Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR ß mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 µM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of ß-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR ß, as well as RXR a, ß and g expression was low in both HC11 and HC11ras cells. In addition, RAR ß mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Hypertension et régulation de l'expression moléculaire de l'angiotensinogène par la ribonucléoprotéine hétérogène nucléaire K

Le diabète est une maladie chronique dont la principale caractéristique est un niveau plasmatique élevé de glucose, qui est causé soit par un défaut dans la production d’insuline, l’action de l’insuline, ou les deux à la fois. Plusieurs études ont démontré que l’hyperglycémie chronique peut mener à la dysfonction et même la défaillance de plusieurs organes, dont le coeur, le système vasculaire, les yeux et les reins, se traduisant par des infarctus du myocarde, des accidents cérébro-vasculaires et des complications rétinales et rénales, respectivement. La néphropathie diabétique (DN) est la principale cause de déficience rénale et affecte près de 25-40% des patients diabétiques. La DN est invariablement associée à un risque élevé d’accident cérébrovasculaire et de dysfonction cardivasculaire. L’angiotensinogène (Agt) est l’unique précurseur de tous les types d’angiotensines. En plus du système rénine-angiotensine (RAS) sytémique, le rein possède son propre système intrarénal et exprime tous les composants du RAS. L’Agt est fortement exprimé dans les cellules du tubule proximal rénal (RPTC) et y est converti en angiotensine II (AngII), le peptide biologiquement actif du RAS. Les patients diabétiques présentent de hauts niveaux d’AngII et une augmentation de l’expression des gènes du RAS, suggérant que l’activation du RAS intrarénal joue un rôle important dans la progression de la DN. Les mécanismes qui contrôlent la régulation du niveau rénal d’Agt par l’hyperglycémie et l’insuline demeurent mal compris. Le but global de cette thèse est de mieux comprendre les mécanismes moléculaires qui contrôlent l’expression du gène Agt chez la souris Akita (un modèle murin de diabète de type 1). Dans cette optique, la première partie de la thèse se concentre sur deux facteurs de transcription de la famille des ribonucléoprotéines nucléaires hétérogènes (hnRNP). Chan et collaborateurs ont déjà identifié 2 protéines nucléaires hnRNP F et hnRNP K, de 48kD et 70kD respectivement. HnRNP F et hnRNP K forment un hétérodimère et se lient à l’élément de réponse à l’insuline (IRE) présent dans le promoteur du gène Agt du rat et inhibent la transcription du gène Agt in vitro. Afin de déterminer si hnRNP F / K sont responsables de l’inhibition de l’expression rénale de Agt par l’insuline in vivo, nous avons étudié des souris Akita males traités ou non avec des implants d’insuline pour une période de 4 semaines. Des souris non-Akita males ont été employées comme contrôles. Les souris Akita développent de l’hypertension et de l’hypertrophie rénale. Le traitement à l’insuline rétablit les niveaux de glucose plasmatiques et la pression systolique (SBP), et atténue l’hypertrophie rénale, l’albuminurie (ratio albumine/créatinine urinaire, ACR) et les niveaux urinaires d’Agt et AngII chez les souris Akita. De plus, le traitement à l’insuline inhibe l’expression rénale du gène Agt, tout en augmentant l’expression des gènes hnRNP F, hnRNP K et ACE2 (enzyme de conversion de l’angiotensine-2). Dans des RPTC in vitro, l’insuline inhibe Agt, mais stimule l’expression de hnRNP F et hnRNP K en présence de hautes concentrations de glucose, et ce via la voie de signalisation MAPK p44/42 (protéine kinase activée par un mitogène). La transfection avec des petits ARN interférents (siRNA) contre hnRNP F et hnRNP K prévient l’inhibition de l’expression d’Agt par l’insuline dans les RPTC. Cette étude démontre bien que l’insuline prévient l’hypertension et atténue les dommages rénaux observés chez les souris Akita diabétiques, en partie grâce à la suppression de la transcription rénale de Agt, via une augmentation de l’expression de hnRNP F et hnRNP K. La seconde partie de cette thèse change de focus et se tourne vers le facteur Nrf2 (nuclear factor erythroid 2-related factor 2). Nrf2 est un facteur de transcription qui contrôle les gènes de la réponse antioxydante cellulaire en réponse au stress oxydant ou aux électrophiles. Le but de cette étude est d’examiner l’impact de la surexpression de la catalase (Cat) dans les RPTC sur l’expression du gène Agt via Nrf2 et sur le développement de l’hypertension et des dommages rénaux résultants chez les souris diabétiques Akita transgéniques (Tg). Nos études ont démontré que la surexpression de Cat dans les souris Akita Cat-Tg normalise la SBP, atténue les dommages rénaux et inhibe l’expression des gènes Nrf2 et Agt dans les RPTC. In vitro, le glucose élevé (HG) et l’oltipraz (un activateur de Nrf2) stimulent l’expression de Nrf2 et Agt, et cet effet peut être bloqué par la trigonelline (inhibiteur de Nrf2), des siRNA contre Nrf2, des antioxydants ou des inhibiteurs pharmacologiques NF-κB et MAPK p38. La suppression de sites de réponse à Nrf2 présents dans le promoteur du gène Agt du rat abolit la stimulation par l’oltipraz. Finalement, des souris males adultes non-transgéniques traitées avec l’oltipraz montrent une augmentation de l’expression de Nrf2 et Agt dans leurs RPTC et cette augmentation peut être normalisée par la trigonelline. Ces données permettent d’identifier un nouveau mécanisme d’action de Nrf2, par la stimulation du gène Agt intrarénal et l’activation du RAS, qui induisent l’hypertension et les dommages rénaux par le glucose élevé et les espèces réactives de l’oxygène chez les souris diabétiques. Nos conclusions permettent de démontrer que l’insuline induit l’expression de hnRNP F et hnRNP K, qui jouent ensuite un rôle protecteur en prévenant l’hypertension. La surexpression de la catalase dans les RPTC vient quant à elle atténuer l’activation de Nrf2 et ainsi réduit la SBP chez les souris Akita.

Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Dominant transformation by mutated human ras genes in vitro requires more than 100 times higher expression than is observed in cancers

The gene-mutation-cancer hypothesis holds that mutated cellular protooncogenes, such as point-mutated proto-ras, “play a dominant part in cancer,” because they are sufficient to transform transfected mouse cell lines in vitro [Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994) Molecular Biology of the Cell (Garland, New York)]. However, in cells transformed in vitro mutated human ras genes are expressed more than 100-fold than in the cancers from which they are isolated. In view of the discrepancy between the very low levels of ras transcription in cancers and the very high levels in cells transformed in vitro, we have investigated the minimal level of human ras expression for transformation in vitro. Using point-mutated human ras genes recombined with different promoters from either human metallothionein-IIA or human fibronectin or from retroviruses we found dominant in vitro transformation of the mouse C3H cell line only with ras genes linked to viral promoters. These ras genes were expressed more than 120-fold higher than are native ras genes of C3H cells. The copy number of transfected ras genes ranged from 2–6 in our system. In addition, nondominant transformation was observed in a small percentage (2–7%) of C3H cells transfected with ras genes that are expressed less than 20 times higher than native C3H ras genes. Because over 90% of cells expressing ras at this moderately enhanced level were untransformed, transformation must follow either a nondominant ras mechanism or a non-ras mechanism. We conclude that the mutated, but normally expressed, ras genes found in human and animal cancers are not likely to “play a dominant part in cancer.” The conclusion that mutated ras genes are not sufficient or dominant for cancer is directly supported by recent discoveries of mutated ras in normal animals, and in benign human tissue, “which has little potential to progress” [Jen, J., Powell, S. M., Papadopoulos, N., Smith, K. J., Hamilton, S. R., Vogelstein, B. & Kinzler, K. W. (1994) Cancer Res. 54, 5523–5526]. Even the view that mutated ras is necessary for cancer is hard to reconcile with (i) otherwise indistinguishable cancers with and without ras mutations, (ii) metastases of the same human cancers with and without ras mutations, (iii) retroviral ras genes that are oncogenic without point mutations, and (iv) human tumor cells having spontaneously lost ras mutation but not tumorigencity.

