A nonsense mutant of the hepatitis B virus large S protein antagonizes multiple tumor suppressor pathways through c-Jun activation domain-binding protein1.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Previous studies have identified recurrent nonsense mutations in the HBV large S (LHBs) gene from the liver from HBV core antigen-positive HCC patients. These nonsense mutants have been shown to be oncogenic in mouse xenograft models using a mouse embryonic fibroblast cell line. Here, we expressed in a liver cell line Huh-7 a carboxy terminally truncated protein from a nonsense mutant of the LHBs gene, sW182* (stop codon at tryptophane-182). Although the sW182* protein appeared not to be very stable in the cultured liver cells, we confirmed that the protein can be highly expressed and retained for a prolonged period of time in the hepatocytes in the mouse liver, indicating its stable nature in the physiological condition. In the Huh-7 cells, the sW182* mutant downregulated tumor suppressors p53 and Smad4. This downregulation was reversed by a proteasome inhibitor MG132, implying the involvement of proteasome-based protein degradation in the observed regulation of the tumor suppressors. On the other hand, we found that c-Jun activation domain-binding protein 1 (Jab1) physically interacts with the sW182*, but not wild-type LHBs. RNA interference (RNAi) of Jab1 restored the levels of the downregulated p53 and Smad4. The sW182* mutant inhibited the promoter activity of downstream target genes of the tumor suppressors. Consistently, Jab1 RNAi reversed the inhibition. These results suggest that the LHBs nonsense mutant antagonizes the tumor suppressor pathways through Jab1 in the liver contributing to HCC development.

Impaired mammary tumor formation and metastasis by the point mutation of a Smad3 linker phosphorylation site.

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-ß acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-ß signaling pathway, is regulated by phosphorylation at several sites. The functional significance of the phosphorylation of the linker region in Smad3 is poorly understood for TNBC. Among the four sites in the Smad3 linker region, threonine-179 (T179) appears to be unique as it serves as the binding site for multiple WW-domain-containing proteins upon phosphorylation, suggesting that this phosphorylation is a key for Smad3 to engage other pathways. Using genome editing, we introduced for the first time a knock-in (KI) mutation in the endogenous Smad3 gene in IV2, a lung-tropic subline of the human MDA-MB-231 TNBC cell line. In the resulting cell line, the Smad3 T179 phosphorylation site is replaced by non-phosphorylatable valine (T179V) with the mutation in both alleles. The T179V KI reduced cell growth rate and mammosphere formation. These phenomena were accompanied by a significant upregulation of p21Cip1 and downregulation of c-Myc. The T179V KI also reduced cell migration and invasion in vitro. In the mouse xenograft models, the T179V KI markedly reduced the establishment of primary tumor in the mammary fat pad and the lung metastasis. Our results using gene editing indicate the cancer-promoting role of Smad3 T179 phosphorylation in the human TNBC cells. Our findings highly suggest that controlling this phosphorylation may have therapeutic potential for TNBC.

Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice.

Previous studies based on cell culture and xenograft animal models suggest that Smad3 has tumor suppressor function for breast cancer during early stages of tumorigenesis. In this report, we show that DMBA (7,12-dimethylbenz[a]anthracene), a chemical carcinogen, induces mammary tumor formation at a significantly higher frequency in the Smad3 heterozygous mice than in the Smad3 wild type mice. This is the first genetic evidence showing that Smad3 inhibits mammary tumor formation in a mouse model. Our findings support the notion that Smad3 has important tumor suppressor function for breast cancer.

Phosphorylation of Smads by Intracellular Kinases.

Smad proteins transduce the TGF-ß family signal at the cell surface into gene regulation in the nucleus. In addition to being phosphorylated by the TGF-ß family receptors, Smads are phosphorylated by a variety of intracellular kinases. The most studied are by cyclin-dependent kinases, the MAP kinase family members, and GSK-3. Phosphorylation by these kinases regulates Smad activities, leading to various biological effects. This chapter describes the methods for analyzing Smad phosphorylation by these kinases.

Identification of transforming hepatitis B virus S gene nonsense mutations derived from freely replicative viruses in hepatocellular carcinoma.

