Ski-interacting Protein Interacts with Smad Proteins to Augment Transforming Growth Factor-β-dependent Transcription

Leong, Gary M.; Subramaniam, Nanthakumar; Figueroa, Jonine D.; Flanagan, Judith; Hayman, Michael J.; Eisman, John A.; Kouzmenko, Alexander

doi:10.1074/jbc.m010815200

Cited by 77 publications

(65 citation statements)

References 35 publications

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“…To test whether RIN1, a GEF for RAB5, regulates T␤R signaling, we measured activation of the SMAD-dependent promoter 3TP (35). TGF-␤ treatment induced reporter gene expression in HeLa cells, which have moderate levels of endogenous RIN1 (17).…”

Section: Resultsmentioning

confidence: 99%

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

Hu¹,

Milstein²,

Bliss³

et al. 2008

Molecular and Cellular Biology

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Transforming growth factor ␤ (TGF-␤) receptor (T␤R) signaling contributes to normal development as well as tumorigenesis. Here we report that RIN1, a RAB5 guanine nucleotide exchange factor (GEF) and down regulator of receptor tyrosine kinases (RTKs), promotes T␤R signaling through enhanced endocytosis. T␤R activation induces SNAI1 (Snail), a transcription repressor that reduces RIN1 expression, providing a negative feedback mechanism to control T␤R trafficking and downstream signaling. Persistent RAS signaling disrupts this equilibrium by stabilizing SNAI1 protein, resulting in strong silencing of RIN1 and stabilization of RTKs. TGF-␤-induced RIN1 silencing in breast cancer cells prolonged sensitivity to hepatocyte growth factor, a ligand for the MET-type RTK, and enhanced growth factor-directed cell motility. We conclude that in some tumor cells T␤R and RAS signals are integrated through the silencing of RIN1, leading to a reduction in RAB5-mediated endocytosis. These findings shed new light on the basis for distinct interpretations of TGF-␤ signaling by normal versus transformed cells.Cell surface receptor internalization controls the intensity and duration of signaling and, as a consequence, regulates phenotypes such as differentiation, mitosis, and migration (36, 44). The clathrin-dependent endocytosis of receptor tyrosine kinases (RTKs), for example, typically results in down regulation and signal termination. The GTPase RAB5 is a key regulator of early endosome formation and trafficking and hence a control point for receptor function. This small GTPase is itself activated by the guanine nucleotide exchange factor (GEF) RIN1 (50) and other GEFs (9) and inhibited by GTPaseactivating proteins (24,39).Transforming growth factor ␤ (TGF-␤), signaling through a receptor serine/threonine kinase complex (T␤RI/T␤RII or T␤R), has antiproliferative effects on normal epithelial cells (48). Some late-stage tumor cells escape this cytostatic effect and instead respond to TGF-␤ with increased proliferation and enhanced motility that promote metastatic spread (16). Activated T␤R phosphorylates associated SMAD2 and SMAD3 proteins that then accumulate in the nucleus, inducing expression of SNAI1 (Snail) (42), a promoter of cell motility in development and tumor progression (3), and other genes. This signaling pathway is enhanced by the recruitment of effector proteins during clathrin-mediated endocytosis (18,27,38,43). An alternate, clathrin-independent, T␤R internalization pathway targets T␤R to a degradation shunt (46). We describe a dynamic role for RIN1 in T␤R endocytosis and regulation of downstream signal intensity. In addition, we provide evidence that T␤R and RTK signals integrate through down regulation of RIN1, contributing to the profound difference in TGF-␤ response between normal and transformed cells. MATERIALS AND METHODSCell culture, transfections, and transductions. MCF10A, MDA-MB-231, HeLa, and HepG2 cell lines were cultured under standard conditions. Transfections were performed using Polyfect (Qiagen) (M...

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Section: Resultsmentioning

confidence: 99%

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

Hu¹,

Milstein²,

Bliss³

et al. 2008

Molecular and Cellular Biology

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“…A TGF-β mediated pathway to this degradation may be the result of a direct Smad2/Smurf2/ ERα complex as ERα has been found directly associated with activated Smads [Yamamoto et al, 2002]. Additionally, the Ski-interacting protein (SKIP) has been shown to interact with both nuclear hormone receptors and TGF-β signaling components potentially serving an adapter role in this regulation [Baudino et al, 1998;Leong et al, 2001]. …”

Section: Discussionmentioning

confidence: 99%

Increased proteasome‐dependent degradation of estrogen receptor‐alpha by TGF‐β1 in breast cancer cell lines

