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Supplementary Figure 1 from Peptidyl-Prolyl Isomerase Pin1 Markedly Enhances the Oncogenic Activity of the Rel Proteins in the Nuclear Factor-κB Family

The peptidyl-prolyl isomerase Pin1 is frequently up-regulated in human cancers in which Rel/nuclear factor-κB (NF-κB) 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-κB. Rel/NF-κB 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-κB, thereby enhancing RelA nuclear localization and stability. Here we show that Pin1 associates with the Rel subunits of NF-κB 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-κB oncoprotein v-Rel, in contrast to a Pin1 mutant in the WW domain involved in interaction with NF-κB. 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-κB–dependent gene expression. Together, these results show that Pin1 is an important regulator of Rel/NF-κB transforming activity and suggest that Pin1 may be a potential therapeutic target in Rel/NF-κB–dependent leukemia/lymphomas. [Cancer Res 2009;69(11):4589–97]

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.

Ductal carcinoma in situ (DCIS) transition to invasive tumor is a big problem for breast cancer. The human MCF10DCIS.com cell line (abbreviated as MCF10DCIS) is derived from the human MCF10A breast epithelial cell line. The MCF10DCIS xenografts are similar to human high grade comedo DCIS. In the xenograft assay, MCF10DCIS cells form DCIS-like lesion, remain as DCIS-like lesion in the first several weeks, and then progress into invasive carcinoma. Thus, it is an excellent model to study human breast cancer transition from DCIS to invasive carcinoma. We investigated the role of CD44 in breast cancer transition from DCIS to invasive carcinoma by knockdown the expression of CD44 in the MCF10DCIS xenograft model. Our results show that knockdown of CD44 expression in the MCF10DCIS cells promotes DCIS transition to invasive carcinoma. Our finding is supported by a previous study that showed CD44 expression is higher in DCIS than in invasive breast carcinomas for human patients. Furthermore, we will present our ongoing studies for the underlying mechanisms. Our findings have important implications for breast cancer. Citation Format: Fang Liu, Jun Zhou, Tanima Kundu-Roy, Joseph Wahler, Jae Young So, Yong Lin, You-Rong Lou, Nicola J. Barnard, Isao Matsuura, Nanjoo Suh. CD44 inhibits the transition of ductal carcinoma in situ to invasive breast carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5052.

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.

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-β acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-β 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.

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-β-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.

Ductal carcinoma in situ (DCIS) transition to invasive tumor is an important issue for breast cancer. The human MCF10DCIS.com cell line (abbreviated as MCF10DCIS) is derived from the human MCF10A breast epithelial cell line. The MCF10DCIS xenografts are similar to human high grade comedo DCIS. In the xenograft assay, MCF10DCIS cells form DCIS-like lesion, remain as DCIS-like lesion in the first few weeks, and then progress into invasive carcinoma. Thus, it is an excellent model to study human breast cancer transition from DCIS to invasive carcinoma. To the best of our knowledge, it is also the only cell model for DCIS transition to invasive carcinoma. We investigated the role of CD44 in breast cancer transition from DCIS to invasive carcinoma by knockdown the expression of CD44 in the MCF10DCIS xenograft model. Our results show that knockdown of CD44 expression in the MCF10DCIS cells enhances DCIS transition to invasive carcinoma. Accordingly, a previous study has shown that CD44 expression is lower in invasive human breast tumors compared with DCIS, especially in luminal A subtype. We have also observed that CD44 expression is lower in the invasive carcinomas compared with DCIS. Furthermore, we provide evidence that CD44 plays a role in maintaining the integrity of the myoepithelial layer. Our study uncovered an unexpected role of CD44 in the prevention of DCIS transition to invasive carcinoma. Our findings have important implications for breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Fang Liu, Jun Zhou, Tanima Kundu-Roy, Joseph Wahler, Jae Young So, Yong Lin, You-Rong Lou, Nicola J. Barnard, Isao Matsuura, Nanjoo Suh. Role of CD44 in the transition of ductal carcinoma in situ to invasive breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1073.

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.