Novel Ras antagonist blocks human melanoma growth

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5–50 μM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

The Multisubstrate Docking Site of the MET Receptor Is Dispensable for MET-mediated RAS Signaling and Cell Scattering

The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association–kinase assay we found that the mutated receptor can still associate and phosphorylate a ∼250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase–extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering.

Ras Pathway Activates Epithelial Na+ Channel and Decreases Its Surface Expression in Xenopus Oocytes

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+ channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together with XK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control, XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12V led to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC′s was also decreased; however, in comparison with progesterone-treated oocytes, XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressed XENaC was increased by XK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting that XK-Ras2AG12V increased the mean activity of XENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

l-Selectin activates the Ras pathway via the tyrosine kinase p56lck

Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827–872 and Butcher, E. C. (1991) Cell 67, 1033–1036]. In this study we show that l-selectin triggering of Jurkat cells using different antibodies or glycomimetics resulted in activation of the src-tyrosine kinase p56lck; tyrosine phosphorylation of intracellular proteins, in particular mitogen-activating protein kinase and l-selectin; and association of Grb2/Sos with l-selectin. This association correlated with an activation of p21Ras, mitogen-activating protein kinase, Rac2, and a transient increase of O2− synthesis. Stimulation of the Ras pathway by l-selectin requires functional p56lck, since p56lck-deficient Jurkat cells (JCaM1.6) do not show tyrosine phosphorylation, association of l-selectin with Grb2/Sos, and activation of Ras upon l-selectin triggering. Transfection of JCaM1.6 cells with p56lck reconstitutes the observed signaling events. Genetic inhibition of Ras or Rac2 prevented Rac2 stimulation and O2− synthesis, respectively. The specificity and the physiological significance of the observed signaling cascade is indicated by stimulation of l-selectin-transfected P815, l-selectin-positive CEM or peripheral blood lymphocytes resulting in the same activation events as in Jurkat cells. Our results point to a signaling cascade from l-selectin via p56lck, Grb2/Sos, Ras, and Rac2 to O2− .

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells.

Benzodiazepine (BZA)-5B, a CAAX farnesyl-transferase inhibitor, was previously shown to block the farnesylation of H-Ras and to reverse the transformed morphology of Rat1 cells expressing oncogenic H-RasV12. Non-transformed Rat1 cells were not affected by BZA-5B, suggesting that they produce a form of Ras whose prenylation is not blocked by this compound. The likely candidate is K-RasB, which differs from H-Ras primarily in the terminal 24 amino acids. In the current study we examined the effect of BZA-5B on the prenylation of a chimeric oncogenic Ras protein designated H/K-RasBV12, consisting of the first 164 amino acids of H-RasV12 followed by the last 24 amino acids of K-RasB. BZA-5B failed to block the prenylation of this chimera and was thus unable to reverse the transformed morphology of Rat1 cells in which it was expressed. Another potent inhibitor of H-Ras farnesylation, L-739,749, also failed to block prenylation of H/K-RasBV12. Similar results were obtained in transfected cells expressing a widely used version of K-RasBV12 containing a 10-amino acid extension at its NH2 terminus. Neither BZA-5B nor L-739,749 reversed the transformed morphology of cells expressing H/K-RasBV12. The resistance of K-RasB to farnesyltransferase inhibition provides a likely explanation for the resistance of nontransformed cells to the growth inhibitory effects of BZA-5B and L-739,749.

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.