BACKGROUND & AIMS: The correlation between chronic hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) has been well-established. But the roles of viral factor remain uncertain. Only HBV X gene and nonsense mutations of S gene (C-terminal truncation of HBV surface protein) have been demonstrated to have transforming activity. Whether they play a significant role in hepatocarcinogenesis is still uncertain. METHODS: Twenty-five HBV-related HCC patients were positive for hepatitis B core antigen (HBcAg) in the cancerous parts of their HCC liver tissues by immunohistochemistry studies, and had available tissue for whole HBV genome sequence analysis. The results were compared with 25 gender and age-matched HBcAg negative HCCs. Plasmids encoding HBV S gene nonsense mutations identified from HBcAg (+) HCC tissue were constructed to investigate their cell proliferation, transformation activity and the oncogenic potentials by xenograft study and in vivo migration assay. RESULTS: HBcAg (+) HCC patients were significantly associated with cirrhosis and small tumor size (≦2 cm) when compared with HBcAg (-) HCC patients. Southern blot analyses revealed freely replicative forms of HBV in the cancerous parts of HBcAg(+) HCC. Three nonsense mutations of S gene (sL95*, sW182*, and sL216*) were identified in the HBcAg(+) HCC tumor tissues. sW182* and sL216* were recurrently found in the 25 HBcAg (-) HCC tumor tissue, too. Functional studies of the above 3 non-sense mutations all demonstrated higher cell proliferation activities and transformation abilities than wild type S, especially sW182*. Tumorigenicity analysis by xenograft experiments and in vitro migration assay showed potent oncogenic activity of sW182* mutant. CONCLUSIONS: This study has demonstrated potent oncogenic activity of nonsense mutations of HBV S gene, suggesting they may play an important role in hepatocarcinogenesis.

Dysregulation of the TGFBI gene is involved in the oncogenic activity of the nonsense mutation of hepatitis B virus surface gene sW182*.

The nonsense mutations of the hepatitis B virus (HBV) surface (S) gene have been reported to have oncogenic potential. We have previously identified several transforming nonsense mutations of the HBV S gene from hepatocarcinoma (HCC) patients. Among them, the sW182* mutant (the stop codon for tryptophan 182) showed the most potent oncogenicity in a mouse xenograft model using stably transfected mouse fibroblast cells. This study is aimed at understanding the molecular mechanisms leading to the oncogenic activity of the sW182* mutant. A gene expression microarray in combination with gene set enrichment analysis (GSEA) revealed differentially expressed gene sets in the sW182* cells, including those related to cell-cycle regulation, deoxyribonucleic acid repair, and genome instability. Of the differentially expressed genes, the transforming growth factor-ß-induced (TGFBI) gene was further validated to be dysregulated in the sW182* cells. This dysregulation was accompanied by hypermethylation of the TGFBI promoter. The level of cyclin D1, a negatively regulated TGFBI target, was highly elevated in the sW182* mutant cells, which is consistent with the potent oncogenicity. Furthermore, frequent abnormal mitosis and multinucleation were observed in the mutant cells. Exogenous expression of TGFBI alleviated the oncogenic activity of the sW182* cells. In human HBV-related HCC cancerous tissue, expression of TGFBI was downregulated in 25 of the 55 (45%) patients examined, suggesting that TGFBI dysregulation could occur in HBV-related HCC development in some cases. These results suggest that dysregulation of TGFBI is involved in the oncogenic activity of the sW182* mutant of the hepatitis B virus S gene.

Microtubule-associated protein tau in bovine retinal photoreceptor rod outer segments: comparison with brain tau.

Recent studies have suggested a possible involvement of abnormal tau in some retinal degenerative diseases. The common view in these studies is that these retinal diseases share the mechanism of tau-mediated degenerative diseases in brain and that information about these brain diseases may be directly applied to explain these retinal diseases. Here we collectively examine this view by revealing three basic characteristics of tau in the rod outer segment (ROS) of bovine retinal photoreceptors, i.e., its isoforms, its phosphorylation mode and its interaction with microtubules, and by comparing them with those of brain tau. We find that ROS contains at least four isoforms: three are identical to those in brain and one is unique in ROS. All ROS isoforms, like brain isoforms, are modified with multiple phosphate molecules; however, ROS isoforms show their own specific phosphorylation pattern, and these phosphorylation patterns appear not to be identical to those of brain tau. Interestingly, some ROS isoforms, under the normal conditions, are phosphorylated at the sites identical to those in Alzheimer's patient isoforms. Surprisingly, a large portion of ROS isoforms tightly associates with a membranous component(s) other than microtubules, and this association is independent of their phosphorylation states. These observations strongly suggest that tau plays various roles in ROS and that some of these functions may not be comparable to those of brain tau. We believe that knowledge about tau in the entire retinal network and/or its individual cells are also essential for elucidation of tau-mediated retinal diseases, if any.