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [3H]cGMP binding with a Kd∼ 50 nm and Pγ inhibits the [3H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [3H]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 Pαβγ, and the amount of [3H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [3H]cGMP-bound Pαβγ, 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 Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change. Structured digital abstract • PDE6 alpha , PDE6 beta and PDE6 gamma physically interact by molecular sieving (View interaction)

2-Chloroethanol (2-CE) has been used on grapevines to accelerate grape growth, and its metabolite, chloroacetaldehyde (CAA), accumulated in the liver and blood from rats intoxicated with 2-CE. Chronic occupational injury might be a possible reason for the 2-CE intoxication. In this study, we used the in vitro and in vivo tests to examine the genotoxicity of 2-CE and CAA. First, 2-CE did not induce chromosome aberration formation in Chinese ovary hamster cells, but CAA did induce chromosome aberration formation, especially the chromosome gap-type aberration after S9 activation. Second, 2-CE at high doses (1/2 LD50), but not at low doses, induced peripheral blood micronucleus formation in mice. CAA induced micronucleus formation at low and high dosages (1/8-1/2 LD50). These results indicated that CAA plays an important role in 2-CE chronic intoxication, and the genotoxic mechanisms of CAA require further study.

CD44 is an important molecule in breast cancer. Although CD44 lacks its own signaling domain, it can associate with and co-stimulate signaling by a number of growth factor receptors, such as Her2 and EGF receptor 1. Knockdown of CD44 significantly reduces mammary tumor-initiating frequency and tumor weight. Disruption of tumor cell surface CD44 function induces apoptosis in metastatic mammary carcinoma cells in vivo. Some studies showed that the breast cancer stem cells are enriched in the CD44highCD24low population. The tumor suppressor p53 is mutated or functionally inactivated in over 70% of cancer. In breast cancer, p53 is mutated in ~35% cases, and ~50% breast cancers contain MDM2 or MDMX overexpression. A previous study showed that p53 repressed the expression of CD44 and that this activity was essential for the tumor-suppressor function of p53. We find that p53 potently represses the expression of CD44 in normal human breast epithelial cells. Mutation of the p53 binding site on the CD44 promoter greatly reduces the ability of p53 to repress CD44 expression. We show that p53 repression of CD44 expression is progressively reduced in a breast cancer progression model. Our ongoing studies are elucidating the underlying mechanisms. Our findings will help for better understanding and treatment of breast cancer. Citation Format: Fang Liu, Zhengxue Liu, Guannan Wang, Tanima Roy-Kundu, Isao Matsuura. Repression of CD44 expression by p53 in a breast cancer progression model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4468.

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.

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.1562CT 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.

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016–15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024–15029), we showed that Pγ is phosphorylated by a previously unknown kinase (Pγ kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pγ loses its ability to interact with GTP/Tα, but gains a 10–15 times higher ability to inhibit GTP/Tα-activated PDE than that of nonphosphorylated Pγ. Thus, we propose that the Pγ phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Tα-activated PDE. Here we demonstrate that all known Pγs preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pγ kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pγ indicates that all known Pγs contain a P-X-T-P-R sequence and that this sequence is required for the Pγ phosphorylation by Pγ kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pγ kinase activity exactly coelutes with Cdk5 and p35. The Pγ kinase activity (∼85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pγ. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pγ in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pγ complexed with GTP/Tα in mature vertebrate retinal photoreceptors.

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.

Smad3 plays an important role in inhibiting cell proliferation and promoting apoptosis. 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. Here 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 tumorigenesis in a mouse model. Our findings provide strong support that Smad3 has important tumor suppressor function for breast cancer. Citation Format: Zhengxue Liu, Tanima R. Kundu, Isao Matsuura, Guannan Wang, Yong Lin, You-Rong Lou, Nicola J. Barnard, Xiao-Fan Wang, Mou-Tuan Huang, Nanjoo Suh, Fang Liu. Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4070.

