Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation.
ABSTRACT: In epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1-/- murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1-/- epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling.
Project description:Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.
Project description:The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure-function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.
Project description:Ras genes are commonly mutated in human cancers of the skin and other tissues. Oncogenic Ras signals through multiple effector pathways, including the Erk1/2 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K) and the Ral guanine nucleotide exchange factor (RalGEF) cascades. In epidermis, the activation of oncogenic Ras induces hyperplasia and inhibits differentiation, features characteristic of squamous cell carcinoma. The downstream effector pathways required for oncogenic Ras effects in epidermis, however, are undefined. In this study, we investigated the direct contribution of Mek1 and Mek2 MAPKKs to oncogenic Ras signaling. The response of murine epidermis to conditionally active oncogenic Ras was unimpaired by deletion of either Mek1 or Mek2 MAPKKs individually. In contrast, Ras effects were entirely abolished by combined deletion of all Mek1/2 alleles, whereas epidermis retaining only one allele of either Mek1 or Mek2 showed intermediate responsiveness. Thus, the effects of oncogenic Ras on proliferation and differentiation in skin show a gene dosage-dependent requirement for the Erk1/2 MAPK cascade at the level of Mek1/2 MAPKKs.
Project description:Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation.
Project description:The signaling pathways that govern the lactotrope-specific differentiated phenotype, and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion, are poorly understood. Moreover, the specific role of MAPK signaling in lactotrope proliferation vs differentiation, whether activated phosphorylated MAPK is sufficient for prolactinoma tumor formation remain unknown. Given that oncogenic Ras mutations and persistently activated phosphorylated MAPK are found in human tumors, including prolactinomas and other pituitary tumors, a better understanding of the role of MAPK in lactotrope biology is required. Here we directly examined the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) continuous addition of exogenous epidermal growth factor. We find that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype in vitro and in vivo with GH4 cell xenograft tumors. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only fails to promote cell proliferation, but also diminishes tumorigenic characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation.
Project description:Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation.
Project description:Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.
Project description:The isolation and culturing of cardiac progenitor cells has demonstrated that growth factor signaling is required to maintain cardiac cell survival and proliferation. In this study, we demonstrate in Xenopus that SHP-2 activity is required for the maintenance of cardiac precursors in vivo. In the absence of SHP-2 signaling, cardiac progenitor cells downregulate genes associated with early heart development and fail to initiate cardiac differentiation. We further show that this requirement for SHP-2 is restricted to cardiac precursor cells undergoing active proliferation. By demonstrating that SHP-2 is phosphorylated on Y542/Y580 and that it binds to FRS-2, we place SHP-2 in the FGF pathway during early embryonic heart development. Furthermore, we demonstrate that inhibition of FGF signaling mimics the cellular and biochemical effects of SHP-2 inhibition and that these effects can be rescued by constitutively active/Noonan-syndrome-associated forms of SHP-2. Collectively, these results show that SHP-2 functions within the FGF/MAPK pathway to maintain survival of proliferating populations of cardiac progenitor cells.
Project description:Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NF?B, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions.
Project description:The mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) is a nonredundant component of stress-activated MAPK signaling modules. Its function in tumorigenesis remains highly controversial with some studies indicating that MKK4 is a tumor suppressor, whereas others have reported a pro-oncogenic role. To clarify the role of MKK4 in cancer, we have created a novel mouse model to test the effect of the specific loss of MKK4 in the epidermis on the formation of papillomas caused by activated ras mutation. We have discovered that skin-specific MKK4-deficient mice are resistant to carcinogen-induced tumorigenesis. One mechanism by which MKK4 promotes cell proliferation and the formation of tumors is by increasing epidermal growth factor receptor expression through the c-Jun NH(2)-terminal protein kinase/c-Jun signaling pathway. Together, our results provide the first genetic demonstration that MKK4 is essential to mediate the oncogenic effect of Ras in vivo, thereby validating MKK4 as a potential drug target for cancer therapy.