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation.

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαß) and two inhibitory subunits (Pγs). Pαß has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαßγγ and its transducin-activated form, Pαßγ, contain two and one cGMP, respectively. Only Pαßγ shows [(3)H]cGMP binding with a K(d) ∼ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαßγ is suppressed during its formation, implying that cGMP binding is not involved in Pαßγγ activation. Once bound to Pαßγ, [(3)H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes' radius of Pαßγ, and the amount of [(3)H]cGMP-bound Pαßγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαßγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαßγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαßγ, but alters neither the level nor its Stokes' radius. These results suggest that Pγ accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pαßγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαßγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.

Effects of chloroacetaldehyde in 2-chloroethanol-induced cardiotoxicity.

Cardiovascular effects have often been found in 2-chloroethanol (2-CE) intoxicated patients, but the 2-CE elicits cardiovascular toxicity mechanism is not clear. Recently, we have found that chloroacetaldehyde (CAA) accumulation in 2-CE-intoxicated rat's blood and play an important role in 2-CE intoxication. In this study, we used an isolated rat atrium model to examine the cardiotoxicity of 2-CE and CAA. Results indicated that 2-CE did not cause tension arrest in isolated rat right atria, but CAA did. 2-CE caused tension inhibition in the isolated rat left atria. In addition, CAA caused significant tension inhibition and contracture in the isolated rat left atria. Nifedipine, an L-type calcium channel blocker, decreased CAA-induced tension inhibition and contracture. Meanwhile, atrial nNOS and calmodulin (CaM) had significantly greater expression in the 2-CE group and the CAA group than control group. Nifedipine could decrease CAA-induced nNOS and CaM expression. 2-CE-induced cardiovascular toxicity might be due to its metabolite CAA. CAA-induced cardiovascular toxicity might be mediated by calcium channel and nifedipine protected against nNOS-triggered cardiovascular effects.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 2: isolation and characterization of the transducin-activated form.

Rod photoreceptor cGMP phosphodiesterase (PDE6) consists of a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma). In the accompanying article, using bovine photoreceptor outer segment homogenates, we show that Pgamma as a complex with the GTP-bound transducin alpha subunit (GTP-Talpha) dissociates from Palphabetagammagamma on membranes, and the Palphabetagammagamma becomes Pgamma-depleted. Here, we identify and characterize the Pgamma-depleted PDE. After incubation with or without guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), Palphabeta complexes are extracted. When a hypotonic buffer is used, Palphabetagammagamma, Palphabetagamma, and a negligible amount of a Palphabeta complex containing Pgamma are isolated with GTPgammaS, and only Palphabetagammagamma is obtained without GTPgammaS. When an isotonic buffer containing Pdelta, a prenyl-binding protein, is used, Palphabetagammagammadelta, Palphabetagammadeltadelta, and a negligible amount of a Palphabeta complex containing Pgamma and Pdelta are isolated with GTPgammaS, and Palphabetagammagammadelta is obtained without GTPgammaS. Neither Palphabeta nor Palphabetagammagamma complexed with GTPgammaS-Talpha is found under any condition we examined. Palphabetagamma has approximately 12 times higher PDE activity and approximately 30 times higher Pgamma sensitivity than those of Palphabetagammagamma. These results indicate that the Pgamma-depleted PDE is Palphabetagamma. Isolation of Palphabetagammagammadelta and Palphabetagammadeltadelta suggests that one C-terminus of Palphabeta is involved in the Palphabetagammagamma interaction with membranes, and that Pgamma dissociation opens another C-terminus for Pdelta binding, which may lead to the expression of high PDE activity. Cone PDE behaves similarly to rod PDE in the anion exchange column chromatography. We conclude that the mechanisms for PDE activation are similar in mammalian and amphibian photoreceptors as well as in rods and cones.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: identification of its inhibitory subunit complexes and their roles.

