KSR stimulates Raf-1 activity in a kinase-independent manner

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we find that murine KSR (mKSR1) translocates from the cytoplasm to the plasma membrane in the presence of activated Ras. At the membrane, mKSR1 modulates Ras signaling by enhancing Raf-1 activity in a kinase-independent manner. The activation of Raf-1 is mediated by the mKSR1 cysteine-rich CA3 domain and involves a detergent labile cofactor that is not ceramide. These findings reveal another point of regulation for Ras-mediated signal transduction and further define a noncatalytic role for mKSR1 in the multistep process of Raf-1 activation.     

MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1.     

Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.     

Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.     

A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function

Ras and Raf-1 are key proteins involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Raf-1 functions downstream of Ras in many signaling pathways. Although Raf-1 directly associates with GTP-bound Ras, an effect of this interaction on Raf-1 activity in vivo has not been established. To examine the biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In this report, we show that a single amino acid mutation in Raf-1 (Arg89 to Leu) disrupted the interaction with Ras in vitro and in the yeast two-hybrid system. This mutation prevented Ras-mediated but not tyrosine kinase-mediated enzymatic activation of Raf-1 in the baculovirus/Sf9 expression system. Furthermore, kinase-defective Raf-1 proteins containing the Arg89-->Leu mutation were no longer dominant-inhibitory or capable of blocking Ras-mediated signal transduction in Xenopus laevis oocytes. These results demonstrate that the association of Raf-1 and Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg89 as a critical residue involved in this interaction. In addition, the finding that tyrosine kinases can stimulate the enzymatic activity of Raf-1 proteins containing a mutation at the Ras-interaction site suggests that Raf-1 can be activated by Ras-independent pathways.     

Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation

OBJECTIVE: To develop a method for the detection of bilateral Horner's syndrome in patients with bilateral interruption of the cervical sympathetic pathway or widespread autonomic neuropathy. METHODS: Darkness pupil diameters and redilatation times during light reflexes have been recorded with infrared TV pupillometry in 65 healthy subjects, 47 patients with unilateral Horner's syndrome, and 20 patients with bilateral Horner's syndrome. The aetiologies of the last group were diabetic autonomic neuropathy (three cases), amyloidosis (four), pure autonomic failure (PAF) (four), dopamine-beta-hydroxylase deficiency (two), and one case each of hereditary sensory and autonomic neuropathy (HSAN) type III, carcinomatous sympathetic neuropathy, familial dysautonomia, multiple system atrophy, Anderson-Fabry disease, and anterior spinal artery thrombosis at C5,6 and one had had bilateral cervical sympathectomies. RESULTS: Darkness diameters on the affected side were below normal in 12 patients with unilateral Horner's syndrome, the measurement yielding only 26% sensitivity for detection of the condition. By contrast, the time taken to reach three quarter recovery in the light reflex (T3/4) was abnormally prolonged (redilatation lag) in 33 of the same eyes. The measurement yielded 70% sensitivity and 95% specificity for detection of the condition. In 20 cases, diagnosed on clinical grounds as having bilateral Horner's syndrome of various aetiologies, pupil diameters were abnormally small on both sides in five and on one side in three patients. Fourteen of these patients had significant redilatation lag in both eyes, five patients in one eye, and one patient had it in neither eye. Measurement of redilatation lag was therefore a more sensitive diagnostic test than pupil diameter in both unilateral and bilateral Horner's syndrome. CONCLUSIONS: Provided that the pupils are not tonic, bilateral Horner's syndrome can be diagnosed on the basis of redilatation lag. It occurs clinically in some generalised autonomic neuropathies and with interruption of the local sympathetic nerve supplies to the two eyes.     

Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity

We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.     

Protein kinase C-epsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity

Several observations indicate that the Raf-1 kinase is a downstream effector of protein kinase C-epsilon (PKC epsilon). We recently have shown that Raf-1 is constitutively activated in PKC epsilon transformed Rat6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Cacace et al., 1996). Cai et al. (1997) demonstrated that PKC epsilon induced proliferation of NIH3T3 cells is independent of Ras or Src, but depends on Raf-1. These authors further suggested that PKC epsilon activates Raf-1 by direct phosphorylation. Here we have investigated the functional interaction between PKC epsilon and Raf-1. PKC epsilon, but not PKC alpha, was found to bind to the Raf-1 kinase domain. The association appeared to be direct, as it could be reconstituted in vitro with purified proteins. Raf-1 and PKC epsilon could be co-precipitated from Sf-9 insect cells and PKC epsilon transformed NIH313 cells (NIH/epsilon). The association was negatively regulated by ATP in vitro and by TPA treatment in NIH/epsilon cells, but not in Sf-9 insect cells. Raf-1 was constitutively activated in NIH/epsilon cells. However, using coexpression experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PKC epsilon. PKC epsilon did not induce translocation of Raf-1 to the membrane. Furthermore, PKC epsilon did not activate Raf-1 nor enhance the kinase activity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase. In contrast, conditioned media from PKC epsilon transformed cells induced a robust activation of Raf-1. This activation could be partially reproduced by recombinant TGFbeta, a growth factors secreted by PKC epsilon transformed Rat6 cells. In conclusion, our results suggest that PKC epsilon stimulates Raf-1 indirectly by inducing the production of autocrine growth factors.     

