Whole exome sequencing is an efficient, sensitive and specific method for determining the genetic cause of short-rib thoracic dystrophies.

Short-rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were

c(4 × 2) and related structural units on the SrTiO3(001) surface: scanning tunneling microscopy, density functional theory, and atomic structure.

Density functional theory is used to simulate high-bias, constant-current scanning tunneling micrographs for direct comparison with experimental images. Coupled to previous spectroscopic data, these simulations are used to determine the atomic structure of Ti-rich nanostructures on strontium titanate (001) surfaces. These nanostructures have three consecutive TiO(x) surface layers and exploit the distinctive structural motif of the c(4 × 2) reconstruction as their main building block. A structural model of a characteristic triline defect is also proposed.

Tumour necrosis factor and PI3-kinase control oestrogen receptor alpha protein level and its transrepression function.

Oestrogen receptor alpha (ERalpha) is an oestrogen-activated transcription factor, which regulates proliferation and differentiation of mammary epithelial cells by activating or repressing gene expression. ERalpha is a critical prognostic indicator and a therapeutic target for breast cancer. Patients with tumours that express higher level of ERalpha have better prognosis than patients with tumours that are ERalpha negative or express lower level of ERalpha. Better prognosis in ERalpha-positive patients is believed to be due to repression of proinvasive gene expression by ERalpha. Oestrogen receptor alpha represses gene expression by transrepressing the activity of the transcription factors such as nuclear factor-kappaB or by inducing the expression of transcriptional suppressors such as MTA3. In this report, we show that ERalpha transrepresses the expression of the proinvasive gene interleukin 6 (IL-6) in ERalpha-negative MDA-MB-231 breast cancer cells stably overexpressing ERalpha. Using these cells as well as ERalpha-positive MCF-7 and ZR-75-1 cells, we show that tumour necrosis factor alpha (TNFalpha) and the phosphatidylinositol-3-kinase (PI3-kinase) modulate transrepression function of ERalpha by reducing its stability. From these results, we propose that TNFalpha expression or PI3-kinase activation lead to reduced levels of ERalpha protein in cancer cells and corresponding loss of transrepression function and acquisition of an invasive phenotype.

Transformation of interleukin-3-dependent cells without participation of Stat5/bcl-xL: cooperation of akt with raf/erk leads to p65 nuclear factor kappaB-mediated antiapoptosis involving c-IAP2.

Tyrosine kinase oncogenes such as p210BCR-ABL activate multiple signal pathways. As a result, it is difficult to infer the functional relevance of a pathway acting alone or in cooperation with another. One or 2 second-tier kinases represented in the p21ras and phosphatidylinositol-3-kinase (PI-3-kinase) pathways (activated RafCAAX and gag-akt, respectively) were expressed in parental H7 interleukin-3 (IL-3)-dependent myeloid cells. IL-3-dependent cells served, independently, as recipients of p210BCR-ABL, which activated p21ras and PI-3-kinase pathways, including raf/erk and akt, respectively, en route to transformation. By contrast, neither RafCAAX nor gag-akt when expressed in parental cells in isolation produced factor-independent cells. On the other hand, H7 cells expressing both RafCAAX and gag-akt (H7gag-akt/RafCAAX) were transformed. Such transformation in H7gag-akt/RafCAAX was accomplished in the absence of active versions of Shc or cbl, and there was no evidence of Stat activity and only modest amounts of bcl-xL, a Stat5 transcriptional target protein, all of which characterized the cells transformed by BCR-ABL. However, H7gag-akt/RafCAAX cells and H7BCR-ABL cells cultured in the absence of IL-3 shared strikingly increased p65 nuclear factor kappaB (NFkappaB) activity. Treatment of cells with a specific NFkappaB inhibitor, parthenolide, led to loss of NFkappaB activity and down-regulation of antiapoptotic c-IAP2. In cells with only gag-akt/RafCAAX, this was sufficient to allow polyADP ribosyltransferase (PARP)-degradative apoptosis, but in cells with p210BCR-ABL, apoptosis was blocked, possibly by a Stat5/bcl-xL-dependent mechanism. Therefore, one hematopoietic antiapoptotic program, among others, available to certain tyrosine kinase oncogenes involves a cooperative response between raf/erk and akt, unambiguous components of p21ras and PI-3-kinase pathways, to induce p65 NFkappaB and c-IAP2.

Cell cycle effects of nonsteroidal anti-inflammatory drugs and enhanced growth inhibition in combination with gemcitabine in pancreatic carcinoma cells.

