Rotraud Wieser

GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex.

The TGF‐beta type II receptor (T beta R‐II) is a transmembrane serine/threonine kinase that, upon ligand binding, recruits and phosphorylates a second transmembrane kinase, T beta R‐I, as a requirement for signal transduction. T beta R‐I is phosphorylated by T beta R‐II in the GS domain, a 30 amino acid region preceding the kinase domain and conserved in type I receptors for other TGF‐beta‐related factors. The functional role of seven serines and threonines in the T beta R‐I GS domain was investigated by mutational analysis. Five of these residues are clustered (TTSGSGSG) in the middle of the GS domain. Mutation of two or more of these residues impairs phosphorylation and signaling activity. Two additional threonines are located near the canonical start of the kinase domain, and their individual mutation to valine strongly inhibits receptor phosphorylation and signaling activity. Replacement of one of these residues, Thr204, with aspartic acid yields a product that has elevated in vitro kinase activity and signals anti‐proliferative and transcriptional responses in the absence of ligand and T beta R‐II. The identification of constitutively active T beta R‐I forms confirms the hypothesis that this kinase acts as a down‐stream signaling component in the TGF‐beta receptor complex, and its activation by T beta R‐II or by mutation is necessary and sufficient for propagation of anti‐proliferative and transcriptional responses.

Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor beta and activin.

Transforming growth factor beta (TGF-beta) and activin bind to receptor complexes that contain two distantly related transmembrane serine/threonine kinases known as receptor types I and II. The type II receptors determine ligand binding specificity, and each interacts with a distinct repertoire of type I receptors. Here we identify a new type I receptor for activin, ActR-IB, whose kinase domain is nearly identical to that of the recently cloned TGF-beta type I receptor, T beta R-I. ActR-IB has the structural and binding properties of a type I receptor: it binds activin only in the presence of an activin type II receptor and forms a heteromeric noncovalent complex with activin type II receptors. In Mv1Lu lung epithelial cells, ActR-IB and T beta R-I signal a common set of growth-inhibitory and transcriptional responses in association with their corresponding ligands and type II receptors. The transcriptional responses include elevated expression of fibronectin and plasminogen activator inhibitor 1. Although T beta R-I and ActR-IB are nearly identical in their kinase domains (90% amino acid sequence identity), their corresponding type II receptor kinase domains are very different from each other (42% amino acid sequence identity). Therefore, signaling of a specific set of responses by TGF-beta and activin correlates with the presence of similar type I kinases in their complex. Indeed, other TGF-beta and activin type I receptors (TSR-I and ActR-I) whose kinase domains significantly diverge from those of T beta R-I and ActR-IB do not substitute as mediators of these growth-inhibitory and extracellular matrix transcriptional responses. Hence, we conclude that the type I receptor subunits are primary specifiers of signals sent by TGF-beta and activin receptor complexes.

Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene

The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c‐raf‐1−/− embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c‐raf‐1−/− fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf‐1−/− fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf‐1‐ deficient fibroblasts are more sensitive than wild‐ type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor‐α. MEK/ERK activation is normal in Raf‐1‐deficient cells and embryos, and is probably mediated by B‐Raf. These results indicate that the essential function of Raf‐1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti‐apoptotic function of Raf‐1.

Signaling activity of transforming growth factor beta type II receptors lacking specific domains in the cytoplasmic region.

The transforming growth factor beta (TGF-beta) type II receptor (T beta R-II) is a transmembrane serine/threonine kinase that contains two inserts in the kinase region and a serine/threonine-rich C-terminal extension. T beta R-II is required for TGF-beta binding to the type I receptor, with which it forms a heteromeric receptor complex, and its kinase activity is required for signaling by this complex. We investigated the role of various cytoplasmic regions in T beta R-II by altering or deleting these regions and determining the signaling activity of the resulting products in cell lines made resistant to TGF-beta by inactivation of the endogenous T beta R-II. TGF-beta binding to receptor I and responsiveness to TGF-beta in these cells can be restored by transfection of wild-type T beta R-II. Using this system, we show that the kinase insert 1 and the C-terminal tail of T beta R-II, in contrast to the corresponding regions in most tyrosine kinase receptors, are not essential to specify ligand-induced responses. Insert 2 is necessary to support the catalytic activity of the receptor kinase, and its deletion yields a receptor that is unable to mediate any of the responses tested. However, substitution of this insert with insert 2 from the activin receptor, ActR-IIB, does not diminish the ability of T beta R-II to elicit these responses. A truncated T beta R-II lacking the cytoplasmic domain still binds TGF-beta, supports ligand binding to receptor I, and forms a complex with this receptor. However, TGF-beta binding to receptor I facilitated by this truncated T beta R-II fails to inhibit cell proliferation, activate extracellular matrix protein production, or activate transcription from a promoter containing TGF-beta-responsive elements. We conclude that the transcriptional and antiproliferative responses to TGF-beta require both components of a heteromeric receptor complex that differs from tyrosine kinase receptors in its mode of signaling.

