Identification of SAS4 and SAS5, two genes that regulate silencing in Saccharomyces cerevisiae.
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In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the alpha-mating phenotype to MATalpha HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia. (+info)
Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine.
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Ras malignant transformation requires posttranslational modification by farnesyltransferase (FTase). Here we report on the design and antitumor activity, in monotherapy as well as in combination therapy with cytotoxic agents, of a novel class of non-thiol-containing peptidomimetic inhibitors of FTase and the closely related family member geranylgeranyltransferase I (GGTase I). The non-thiol-containing FTI-2148 is highly selective for FTase (IC50, 1.4 nM) over GGTase I (IC50, 1700 nM), whereas GGTI-2154 is highly selective for GGTase I (21 nM) over FTase (IC50, 5600 nM). In whole cells, the corresponding methylester prodrug FTI-2153 is >3000-fold more potent at inhibiting H-Ras (IC50, 10 nM) than Rap1A processing, whereas GGTI-2166 is over 100-fold more selective at inhibiting Rap1A (IC50, 300 nM) over H-Ras processing. Furthermore, FTI-2153 was highly effective at suppressing oncogenic H-Ras constitutive activation of mitogen-activated protein kinase and human tumor growth in soft agar. FTI-2148 suppressed the growth of the human lung adenocarcinoma A-549 cells in nude mice by 33, 67, and 91% in a dose-dependent manner. Combination therapy of FTI-2148 with either cisplatin, gemcitabine, or Taxol resulted in a greater antitumor efficacy than monotherapy. GGTI-2154 in similar antitumor efficacy experiments is less potent than FTI-2148 and inhibits tumor growth by 9, 27, and 46%. Combination therapy of GGTI-2154 with cisplatin, gemcitabine, or Taxol is also more effective. Finally, FTI-2148 and GGTI-2154 are 30- and 33-fold more selective and 30- and 16-fold more potent in whole cells than our previously reported thiol-containing FTI-276 and GGTI-297, respectively. Thus, our results demonstrate that this highly potent and selective novel class of non-thiol-containing peptidomimetics inhibits human tumor growth in whole animals and that combination therapy with cytotoxic agents is more beneficial than monotherapy. (+info)
Yeast Ku protein plays a direct role in telomeric silencing and counteracts inhibition by rif proteins.
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Yku70p/Yku80p, the yeast Ku protein homologue, is a DNA end-binding heterodimer involved in non-homologous end joining. It also binds to telomeres, where it plays an important role in the maintenance of telomeric DNA structure [1] [2] [3] [4] [5]. Ku protein, together with Rap1p, a telomeric DNA (TG(1-3) repeat)-binding protein, is also required to initiate transcriptional silencing, or telomere-position effect (TPE). Here, we provide evidence for a direct role of Ku in TPE, which is most likely to be in either the recruitment or activation of Sir4 protein at the telomere. Surprisingly, however, the essential role of Ku in TPE is to overcome the inhibitory effect of two Rap1p-interacting proteins, Rif1p and Rif2p, both of which also play an important role in telomere length regulation [6] [7]. Previous studies showed that Rif and Sir proteins compete for binding to the carboxyl terminus of Rap1p [7] [8] [9]. In the absence of this competition, for example, when RIF genes are mutated, Ku is no longer necessary for TPE, whereas the Rap1p carboxyl terminus is still absolutely required. We show that Rif1p is localized to telomeres, indicating that its inhibitory effect on TPE is direct. Our data implicate a role for Ku in the competition between Sir and Rif proteins for access to the telomeric array of Rap1p molecules, which results in a balance between telomeric silencing and telomere length control. (+info)
A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.
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The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases. (+info)
RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans.
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A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution. (+info)
PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2.
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The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain. (+info)
Sequential regulation of the small GTPase Rap1 in human platelets.
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Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with alpha-thrombin. For this activation, a phospholipase C-mediated increase in intracellular calcium is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Indeed, the PKC activator phorbol 12-myristate 13-acetate induced Rap1 activation, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin alpha(IIb)beta(3), a downstream target of PKC, with monoclonal antibody LIBS-6 also induced Rap1 activation. However, studies with alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann's thrombasthenia type 1 show that alpha(IIb)beta(3) is not essential for Rap1 activation. Interestingly, induction of platelet aggregation by thrombin resulted in the inhibition of Rap1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, alpha-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin alpha(IIb)beta(3). Inactivation of Rap1 is mediated by an aggregation-dependent process that correlates with the translocation of Rap1 to the cytoskeletal fraction. (+info)
Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase.
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Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions. (+info)