A Three-Dimensional FRET Analysis to Construct an Atomic Model of the Actin–Tropomyosin–Troponin Core Domain Complex on a Muscle Thin Filament

It is essential to knowthe detailed structure of the thin filament to understand the regulation mechanism of striated muscle contraction. Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm)–troponin (Tn) core domain complex. We generated single-cysteine mutants in the 167–195 region of Tm and in TnC, TnI, and the β-TnT 25-kDa fragment, and each was attached with an energy donor probe. An energy acceptor probe was located at actin Gln41, actin Cys374, or the actin nucleotide-binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca2+. Using the atomic coordinates for F-actin, Tm, and the Tn core domain, we searched all possible arrangements for Tm or the Tn core domain on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of Tm segment 167– 195 and the Tn core domain on F-actin with andwithout Ca2+. The bulk of the Tn core domain is located near actin subdomains 3 and 4. The central helix of TnC is nearly perpendicular to the F-actin axis, directing the N-terminal domain of TnC toward the actin outer domain. The C-terminal region in the I–T arm forms a four-helix-bundle structure with the Tm 175–185 region. After Ca2+ release, the Tn core domainmoves toward the actin outer domain and closer to the center of the F-actin axis

Tuberous sclerosis complex tumor suppressor–mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells

Palmitoylation of the canine histamine H2 receptor occurs at Cys305 and is important for cell surface targeting

AbstractTo determine the presence and functional role of the histamine H2 receptor (H2R) palmitoylation, a receptor with a Cys305 to Ala (A305 receptor) mutation was generated. Wild-type (WT) and A305 receptors were tagged at their N-termini with a hemagglutinin (HA) epitope. WT, but not A305, receptors incorporated [3H]palmitate by metabolic labeling, indicating that the H2R is palmitoylated at Cys305. Immunocytochemistry of WT and A305 receptors expressed in COS7 cells revealed WT receptors to be distributed at the plasma membrane, while the majority of A305 receptors were localized intracellularly with only a small portion being at the plasma membrane. However, the affinity of the A305 receptor for tiotidine was comparable to that of the WT receptor. In addition, when the amounts of cell surface receptors as determined by anti-HA antibody binding were equivalent, A305 receptors mediated production of more cAMP than WT receptors. Preincubation of COS7 cells expressing each receptor with 10−5 M histamine for 30 min reduced subsequent cAMP production in response to histamine via the receptors to similar extents, indicating that palmitoylation is not necessary for desensitization. In addition, cell surface A305 receptors were capable of being internalized from the cell surface at a rate and extent similar to those of WT receptors. Finally, CHO cell lines stably expressing either WT or A305 receptors were incubated with 10−5 M histamine for 1, 6, 12 and 24 h. Total amounts of WT and A305 receptors, as determined by tiotidine binding, were reduced by incubation, indicating downregulation. Downregulation of the A305 receptor was more extensive than that of the WT receptor. Thus, palmitoylation of the H2R might be important for targeting to the cell surface and stability

RB1CC1 Together with RB1 and p53 Predicts Long-Term Survival in Japanese Breast Cancer Patients

RB1-inducible coiled-coil 1 (RB1CC1) plays a significant role in the enhancement of the retinoblastoma tumor suppressor (RB1) pathway and is involved in breast cancer development. However, RB1CC1's role in clinical progression of breast cancer has not yet been evaluated, so, as a first step, it is necessary to establish its usefulness as a tool to evaluate breast cancer patients. In this report, we have analyzed the correlation between abnormalities in the RB1CC1 pathway and long-term prognosis, because disease-specific death in later periods (>5 years) of the disease is a serious problem in breast cancer. Breast cancer tissues from a large cohort in Japan were evaluated by conventional immunohistochemical methods for the presence of the molecules involved in the RB1CC1 pathway, including RB1CC1, RB1, p53, and other well-known prognostic markers for breast cancer, such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The correlation between the immunohistochemical results and clinical outcomes of 323 breast cancer patients was analyzed using a Kaplan-Meier log-rank test and a multivariate Cox proportional hazards regression analysis. Absence of nuclear RB1CC1 expression was associated with the worst prognosis (Log-rank test, Chi-Square value = 17.462, p<0.0001). Dysfunction of either one of RB1CC1, RB1, or p53 was associated with the highest risk for cancer-specific death, especially related to survival lasting more than 5 years (multivariate Cox proportional hazard ratio = 3.951, 95% Confidence Interval = 1.566–9.967, p = 0.0036). Our present data demonstrate that the combined evaluation of RB1CC1, RB1 and p53 by conventional immunohistochemical analysis provides an accurate prediction of the long-term prognoses of breast cancer patients, which can be carried out as a routine clinical examination

TGF-β regulates isoform switching of FGF receptors and epithelial–mesenchymal transition

Both TGF-β and FGF signalling regulate the epithelial–mesenchymal transition. Here, TGF-β is found to promote myofibroblast differentiation, while concomitant FGF pathway activation instead drives cells towards an invasive mesenchymal fate