Characterization of Raf-1 activation in mitosis

We have used site-directed mutagenesis to explore the mechanisms underlying Raf-1 activation in mitosis, and we have excluded most previously characterized activating interactions. Our results indicate that the primary locus of activation lies in the carboxyl-half of the molecule, although the extent of activation can be influenced by the amino-proximal region, particularly by the Raf-1 zinc finger. We also found that Raf-1 is hyperphosphorylated in mitosis at multiple sites within residues 283-302 and that these hyperphosphorylations are not required for activation. In addition, neither Mek1 nor Mek2 are stably activated in coordination with Raf-1 in nocodazole-arrested cells. Overall, the data suggest that the mechanism(s) responsible for activating Raf-1 during mitosis, and the subsequent downstream effects, are distinct from those involved in growth factor stimulation.     

TSG-6 interacts with hyaluronan and aggrecan in a pH-dependent manner via a common functional element: implications for its regulation in inflamed cartilage

Glycogen from the thermophilic eubacterium Thermus thermophilus has been characterized by enzymatic, chemical and spectroscopic analysis. With an average chain length of seven glucose units, the glycogen from T. thermophilus is one of the most highly branched glycogens known. In contrast to other bacterial species, in T. thermophilus, accumulation of glycogen appears not be affected by low nitrogen concentration. For the first time, alpha-glucan phosphorylase activity and glycogen content were measured throughout the growth cycle of T. thermophilus in order to gain insight into glycogen metabolism. In contrast to the situation that prevails in Escherichia coli, additional carbon sources had no effect on alpha-glucan phosphorylase activity in T. thermophilus. Maximal activity of the thermophilic enzyme was found in the early logarithmic phase of growth, suggesting a function of the alpha-glucan phosphorylase in T. thermophilus as an outgrowth-specific enzyme.     

The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr

bcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl.     

Mechanisms regulating Raf-1 activity in signal transduction pathways

Raf-1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf-1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein-protein interactions play in regulating the biological and biochemical activity of Raf-1. Our studies have provided evidence that the activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Recently, we reported that Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. Our findings have also shown that phosphorylation events play a role in regulating Raf-1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (14-3-3). Results from our work suggest that Raf-1 is regulated at multiple levels by several distinct mechanisms.     

Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

ALL-1 interacts with unr, a protein containing multiple cold shock domains

The ALL-1 gene is involved in human acute leukemia through chromosome translocations and fusion to partner genes, or through partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which transregulates the homeotic genes of the Antennapedia and bithorax complexes controlling body segment identity. ALL-1 encodes a very large protein of 3968 amino acids which presumably interacts with many proteins. Here we applied yeast two hybrid screening to identify proteins interacting with the N-terminal segment of ALL-1. One protein obtained in this way was the product of the unr gene. This protein consists of multiple repeats homologous to the cold shock domain (CSD), a motif common to some bacterial and eukaryotic nucleic acids-binding proteins. The minimal region on unr required for the interaction with ALL-1 included two CSD and two intervening polypeptides. The interaction was confirmed by in vitro binding studies, and by coimmunoprecipitation from COS cells overexpressing the relevant segments of the two proteins. These results suggest that unr is involved in an interaction of ALL-1 with DNA or RNA.     

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.     

The oestrogen receptor regulates NFkappaB and AP-1 activity in a cell-specific manner

It has recently been identified the PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2 represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di- and tripeptides and of selected peptidomimetics. Here the functional expression of PEPT2 in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter. Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85-87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide 3H-D-Phe-Ala with an app. K0.5 of 0.143 +/- 0.016 mM. Inhibition of 3H-D-Phe-Ala uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPT2 expressing cell clone in which transport function can be monitored by a simple yeast growth test was established. In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast. The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPT2 or for selection of mutants generated by random mutagenesis.     

Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.