Petrel

Brueggemeier

2002

J of Cellular Biochemistry

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Normal mammary epithelial cells are rapidly induced to G 1 arrest by the widely expressed cytokine, transforming growth factor beta (TGF-β1). Studies in established breast cancer cell lines that express the estrogen receptor alpha (ERα) have demonstrated loss of this responsiveness. This inverse correlation suggests interpathway signaling important to cell growth and regulation. The adenocarcinoma breast cell line BT474, which was not growth arrested by TGF-β1, was used as a model of estrogen-inducible growth to explore interpathway crosstalk. Although BT474 cells were not growth-arrested by TGF-β1 as determined by flow cytometry analysis and 5′-bromo-3′-deoxyuridine incorporation into DNA, estrogen receptor protein levels were attenuated by 100 pM TGF-β1 after 6 h. This decrease in ERα reached 50% of untreated control levels by 24 h of treatment and was further supported by a 50% decrease in estrogen-inducible DNA synthesis. Inspection of ERα transcripts suggested that this decrease was primarily the result of altered ERα protein stability or availability. Use of the proteasome inhibitor, MG132, abolished all effects on ERα by TGF-β1. Collectively, this data supports a role for TGF-β1 in regulating the growth of otherwise insensitive breast cancer cells through modulation of ERα stability.Keywords breast cancer; estrogen receptor alpha; transforming growth factor beta; ubiquitin; proteasome; cell proliferation Breast cancer is a leading cause of death among women worldwide and an estimated 203,500 new cases will be diagnosed in the US alone in 2002 [American Cancer Society, 2001]. Systemic and locally produced estrogens are intimately associated with increased risk for breast cancer development. Early menarche and late menopause increase the lifetime exposure to estrogens and as much as a 20% decrease in breast cancer risk is observed for each year this exposure is shortened [Kelsey, 1979]. By the same token, increased and sustained physical activity can reduce the total lifetime ovulatory period and has been shown to confer a significant decrease in breast cancer risk [Bernstein et al., 1994]. Numerous epidemiological studies of breast cancer risk in postmenopausal women have shown higher mean serum estrogen concentrations than disease-free control subjects [Thomas et al., 1997]. These and additional risk factors share a common theme, i.e., increased lifetime tissue exposure to estrogens. Thus, estrogen exposure is a central and reoccurring phenomenon in breast cancer etiology.Although many risk factors have been identified, the molecular mechanisms underlying the initiation and progression of breast cancer are still elusive. Estrogens such as 17β-estradiol (E 2 ) play a key role in the development and maintenance of the mature mammary gland. The most well characterized cellular receptor for this steroid hormone is the estrogen receptor alpha (ERα) [Moggs and Orphanides, 2001]. This receptor is expressed in a tissue specific manner and regulates growth and differentiation in response to hormonal stimul...

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“…Skip is particularly interesting as its overexpression partially overcomes Ski/Sno-dependent repression. However, Ski/Sno overexpression attenuates Skip augmentation of TGF-b-dependent transcription (Leong et al, 2001). Considering both, resistant to TGF-b and SKI overexpression, we predict that SKI counteracts Skip activity in malignant melanoma tumors.…”

Section: Perspectives and Future Directionsmentioning

confidence: 90%

Repression of TGF-β signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis

Medrano

2003

Oncogene

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Transforming growth factor-b (TGF-b) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. TGF-b-mediated growth inhibition is gradually lost during melanoma tumor progression, but there are no measurable defects at the receptor level. Furthermore, melanoma cells release high levels of TGF-b to the microenvironment, which upon activation induces matrix deposition, angiogenesis, survival, and transition to more aggressive phenotypes. The SKI and SnoN protein family associate with and repress the activity of Smad2, Smad3, and Smad4, three members of the TGF-b signaling pathway. SKI also facilitates cellcycle progression by targeting the RB pathway by at least two ways: it directly associates with RB and represses its activity when expressed at high levels, and indirectly, it represses Smad-mediated induction of p21 Waf-1 . This results in increased CDK2 activity, RB phosphorylation, and inactivation. Therefore, high levels of SKI result in lesions to the RB pathway in a manner similar to p16 INK4a loss. SKI mRNA and protein levels dramatically increase during human melanoma tumor progression. In addition, the SKI protein shifts from nuclear localization in intraepidermal melanoma cells to nuclear and cytoplasmic in invasive and metastatic melanomas. Here, I discuss the basis for repression of intracellular TGF-b signaling by SKI, some additional activities of this protein, and propose that by disrupting multiple tumor suppressor pathways, SKI functions as a melanoma oncogene.

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Ski-interacting Protein Interacts with Smad Proteins to Augment Transforming Growth Factor-β-dependent Transcription

Abstract: Transforming growth factor-␤ (TGF-␤

Cited by 77 publications

References 35 publications

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

Increased proteasome‐dependent degradation of estrogen receptor‐alpha by TGF‐β1 in breast cancer cell lines

Repression of TGF-β signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis

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