Monica A. Naujokas

The Tyrosine Phosphatase SHP-2 Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase

ABSTRACT Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cγ, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

The Gab1 PH Domain Is Required for Localization of Gab1 at Sites of Cell-Cell Contact and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase

ABSTRACT Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.

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.

A Conserved Inositol Phospholipid Binding Site within the Pleckstrin Homology Domain of the Gab1 Docking Protein Is Required for Epithelial Morphogenesis

Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4,5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.

Pak4, a Novel Gab1 Binding Partner, Modulates Cell Migration and Invasion by the Met Receptor

ABSTRACT Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, induces epithelial cell dispersal, invasion, and morphogenesis, events that require remodeling of the actin cytoskeleton. The scaffold protein Gab1 is essential for these biological responses downstream from Met. We have identified p21-activated kinase 4 (Pak4) as a novel Gab1-interacting protein. We show that in response to HGF, Gab1 and Pak4 associate and colocalize at the cell periphery within lamellipodia. The association between Pak4 and Gab1 is dependent on Gab1 phosphorylation but independent of Pak4 kinase activity. The interaction is mediated through a region in Gab1, which displays no homology to known Gab1 interaction motifs and through the guanine exchange factor-interacting domain of Pak4. In response to HGF, Gab1 and Pak4 synergize to enhance epithelial cell dispersal, migration, and invasion, whereas knockdown of Pak4 attenuates these responses. A Gab1 mutant unable to recruit Pak4 fails to promote epithelial cell dispersal and an invasive morphogenic program in response to HGF, demonstrating a physiological requirement for Gab1-Pak4 association. These data demonstrate a novel association between Gab1 and Pak4 and identify Pak4 as a key integrator of cell migration and invasive growth downstream from the Met receptor.

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.

Distinct tyrosine autophosphorylation sites mediate induction of epithelial mesenchymal like transition by an activated ErbB-2/Neu receptor.

Tight control of cell proliferation and morphogenesis is required to ensure normal tissue patterning and prevent cancer formation. Overexpression of the ErbB-2/Neu receptor tyrosine kinase is associated with increased progression in human breast cancer, yet in breast explant cultures, the ErbB-2/Neu receptor contributes to alveolar differentiation. To examine the consequence of deregulated ErbB-2/Neu activation on epithelial morphogenesis, we have expressed a constitutively activated mutant of ErbB-2/Neu in a Madin–Darby canine kidney (MDCK) epithelial cell model. Using two-dimensional cultures we demonstrate that activated ErbB-2/Neu induces breakdown of cell–cell junctions, increased cell motility and dispersal of epithelial colonies. This correlates with reorganization of the actin cytoskeleton and focal adhesions and loss of insoluble cell–cell junction complexes involving E-cadherin. Interestingly, a constitutively activated ErbB-2/Neu receptor promotes an invasive morphogenic program in MDCK cells in a three-dimensional matrix. We show that two tyrosines in the carboxy-terminal tail of ErbB-2/Neu, involved in the phosphorylation of the Shc adapter protein, are each sufficient to promote epithelial-mesenchymal like transition and enhanced cell motility in two-dimensional culture and cell invasion rather than a morphogenic response in matrix culture. This provides a model system to investigate ErbB-2/Neu induced signaling pathways required for epithelial cell dispersal and invasion versus morphogenesis.

Enhanced Transformation by a Plasma Membrane-Associated Met Oncoprotein: Activation of a Phosphoinositide 3′-Kinase-Dependent Autocrine Loop Involving Hyaluronic Acid and CD44

ABSTRACT A Met-hepatocyte growth factor receptor oncoprotein, Tpr-Met, generated by chromosomal rearrangement, fuses a protein dimerization motif with the cytoplasmic domain of the Met receptor, producing a cytosolic, constitutively activated tyrosine kinase. Although both the Met receptor and the Tpr-Met oncoprotein associate with the same substrates, activating mutations of the Met receptor in hereditary papillary renal carcinomas have different signaling requirements for transformation than Tpr-Met. This suggests differential activation of membrane-localized pathways by oncogenic forms of the membrane-bound Met receptor but not by the cytoplasmic Tpr-Met oncoprotein. To establish which pathways might be differentially regulated, we have localized the constitutively activated Tpr-Met oncoprotein to the membrane using the c-src myristoylation signal. Membrane localization enhances cellular transformation, focus formation, and anchorage-independent growth and induces tumors with a distinct myxoid phenotype. This correlates with the induction of hyaluronic acid (HA) and the presence of a distinct form of its receptor, CD44. A pharmacological inhibitor of phosphoinositide 3′ kinase (PI3′K), inhibits the production of HA, and conversely, an activated, plasma membrane-targeted form of PI3′K is sufficient to enhance HA production. Furthermore, the multisubstrate adapter protein Gab-1, which couples the Met receptor with PI3′K, enhances Met receptor-dependent HA synthesis in a PI3′K-dependent manner. These results provide a positive link to a role for HA and CD44 in Met receptor-mediated oncogenesis and implicate PI3′K in these events.

Crk Synergizes with Epidermal Growth Factor for Epithelial Invasion and Morphogenesis and Is Required for the Met Morphogenic Program

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer

Significance Triple-negative breast cancers lack targeted therapies and are subdivided into molecular subtypes, including basal and claudin-low. Preclinical models representing these subtypes are limited. We have developed a murine model in which mammary gland expression of a receptor tyrosine kinase (MET) and loss of tumor suppressor gene p53 (Trp53), synergize to promote tumors with pathological and molecular features of claudin-low breast cancer. These tumors require MET signaling for proliferation, as well as mesenchymal characteristics, which are key features of claudin-low biology. This work associates MET expression and p53 loss with claudin-low breast cancers and highly proliferative breast cancers of poor outcome. Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Metmt) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Metmt mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Metmt, significantly increased tumor penetrance over Metmt or Trp53 loss alone. Unlike Metmt tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Metmt tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-to-mesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.