Caroline Saucier

Met/Hepatocyte Growth Factor Receptor Ubiquitination Suppresses Transformation and Is Required for Hrs Phosphorylation

ABSTRACT 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.

The 5-HT1A receptor : signaling, desensitization, and gene transcription

The hypothesis that antianxiety or antidepressant agents (e.g., 5-HT1A agonists, 5-HT uptake blockers) exert their clinical action via enhancement of serotonergic neurotransmission due to desensitization of 5-HT1A autoreceptors predicts that regulation of this receptor plays a crucial role in the therapeutic actions of these agents. A multidisciplinary strategy is described for the characterization of the 5-HT1A receptor at the level of cellular signaling mechanisms and genetic regulation, using heterologous expression of the cloned receptor in cell lines, site-directed mutagenesis, isolation of receptor-positive neuronal cell lines, and promoter analysis of the 5-HT1A receptor gene. These analyses will yield new insights into the possible mechanisms down-regulation of 5-HT1A receptor signaling, and may suggest novel sties of inherent defect involved in anxiety syndromes or major depression.

Use of signal specific receptor tyrosine kinase oncoproteins reveals that pathways downstream from Grb2 or Shc are sufficient for cell transformation and metastasis.

Many human cancers have been associated with the deregulation of receptor tyrosine kinases (RTK). However, the individual contribution of receptor-associated signaling proteins in cellular transformation and metastasis is poorly understood. To examine the role of RTK activated signal transduction pathways to processes involved in cell transformation, we have exploited the oncogenic derivative of the Met RTK (Tpr–Met). Unlike other RTKs, twin tyrosine residues in the carboxy-terminal tail of the Met oncoprotein and receptor are required for all biological and transforming activities, and a mutant lacking these tyrosines is catalytically active but non transforming. Using this mutant we have inserted oligonucleotide cassettes, each encoding a binding site for a specific signaling protein derived from other RTKs. We have generated variant forms of the Tpr–Met oncoprotein with the ability to bind individually to the p85 subunit of PI3′K, PLCγ, or to the Grb2 or Shc adaptor proteins. Variants that recruit the Shc or Grb2 adaptor proteins generated foci of morphologically transformed fibroblast cells and induced anchorage-independent growth, scattering of epithelial cells and experimental metastasis. In contrast, variants that bind and activate PI3′K or PLCγ failed to generate readily detectable foci. Although cell lines expressing the PI3′K variant grew in soft-agar, these cells were non metastatic. Using this unique RTK oncoprotein model, we have established that Grb2 or Shc dependent signaling pathways are sufficient for cell transformation and metastatic spread.

Endogenous serotonin-2A and -2C receptors in Balb/c-3T3 cells revealed in serotonin-free medium: Desensitization and down-regulation by serotonin

We studied the endogenous expression of the serotonin-2A (5-hydroxytryptamine2A, 5-HT2A) 5-HT2C, and a splice-variant of the 5-HT2C receptor in murine Balb/c-3T3 fibroblast cells that is revealed when these cells are maintained in medium containing 5-HT-free serum. RNA editing of the 5-HT2C receptor was exclusively at a single brain-specific site. Addition of 5-HT (EC50 = 23 +/- 2.9 nM) induced an immediate release of calcium from an ionomycin-sensitive intracellular store by coupling to a pertussis toxin-insensitive pathway. The 5-HT-induced calcium mobilization displayed a 5-HT-2-like pharmacology, and ligand binding analyses indicated the presence of specific binding sites (27.5 +/- 2 fmol/mg protein) with a 5-HT2A-like pharmacology. Although the 5-HT2A receptor site was predominant, the smaller component of 5-HT2C receptors alone was sufficient to mediate a maximal calcium response. The 5-HT-induced increase in [Ca2+]i was reversibly inhibited by >75% following a 12-hr pretreatment (T1/2 = 2 hr) with 5-HT (EC50 = 400 nM). Extended treatment (24-96 hr) with 5-HT induced a complete functional desensitization that was associated with a partial (60%) reduction in 5-HT2 receptor number, implicating both receptor down-regulation and post-receptor mechanisms in 5-HT-induced desensitization. Long-term (hours to days) treatment with 5-HT did not modulate DNA synthesis, cell proliferation, or transformation in Balb/c-3T3 cells. These results demonstrate that Balb/c-3T3 cells express endogenous 5-HT2 receptors that are desensitized by the 5-HT present in normal serum, illustrating the importance of growth conditions in the identification of receptor responsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a safety valve to prevent abnormal cell growth during sustained 5-HT2 receptor activation.