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma) and is regulated by the alpha subunit of transducin (Talpha). Here, we show an overall mechanism for PDE regulation by identifying Pgamma complexes in OS homogenates prepared with an isotonic buffer. Before Talpha activation, three Pgamma complexes exist in the soluble fraction. Complex a, a minor complex, contains Palphabeta, Talpha, and a protein named Pdelta. Complex b, Palphabetagammagamma( b ), has a PDE activity similar to that of membranous Palphabetagammagamma, Palphabetagammagamma( M ), and its level, although its large portion is Pdelta-free, is estimated to be 20-30% of the total Palphabetagammagamma. Complex c, (Pgamma.GDP-Talpha) (2) ( c ) , appears to be a dimer of Pgamma.GDP-Talpha. Upon Talpha activation, (1) complex a stays unchanged, (2) Palphabetagammagamma( b ) binds to membranes, (3) the level of (Pgamma.GDP-Talpha) (2) ( c ) is reduced as its GTP-form is produced, (4) complex d, Pgamma.GTP-Talpha( d ), is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Palphabetagammagamma, Palphabetagammagamma( M ) and/or Palphabetagammagamma( b ), becomes Pgamma-depleted. These observations indicate that Pgamma as a complex with GTP-Talpha dissociates from Palphabetagammagamma on membranes and is released to the soluble fraction and that Pgamma-depleted PDE is the GTP-Talpha-activated PDE. After GTP hydrolysis, both (Pgamma.GDP-Talpha) (2) ( c ) and Pgamma.GDP-Talpha( d ), without liberating Pgamma, deactivate Pgamma-depleted PDE. The preferential order to be used for the deactivation is membranous Pgamma.GDP-Talpha( d ), solubilized Pgamma.GDP-Talpha( d ) and (Pgamma.GDP-Talpha) (2) ( c ) . Release of Pgamma.GTP-Talpha complexes to the soluble fraction is relevant to light adaptation.

Functional interaction between Smad3 and S100A4 (metastatin-1) for TGF-beta-mediated cancer cell invasiveness.

TGF-beta (transforming growth factor-beta) induces a cytostatic response in most normal cell types. In cancer cells, however, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we show that S100A4, a metastasis-associated protein, also called metastatin-1, can physically and functionally interact with Smad3, an important mediator of TGF-beta signalling. In agreement with its known property, S100A4 binds to Smad3 in a Ca2+-dependent manner. The S100A4-binding site is located in the N-terminal region of Smad3. S100A4 can potentiate transcriptional activity of Smad3 and the related Smad2. When exogenously expressed in MCF10CA1a.cl1, an MCF10-derived breast cancer cell line, S100A4 increases TGF-beta-induced MMP-9 (matrix metalloproteinase-9) expression. On the other hand, depletion of S100A4 by siRNA (small interfering RNA) from the MDA-MB231 cell line results in attenuation of MMP-9 induction by TGF-beta. Consistent with these observations, S100A4 increases cell invasion ability induced by TGF-beta in MCF10CA1a.cl1 cells, and depletion of the protein in MDA-MB-231 cells inhibits it. Because expression of both S100A4 and TGF-beta is highly elevated in many types of malignant tumours, S100A4 and Smad3 may co-operatively increase metastatic activity of some types of cancer cells.

Pin1 promotes transforming growth factor-beta-induced migration and invasion.

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Novel IL31RA gene mutation and ancestral OSMR mutant allele in familial primary cutaneous amyloidosis.

Primary cutaneous amyloidosis (PCA) is an itchy skin disorder associated with amyloid deposits in the superficial dermis. The disease is relatively common in Southeast Asia and South America. Autosomal dominant PCA has been mapped earlier to 5p13.1-q11.2 and two pathogenic missense mutations in the OSMR gene, which encodes the interleukin-6 family cytokine receptor oncostatin M receptor beta (OSMRbeta), were reported. Here, we investigated 29 Taiwanese pedigrees with PCA and found that 10 had heterozygous missense mutations in OSMR: p.D647V (one family), p.P694L (six families), and p.K697T (three families). The mutation p.P694L was associated with the same haplotype in five of six families and also detected in two sporadic cases of PCA. Of the other 19 pedigrees that lacked OSMR pathology, 8 mapped to the same locus on chromosome 5, which also contains the genes for 3 other interleukin-6 family cytokine receptors, including interleukin-31 receptor A (IL31RA), which can form a heterodimeric receptor with OSMRbeta through interleukin-31 signaling. In one family, we identified a point mutation in the IL31RA gene, c.1562C>T that results in a missense mutation, p.S521F, which is also sited within a fibronectin type III-like repeat domain as observed in the OSMR mutations. PCA is a genetically heterogeneous disorder but our study shows that it can be caused by mutations in two biologically associated cytokine receptor genes located on chromosome 5. The identification of OSMR and IL31RA gene pathology provides an explanation of the high prevalence of PCA in Taiwan as well as new insight into disease pathophysiology.