Bortezomib in relapsed multiple myeloma: response rates and duration of response are independent of a chromosome 13q-deletion

Studies of bortezomib in patients with relapsed multiple myeloma (MM) suggested that bortezomib may be active even in the presence of adverse prognostic factors. We therefore evaluated 62 patients with relapsed/refractory MM who were treated with single-agent bortezomib, and addressed the question whether or not the negative prognostic impact of unfavorable cytogenetic abnormalities may be overcome by bortezomib. By interphase fluorescence in situ hybridization (FISH), a deletion of chromosome 13q14 [del(13q14)] was present in 33 patients (53%). Overall response rates to bortezomib were similar in patients with and without del(13q14) (45 versus 55%; P=0.66), and rates of complete remission (CR) near CR were also not different between the two patient populations (18 versus 14%). Three patients had a t(4;14)(p16;q32) in addition to del(13q14), and all of them had a >50% paraprotein reduction. Median duration of response was 12.3 months in patients with del(13q14) compared with 9.3 months in patients with normal 13q-status (P=0.25), and survival was also not different between the two patient populations. Patients not benefiting from single-agent bortezomib were characterized by the combined presence of a del(13q14) and low serum albumin (median survival 4.6 months). Our results provide evidence for remarkable activity of bortezomib in MM with del(13q14). Patients who do not respond to bortezomib and consecutively have short time to treatment failure and overall survival can be identified by low serum albumin in addition to del(13q14) and should be considered for bortezomib combinations.

NEGATIVE REGULATION OF TRANSCRIPTION OF THE SACCHAROMYCES CEREVISIAE CATALASE T (CTT1) GENE BY CAMP IS MEDIATED BY A POSITIVE CONTROL ELEMENT

Transcription of the CTT1 (catalase T) gene of Saccharomyces cerevisiae is controlled by oxygen via heme, by nutrients via cAMP and by heat shock. Nitrogen limitation triggers a rapid, cycloheximide‐insensitive derepression of the gene. Residual derepression in a cAMP‐nonresponsive mutant with attenuated protein kinase activity (bcy1 tpk1w tpk2 tpk3) demonstrates the existence of an alternative, cAMP‐independent nutrient signaling mechanism. Deletion analysis using CTT1‐lacZ fusion genes revealed the contribution of multiple control elements to derepression, not all of which respond to the cAMP signal. A positive promoter element responding to negative control by cAMP was inactivated by deletion of a DNA region between base pairs ‐340 and ‐364. Upstream fragments including this element confer negative cAMP control to a LEU2‐lacZ fusion gene. Northern analysis of CTT1 expression in the presence or absence of heme, in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat‐shocked cells (37 degrees C) shows that CTT1 is only induced to an appreciable extent when at least two of the three factors contributing to its expression (oxidative stress signaled by heme, nutrient starvation (low cAMP) and heat stress) activate the CTT1 promoter.

Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids. This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases. This study shows that expression of the S. cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients. Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene. Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway. Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway. Mutants characterized in this investigation fall into five complementation groups. Both cdc25 and ras2 alleles were identified among these mutants.

Both IGH translocations and chromosome 13q deletions are early events in monoclonal gammopathy of undetermined significance and do not evolve during transition to multiple myeloma.

Molecular and genetic events associated with the transition from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) are still poorly characterized. We investigated serial bone marrow specimens from 11 patients with MGUS who eventually progressed to MM (MM post-MGUS) by interphase fluorescence in situ hybridization for immunoglobulin heavy-chain gene (IgH) translocations and chromosome 13q deletions (del(13q)). In nine patients, IgH translocations were present both in MGUS and MM post-MGUS plasma cells, including three t(11;14)(q13;q32) and one t(4;14)(p16;q32), which was observed already 92 months prior to MM. Similarly, all five MM patients with del(13q) had this aberration already at the MGUS stage. Two patients without IgH translocation and del(13q) had chromosomal gains suggesting hyperdiploidy, but IgH translocations and/or del(13q) did not emerge at MM post-MGUS. IgH translocations and del(13q) are early genetic events in monoclonal gammopathies, suggesting that additional events are required for the transition from stable MGUS to progressive MM.

The transforming growth factor-beta signaling pathway in tumorigenesis.

Transforming growth factor-β is believed to play a dual role in carcinogenesis. Through its ability to inhibit cellular proliferation it suppresses tumor development in its early stages, but in the course of tumor progression malignant cells often acquire resistance to growth inhibition by transforming growth factor-β and themselves secrete large amounts of this cytokine. Transforming growth factor-β furthers malignant progression in two ways: for one, it acts on nontransformed cells present in the tumor mass to suppress antitumor immune responses and to augment angiogenesis. Secondly, it promotes invasion and the formation of metastases in a cell-autonomous manner that requires transforming growth factor-β signaling activity, albeit at reduced levels, to be present in the tumor cells themselves.

The leukaemia‐associated transcription factors EVI‐1 and MDS1/EVI1 repress transcription and interact with histone deacetylase

EVI‐1 and its variant form, MDS1/EVI1, have been reported to act in an antagonistic manner and be differentially regulated in samples from patients with acute myeloid leukaemia and rearrangements of the long arm of chromosome 3. Here, we show that both EVI‐1 and MDS1/EVI1 can repress transcription from a reporter construct containing EVI‐1 binding sites and interact with histone deacetylase in mammalian cells. This interaction can be recapitulated in vitro and is mediated by a previously characterized transcription repression domain, whose activity is alleviated by the histone deacetylase inhibitor trichostatin A.