Increased cyclooxygenase-2 (COX-2) expression in human pancreatic adenocarcinomas, as well as the growth-inhibitory effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro, suggests that NSAIDs may be an effective treatment for pancreatic cancer. Gemcitabine is currently the most effective chemotherapeutic drug available for patients with pancreatic cancer, but is only minimally effective against this aggressive disease. Clearly, other treatment options must be identified. To design successful therapeutic strategies involving compounds either alone or in combination with others, it is necessary to understand their mechanism of action. In the present study, we evaluated the effects of three NSAIDs (sulindac, indomethacin, and NS-398) or gemcitabine in two human pancreatic carcinoma cell lines, BxPC-3 (COX-2-positive) and PaCa-2 (COX-2-negative), previously shown to be growth-inhibited by these NSAIDs. Effects on cell cycle and apoptosis were investigated by flow cytometry or Western blotting. Treatment with NSAIDs or gemcitabine altered the cell cycle phase distribution as well as the expression of multiple cell cycle regulatory proteins in both cell lines, but did not induce substantial levels of apoptosis. Furthermore, we demonstrated that the combination of the NSAID sulindac or NS-398 with gemcitabine inhibited cell growth to a greater degree than either compound alone. These results indicate that the antiproliferative effects of NSAIDs and gemcitabine in pancreatic tumor cells are primarily due to inhibition of cell cycle progression rather than direct induction of apoptotic cell death, regardless of COX-2 expression. In addition, NSAIDs in combination with gemcitabine may hold promise in the clinic for the treatment of pancreatic cancer.

Phosphorylation site specificity of the Pak-mediated regulation of Raf-1 and cooperativity with Src.

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pak-mediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor- and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell.

Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2.

Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.

Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association.

The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.

Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338.

We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.

Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak.

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85Delta, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.

Cyclooxygenase-2 expression in human pancreatic adenocarcinomas.

Cyclooxygenase-2 (COX-2) expression is up-regulated in several types of human cancers and has also been directly linked to carcinogenesis. To investigate the role of COX-2 in pancreatic cancer, we evaluated COX-2 protein expression in primary human pancreatic adenocarcinomas (n = 23) and matched normal adjacent tissue (n = 11) by immunoblot analysis. COX-2 expression was found to be significantly elevated in the pancreatic tumor specimens compared with normal pancreatic tissue. To examine whether the elevated levels of COX-2 protein observed in pancreatic tumors correlated with the presence of oncogenic K-ras, we determined the K-ras mutation status in a subset of the tumors and corresponding normal tissues. The presence of oncogenic K-ras did not correlate with the level of COX-2 protein expressed in the pancreatic adenocarcinomas analyzed. These observations were also confirmed in a panel of human pancreatic tumor cell lines. Furthermore, in the pancreatic tumor cell line expressing the highest level of COX-2 (BxPC-3), COX-2 expression was demonstrated to be independent of Erk1/2 activation. The lack of correlation between COX-2 and oncogenic K-ras expression suggests that Ras activation may not be sufficient to induce COX-2 expression in pancreatic tumor cells and that the aberrant activation of signaling pathways other than Ras may be required for up-regulating COX-2 expression. We also report that the COX inhibitors sulindac, indomethacin and NS-398 inhibit cell growth in both COX-2-positive (BxPC-3) and COX-2-negative (PaCa-2) pancreatic tumor cell lines. However, suppression of cell growth by indomethacin and NS-398 was significantly greater in the BxPC-3 cell line compared with the PaCa-2 cell line (P = 0.004 and P < 0.001, respectively). In addition, the three COX inhibitors reduce prostaglandin E(2) levels in the BxPC-3 cell line. Taken together, our data suggest that COX-2 may play an important role in pancreatic tumorigenesis and therefore be a promising chemotherapeutic target for the treatment of pancreatic cancer.

Lack of elevated MAP kinase (Erk) activity in pancreatic carcinomas despite oncogenic K-ras expression.

Activating mutations within the K-ras gene have been found in up to 90% of pancreatic carcinomas. Although multiple Ras effector pathways have been identified, the Raf protein kinases which are upstream regulators of the mitogen-activated protein kinases (MAPK/Erk) are believed to be the primary mitogenic effectors. Constitutive upregulation of this pathway by oncogenic ras is thought to promote cellular transformation. To explore the biological effects of mutated K-ras, we analyzed the Ras signaling pathway in a panel of cell lines derived from human pancreatic carcinomas. We found that despite high levels of Ras-GTP in each cell line expressing mutant K-ras, elevated levels of active Erk1 and Erk2 were not detectable under conditions of exponential growth or serum-starvation. Depending upon the cell line, the block in Erk signaling was observed to occur at either the level of Raf or Erk. Increased levels of active Erk1 and Erk2 were detected in only 2 out of 10 normal tissue-matched primary pancreatic tumors with mutated K-ras. Our results suggest that Erk signaling is not aberrantly upregulated in pancreatic cancers containing oncogenic K-ras mutations. The lack of Erk activation observed in both cell lines and primary tumor tissue suggests that constitutive Erk activation may not be required for tumor maintenance or progression in K-ras transformed pancreatic cells. We hypothesize that other Ras-dependent signaling pathways or an unidentified Raf/Mek-dependent pathway may be important for carcinogenesis in the pancreas. These findings may have important implications for drug treatment strategies which currently target the MAP kinase branch of the Ras signaling pathway.

The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338.

