mitogen-activated protein kinase (MAPK). Indeed, upon activation by growth factor-stimulated receptors, activated Ras complexes with and promotes Raf kinases, which in turn activate MAPK kinases (MEK1 and MEK2), resulting in activation of extracellular signal-regulated kinases (ERK1 and ERK2). 3 Activated ERKs then translocate into the nucleus where they phosphorylate and activate nuclear transcription factors, such as Elk-1, ATF-2 and ETS1/2 resulting in immediate-early gene induction. 4 Studies on cultured intestinal epithelial cells and many othe...

The hypothesis that antianxiety or antidepressant agents (e.g., Serotonin uptake inhibitors (e.g., fluoxetine, paroxetine) and selective 5-HTIA receptor partial agonists (e.g., buspirone, ipsapirone) are effective in the treatment of major depression and generalized anxiety disorder but require several weeks of drug treatment to observe clinical improvement (Blier et al. 1990; Charney et al. 1990). Acting via distinct mechanisms, these compounds share the property of enhancing serotonergic neurotransmission by selectively downregulating 5-HTIA receptors located on the serotonergic cell body (Azmitia 1994). Serotonin 1A receptors act as inhibitory autoreceptors on serotonergic neurons of the raphe nuclei, whereas 5-HTIB receptors act as inhibitory presynaptic receptors at serotonergic nerve terminals. Both of these 5-HT receptors participate in a negative feedback loop to inhibit serotonergic activity (Figure 1). In experimental animals, long-term treatment with antidepressants induces selective loss of 5-HTIA autoreceptors without altering the responsiveness of postsynaptic 5-HTIA receptors (Welner et al. 1989;Blier et al. 1990; Fanelli and McMonagle-Strucko 1992). Uptake blockers may mediate this action due to chronic elevation of 5-HT levels at the synaptic deft, leading to autoreceptor downregulation. Serotonin 1A receptor agonists appear to have a direct action on the autoreceptor to induce its desensitization. Desensitization of the autoreceptor disinhibits the serotonergic neuron, enhancing the rate of action potential h_ring to augment serotonergic neurotransmission. Increase in serotonergic neurotransmission correlates with the antidepressant or antianxiety activity of these therapeutic compounds. Interestingly, chronic enhancement of serotonergic neurotransmission induced by uptake blockers is potentiated by 5-HTIA receptor agonists, suggesting the importance of autoreceptor desensitization in antidepressant action (Hjorth 1993). The cellular mechanisms involved in the longterm regulation of the 5-HTIA receptor remain uncharacterized, although the time course of therapeutic drug action suggests an effect of these compounds on gene transcription of components in the 5-HT1A receptor signaling system: either the receptor (Welner et al. 1989; 0893-133X/96/$15.00 SSDI 0893-133X(94)00044-Z

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