Peptidyl-prolyl isomerase Pin1 markedly enhances the oncogenic activity of the rel proteins in the nuclear factor-kappaB family.

The peptidyl-prolyl isomerase Pin1 is frequently up-regulated in human cancers in which Rel/nuclear factor-kappaB (NF-kappaB) is constitutively activated, but its role in these cancers remains to be determined, and evidence is still lacking to show that Pin1 contributes to cell transformation by Rel/NF-kappaB. Rel/NF-kappaB transcriptional and oncogenic activities are modulated by several posttranslational modifications and coregulatory proteins, and previous studies showed that cytokine treatment induces binding of Pin1 to the RelA subunit of NF-kappaB, thereby enhancing RelA nuclear localization and stability. Here we show that Pin1 associates with the Rel subunits of NF-kappaB that are implicated in leukemia/lymphomagenesis and modulates their transcriptional and oncogenic activities. Pin1 markedly enhanced transformation of primary lymphocytes by the human c-Rel protein and also increased cell transformation by the potent viral Rel/NF-kappaB oncoprotein v-Rel, in contrast to a Pin1 mutant in the WW domain involved in interaction with NF-kappaB. Pin1 promoted nuclear accumulation of Rel proteins in the absence of activating stimuli. Importantly, inhibition of Pin1 function with the pharmacologic inhibitor juglone or with Pin1-specific shRNA led to cytoplasmic relocalization of endogenous c-Rel in human lymphoma-derived cell lines, markedly interfered with lymphoma cell proliferation, and suppressed endogenous Rel/NF-kappaB-dependent gene expression. Together, these results show that Pin1 is an important regulator of Rel/NF-kappaB transforming activity and suggest that Pin1 may be a potential therapeutic target in Rel/NF-kappaB-dependent leukemia/lymphomas.

Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

Identification and characterization of ERK MAP kinase phosphorylation sites in Smad3.

Smad3 is phosphorylated by ERK MAP kinase upon EGF treatment. We have mapped the ERK phosphorylation sites to Ser 207, Ser 203, and Thr 178 in Smad3. We show that, upon EGF treatment, Smad3 is rapidly phosphorylated in these sites, peaking at approximately 15-30 min and that MEK1 inhibitors PD98059 and U0216 inhibit Smad3 phosphorylation induced by EGF. Ser 207 is the best ERK site in Smad3. Its phosphorylation shows the highest EGF induction in Smad3. It is also a very sensitive site to EGF treatment, significantly responding to low concentrations of EGF. These three sites are also phosphorylated by recombinant ERK2 in vitro. We have compared the kinetic parameters of Smad3 with those of ELK1 and MBP for ERK2. We further show that mutation of the ERK phosphorylation sites increases the ability of Smad3 to stimulate a Smad target gene, suggesting that ERK phosphorylation inhibits Smad3 activity.

Inhibition of Smad antiproliferative function by CDK phosphorylation.

Rb family members were the only demonstrated substrates of CDK4 until it was shown recently that Smad3, which plays a key role in mediating TGF-beta antiproliferative responses, is phosphorylated by both CDK4 and CDK2 in vivo and in vitro. CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. The Rb pathway is disrupted in almost all human cancers. Most cancers contain high levels of CDK activity due to frequent inactivation of the p16 tumor suppressor or overexpression of cyclin D1. Therefore, disruption of the Rb pathway not only inactivates Rb, but also likely diminishes Smad activity, which may contribute to tumorigenesis and resistance to the TGF-beta growth-inhibitory effects in cancers. Although genetic mutation of Smad3 has not been reported, CDK phosphorylation of Smad3 may provide an epigenetic mechanism for inhibition of the tumor suppressive function of Smad3 during the early stages of tumorigenesis.

The Smad3 linker region contains a transcriptional activation domain.

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.