Chimeric proteins from chicken and yeast calmodulin were prepared, and roles of three structural domains, N-domain (1-72), central helix (73-87) and C-domain (88-148), were evaluated. Mutants with the chicken-type C-domain activated the cyclic nucleotide phosphodiesterase with small values of Kact (the concentration of calmodulin giving a half maximal activation), a property of chicken calmodulin. On the other hand mutants with the yeast-type C-domain activated the phosphodiesterase with μM range of Kact, a property of yeast calmodulin. In activation of myosin light chain kinase, introduction of the yeast-type C-domain into chicken calmodulin increased the Kact value by more than 1000-fold with a dramatic decrease in the maximum activity (Vmax). On the other hand introduction of the chicken-type C-domain led to a profile with lower Kact and higher Vmax. Minor effects on Vmax and Kact were observed by substitution of the central helix. Although various small contributions of the N-domain were observed, a common role of the chicken-type C-domain was suggested to catch and maintain the high-affinity interaction with target enzymes.

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [(3)H]cGMP binding with a K(d) ∼ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [(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 Pαβγ, and the amount of [(3)H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαβγ, 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 Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.

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.

The peptidyl-prolyl isomerase Pin1 is frequently up-regulated in human cancers in which Rel/nuclear factor-κB (NF-κB) 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-κB. Rel/NF-κB 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-κB, thereby enhancing RelA nuclear localization and stability. Here we show that Pin1 associates with the Rel subunits of NF-κB 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-κB oncoprotein v-Rel, in contrast to a Pin1 mutant in the WW domain involved in interaction with NF-κB. 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-κB–dependent gene expression. Together, these results show that Pin1 is an important regulator of Rel/NF-κB transforming activity and suggest that Pin1 may be a potential therapeutic target in Rel/NF-κB–dependent leukemia/lymphomas. [Cancer Res 2009;69(11):4589–97]

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.

Smad3 plays an important role in inhibiting cell proliferation and promoting apoptosis in multiple tissues, including the breast tissue. At late stages, Smad3 promotes breast cancer progression and metastasis. We previously showed that the proline-rich linker region of Smad3 is phosphorylated by several kinases, such as by CDKs and MAP kinases. We have mapped these phosphorylation sites. We show that mutation of the phosphorylation sites in the Smad3 linker region increases the ability of Smad3 to inhibit cell proliferation and promote apoptosis. Interestingly, mutation of the Smad3 linker phosphorylation sites also increases the ability of Smad3 to activate genes that promote metastasis. Accordingly, mutation of the Smad3 linker phosphorylation sites markedly inhibits tumorigenicity but promotes metastasis of breast cancer cell lines. Using human breast cancer tissue microarrays, we further show that Smad3 linker phosphorylation is progressively reduced along breast cancer progression. Taken together, our findings strongly suggest that Smad3 linker phosphorylation promotes tumorigenesis but inhibits metastasis. Our findings have important implications for cancer therapy. Citation Format: Fang Liu, Wen Xie, Zhengxue Liu, Tanima Roy, Eunjin Bae, Lalage M. Wakefield, Seong-Jin Kim, Akira Ooshima, Michael Reiss, Isao Matsuura. Role of Smad3 linker phosphorylation in breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4940. doi:10.1158/1538-7445.AM2015-4940

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.

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-β 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 essentially in all types of human solid tumors. 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-β 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.

Transforming growth factor-beta (TGF-beta) potently inhibits cell cycle progression at the G1 phase. Smad3 has a key function in mediating the TGF-beta growth-inhibitory response. Here we show that Smad3 is a major physiological substrate of the G1 cyclin-dependent kinases CDK4 and CDK2. Except for the retinoblastoma protein family, Smad3 is the only CDK4 substrate demonstrated so far. We have mapped CDK4 and CDK2 phosphorylation sites to Thr 8, Thr 178 and Ser 212 in Smad3. Mutation of the CDK phosphorylation sites increases Smad3 transcriptional activity, leading to higher expression of the CDK inhibitor p15. Mutation of the CDK phosphorylation sites of Smad3 also increases its ability to downregulate the expression of c-myc. Using Smad3(-/-) mouse embryonic fibroblasts and other epithelial cell lines, we further show that Smad3 inhibits cell cycle progression from G1 to S phase and that mutation of the CDK phosphorylation sites in Smad3 increases this ability. Taken together, these findings indicate that CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity, diminishing Smad3 activity by CDK phosphorylation may contribute to tumorigenesis and TGF-beta resistance in cancers.

