SKI promotes Smad3 linker phosphorylations associated with the tumor-promoting trait of TGFbeta

Abstract

The transcriptional co-regulator SKI is a potent inhibitor of TGFbeta-growth inhibitory signals. SKI binds to receptor-activated Smads in the nucleus, forming repressor complexes containing HDACs, mSin3, NCoR, and other protein partners. Alternatively, SKI binds to activated Smads in the cytoplasm, preventing their nuclear translocation. SKI is necessary for anchorage-independent growth of melanoma cells in vitro, and most important, for human melanoma xenograft growth in vivo. We recently identified a novel role of SKI in TGFbeta signaling. SKI promotes the switch of Smad3 from repressor of proliferation to activator of oncogenesis by facilitating phosphorylations in the linker domain. High levels of endogenous SKI are required by the tumor promoting trait of TGFbeta to induce expression of the plasminogen-activator inhibitor-1 (PAI-1), sustained expression of C-Myc and for aborting upregulation of p21(Waf-1). Here we discuss how SKI diversifies and amplifies its functions by associating with multiple protein partners and by promoting Smad3 linker phosphorylation(s) in response to TGFbeta signaling in melanoma cells.

Figure 1

Phosphorylations of Smad3L in melanoma are SKI-dependent. (A) A western blot shows that endogenous or overexpressed SKI in UCD-Mel-N promotes high levels of Smad3C and Smad3L phosphorylations after treatment with TGFβ for 1 hr. The western blot was probed with anti-SKI, anti-SmadC^Ser423/425, anti-Smad3L^Ser208/213 rabbit IgG affinity purified (Immuno-Biological laboratories, Gunma Japan), anti-Smad3 and anti-β-actin antibodies. The Abs were used at concentrations suggested by the manufacturers (reviewed in ref. 2). (B) Immunofluorescence analysis of Smad3 localization in melanoma cells with downregulated SKI (UCD-RNAi-SKI). (C and D) In the presence of SKI, Smad3 had a variable intensity under basal conditions; some cells showed intense nuclear staining while others displayed nucleo/cytoplasmic distribution, or a punctate, cytoplasmic localization. After treatment with TGFβ, Smad3 co-localized with SKI in a large number of the cells. Arrows indicate Smad3 immunoreactivity in metaphase cells. Note that SKI/Smad3 co-localization was prominent in anaphase (arrows) and late anaphase (arrowhead) regardless of any addition of TGFβ. The co-localization diminished or was undetectable in telophase when cytokinesis has begun (small white arrows).

Figure 2

SKI functions as a sensor and modifier of TGFβ. (A and B) Gel-filtration analysis and western blotting demonstrated that SKI moves from fractions containing high molecular weight to intermediate molecular weight complexes after TGFβ treatment. Preparation of cell extracts and gel filtration chromatography were performed following protocols described in ref. . (C and D) Downregulation of SKI changes the composition of basal and TGFβ-induced Smad2/3 complexes. Western blots of cell extracts fractionated by HPLC show the characteristic doublets of TGFβ-activated Smad2 and Smad3 that localize in intermediate mass complex(es). A star indicates position of input SKI from wild-type melanoma cells. Cells were lysed in a 10 mM Tris-Cl (pH 8.0) buffer containing 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS 140 mM NaCl, 1 mM PMSF plus a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail set II (Calbiochem). The polyclonal anti-pSmad2L (S^250–255) and anti-Smad3L (S^208–213) antibodies, and monoclonal G8 anti-Ski Ab were used as described previously. The following commercial Abs were used following manufacturer’s instructions: polyclonal Abs Smad2 (Invitrogen, 51–1300); Smad3 (Invitrogen, 51–1500), polyclonal SKI Ab (Santa Cruz, sc-9140), HDAC1 (Santa Cruz, sc-7872), mSin3 (Santa Cruz, sc-994); MeCP2 (Millipore, 07–013), pSmad2C (Ser^465–467) (Abcam, Ab5451), pSmad3C (Ser^423–425) (Cell Signaling, Ab 3108) and monoclonal Abs RB (Lab Vision, Ab1-1F8) and Brm (Becton-Dickinson, BD 610389).

Figure 3

The multiple functions of SKI in melanoma are mediated by protein-protein interactions and protein-partner phosphorylations (Smad3L phosphorylations). See text for an explanation of the model presented in this figure.