Identification of an Endogenous 5‐Hydroxytryptamine2A Receptor in NIH‐3T3 Cells: Agonist‐Induced Down‐Regulation Involves Decreases in Receptor RNA and Number

Abstract: NIH‐3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5‐hydroxytryptamine (5‐HT) when cultured in 5‐HT‐free medium, became responsive to 5‐HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH‐3T3 cells endogenously express a 5‐HT2A receptor that, when activated, mobilizes calcium from ionomycin‐sensitive intracellular stores via coupling to a pertussis toxin‐insensitive pathway. Using reverse transcriptase‐PCR cloning and northern blot analysis, the presence of 5‐HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH‐3T3 cells. Responsiveness of the endogenous 5‐HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half‐time = 2 h) with 1 µM 5‐HT and resensitized on removal of 5‐HT. In contrast to NIH‐3T3 cells transfected with 5‐HT2A receptor cDNA under control of a viral promoter, the long‐term agonist‐induced functional desensitization in nontransfected NIH‐3T3 cells was paralleled by a decrease in both 5‐HT2A receptor density and RNA level. These results show that NIH‐3T3 cells express an endogenous 5‐HT2A receptor that is desensitized by agonist via down‐regulation of both receptor number and mRNA. The NIH‐3T3 cells provide a novel system for understanding 5‐HT2A receptor regulation.

Oncogenic Met receptor induces ectopic structures in Xenopus embryos

When aberrantly expressed or activated, the Met receptor tyrosine kinase is involved in tumor invasiveness and metastasis. In this study, we have used the Xenopus embryonic system to define the role of various Met proximal-binding partners and downstream signaling pathways in regulating an induced morphogenetic event. We show that expression of an oncogenic derivative of the Met receptor (Tpr-Met) induces ectopic morphogenetic structures during Xenopus embryogenesis. Using variant forms of Tpr-Met that are engineered to recruit a specific signaling molecule of choice, we demonstrate that the sole recruitment of either the Grb2 or the Shc adaptor protein is sufficient to induce ectopic structures and anterior reduction, while the recruitment of PI-3Kinase (PI-3K) is necessary but not sufficient for this effect. In contrast, the recruitment of PLCγ can initiate the induction, but fails to maintain or elongate supernumerary structures. Finally, evidence indicates that the Ras/Raf/MAPK pathway is necessary, but not sufficient to induce these structures. This study also emphasizes the importance of examining signaling molecules in the regulatory context that is provided by receptor/effector interactions when assessing a role in cell growth and differentiation.

Oncogenic Met receptor induces cell-cycle progression in Xenopus oocytes independent of direct Grb2 and Shc binding or Mos synthesis, but requires phosphatidylinositol 3-kinase and Raf signaling.

Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell‐cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal‐binding partners and downstream signaling pathways in cell‐cycle regulation. We show that cell‐cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr‐Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C‐terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell‐cycle progression and activation of MAPK and JNK, while the binding of phospholipase‐Cγ or phosphatidylinositol 3‐kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3‐kinase, phospholipase‐Cγ, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK‐dependent and independent signaling contribute to Met receptor‐mediated cell‐cycle progression, but phospholipase‐Cγ or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3‐kinase signaling are required for cell‐cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors. J. Cell. Physiol. 207: 271–285, 2006.