Smad proteins play pivotal roles in mediating the transforming growth factor β (TGF-β) transcriptional responses. We show in this report that PIAS3, a member of the protein inhibitor of activated STAT (PIAS) family, activates TGF-β/Smad transcriptional responses. PIAS3 interacts with Smad proteins, most strongly with Smad3. PIAS3 and Smad3 interact with each other at the endogenous protein level in mammalian cells and also in vitro , and the association occurs through the C-terminal domain of Smad3. We further show that PIAS3 can interact with the general coactivators p300/CBP, the first evidence that a PIAS protein can associate with p300/CBP. In contrast, PIASy, which inhibits Smad transcriptional activity and other transcriptional responses, is unable to interact with p300/CBP. The RING domain of PIAS3 is essential for interaction with p300/CBP, and a RING domain mutant PIAS3, which cannot bind p300/CBP, no longer activates TGF-β/Smad-dependent transcription. Furthermore, we show that PIAS3, Smad3, and p300 can form a ternary complex, which is markedly increased by TGF-β treatment. Taken together, our studies indicate that on TGF-β treatment, PIAS3 can form a complex with Smads and p300/CBP and activate Smad transcriptional activity.

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 ± 0.20 mm), GCAP2 enhanced the retGC activity 10–13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3–4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 ± 2.7 nm). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nm). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nm). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.

A synthetic peptide corresponding in sequence to residues 6-20 of p34cdc2, cdc2(6-20), and a substitution analogue, cdc2(6-20)F15K19 , which contains Thr-14 as the only phosphorylation target were used as substrates to identify a novel protein kinase in bovine thymus cytosol. The kinase catalyzed the phosphorylation of Thr-14 in both peptides and was purified extensively on the basis of its peptide phosphorylation activity. Upon SDS-polyacrylamide gel electrophoresis analyses, the purified samples consistently displayed a prominent 43-kDa protein band which could undergo in gel autophosphorylation, thus suggesting that this band represented the kinase protein. The suggestion was supported further by the observation that both cdc2(6-20) peptide phosphorylation and the autophosphorylation reaction of the 43-kDa protein were inhibited by millimolar concentrations of cAMP. The kinase was found to inactivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and neuronal Cdk5 activator (Nck5a), under phosphorylation conditions. The phosphorylation of Nclk by the purified thymus kinase occurred on Cdk5. The monomeric form of Cdk5 was also phosphorylated by the kinase. Phosphoamino acid and phosphopeptide analysis of the phosphorylated Nclk revealed that Thr-14 of Cdk5 was the sole site of protein phosphorylation. The results suggest that this thymus kinase is a novel Cdk inhibitory protein kinase, distinct from the recently cloned dual functional and membrane-associated Cdc2 inhibitory kinase, Myt1 (Mueller, P. R., Coleman, T. R., Kumagai, A., and Durphy, W. G. (1995) Science 270, 86-90).

Concentration distributions of p-aminoazobenzene (PAAB) were measured by the use of repeated laminates and compared with those predicted by the theoretical treatment1). Film rolls composed of two different component films were used in the measurements.Over all diffusion coefficients Dov through the repeated laminates were calculated either from the observed concentration distributions in both component films or from that in one component film by employing the methods described previously1). Both agreed well with each other.With known Dov together with D through the partner component film, a method of calculation of diffusion coefficient D through a component film in a given repeated laminate was presented.In repeated laminates composed of nylon 6 with polypropyrene (Pp) or cellophane (Ce), values of D for PAAB through Pp and Ce were estimated using the method.

Equations for permeation through laminated composites (slabs) made by two components have been derived under the same conditions as in previous papers4, 5) in which the diffusion coefficient Di is constant and the equilibrium sorption Csi follows Henry's law in each component layer i.Both the concentration distribution in the various laminated composites and permeation rate through them are calculated, by substituting Di and Csi of p-nitroaniline in each component film (nylon 6, cellulose diacetate, cellulose triacetate) into the equations. Good agreement is obtained between the calculations and experiments for the permeation in such systems.Variations of over all diffusion coefficients (DPov, DLov) obtained by the permeation rate and time lag, respectively, are demonstrated against the volume fraction of a component in the composites. It is revealed that the DPov depends on only the volume fraction, whereas the DLov, depends on also the order of components in the composites.