The pathway involving the signalling protein p21Ras propagates a range of extracellular signals from receptors on the cell membrane to the cytoplasm and nucleus. The Ras proteins regulate many effectors, including members of the Raf family of protein kinases. Ras-dependent activation of Raf-1 at the plasma membrane involves phosphorylation events, protein-protein interactions and structural changes. Phosphorylation of serine residues 338 or 339 in the catalytic domain of Raf-1 regulates its activation in response to Ras, Src and epidermal growth factor. Here we show that the p21-activated protein kinase Pak3 phosphorylates Raf-1 on serine 338 in vitro and in vivo. The p21-activated protein kinases are regulated by the Rho-family GTPases Rac and Cdc42. Our results indicate that signal transduction through Raf-1 depends on both Ras and the activation of the Pak pathway. As guanine-nucleotide-exchange activity on Rac can be stimulated by a Ras-dependent phosphatidylinositol-3-OH kinase, a mechanism could exist through which one Ras effector pathway can be influenced by another.

In vitro inhibition of Ras-Raf association by short peptides.

Seven amino acid peptides were tested as in vitro inhibitors of oncogenic Ras-Raf association. The sequences of these peptides were derived from the H-Ras effector region (amino acids 25 to 51) and the Ras binding domain of Raf-1 (amino acids 64 to 105). Eleven out of the twenty-one Ras 7-mers tested inhibited formation of the Ras-Raf complex by at least 20% at 100 microM. The most potent of these inhibitory peptides contained the effector residues 32 to 37 or 40 to 45. Of the Raf-1 peptides tested, only the 94-ECCAVFR-100 and 95-CCAVFRL-101 peptides were significant inhibitors of Ras-Raf binding. The 95-101 Raf peptide had an IC50 value of 7 microM and also inhibited Ras-RalGDS binding. Analysis of the 95-101 peptide showed that its inhibitory activity required at least one cysteine followed by several hydrophobic residues. Our results demonstrate the feasibility of using small molecules as inhibitors of Ras protein-protein interactions.

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase. Creating the perfect farnesyltransferase.

Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly. The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type. The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates. The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL. The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate. These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated. The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures. Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.

Characterization of the Ras binding domain of the RalGDS-related protein, RLF.

The Ras binding domain (RBD) of Rlf, a member of the RalGDS family of proteins, was characterized. Using an ELISA-based technique, the relative binding affinity of Rlf for a variety of mutant Ras proteins was determined. Rlf had significantly different binding characteristics than the Raf-1 RBD. The minimal effective Ras binding domain was defined as residues 657-778 using N- and C-terminal deletions of Rlf. Using the PHD algorithm, the secondary structure of this domain was predicted to be similar to the ubiquitin superfold previously identified in the Raf-1 RBD. When the predicted secondary structure of the Rlf-RBD was aligned with the known secondary structure of the Raf-RBD, amino acids in Raf-1 essential for Ras binding were found to also be conserved in Rlf. Consistent with this observation, alanine substitution of one of these residues (K687) in Rlf significantly reduced affinity for Ras-GTP.

An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ.

The function of the c-Raf-1 zinc finger domain in the activation of the Raf kinase was examined by the creation of variant zinc finger structures. Mutation of Raf Cys 165 and Cys 168 to Ser strongly inhibits the Ras-dependent activation of c-Raf-1 by epidermal growth factor (EGF). Deletion of the Raf zinc finger and replacement with a homologous zinc finger from protein kinase C gamma (PKC gamma) (to give gamma/Raf) also abrogates EGF-induced activation but enables a vigorous phorbol myristate acetate (PMA)-induced activation. PMA activation of gamma/Raf does not require endogenous Ras or PKCs and probably occurs through a PMA-induced recruitment of gamma/Raf to the plasma membrane. The impaired ability of EGF to activate the Raf zinc finger variants in situ is attributable, at least in part, to a major decrement in their binding to Ras-GTP; both Raf zinc finger variants exhibit decreased association with Ras (V12) in situ upon coexpression in COS cells, as well as diminished binding in vitro to immobilized, processed COS recombinant Ras(V12)-GTP. In contrast, Raf binding to unprocessed COS or prokaryotic recombinant Ras-GTP is unaffected by Raf zinc finger mutation. Thus, the Raf zinc finger contributes an important component to the overall binding to Ras-GTP in situ, through an interaction between the zinc finger and an epitope on Ras, distinct from the effector loop, that is present only on prenylated Ras.

Mutational analysis of the beta-subunit of yeast geranylgeranyl transferase I.

The gene CAL1 (also known as CDC43) of Saccharomyces cerevisiae encodes the beta subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded by cal1-1, and cdc43-2 to cdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. The cal1-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rho1p was observed in the cal1-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal1-1 is most efficiently suppressed by overproduction of Rho1p. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal1-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.

p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues.

Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids. Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation. Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure. The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions. However, Arg786, Lys831, and Arg925 were observed to be essential for binding to Ras-GTP but not for protein structure. RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras. This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins. Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.