Simian Virus 40 Gene A Regulates the Association Between a Highly Phosphorylated Protein and Chromatin and Ribosomes in Simian Virus 40-Transformed Cells

The species of proteins associated with chromatin and ribosomes of simian virus 40 (SV40)-transformed and untransformed monkey, mouse, and rat cells have been compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after phosphorylation of these proteins either in vivo or in vitro. In vitro phosphorylation was carried out by protein kinase associated with these organelles and [gamma-(32) P]ATP as the phosphoryl donor. The reaction products contained both phosphoserine and phosphothreonine in approximately equal amounts. The electrophoretic analysis of the phosphorylated proteins revealed that the highly phosphorylated protein with a molecular weight of approximately 90,000 (90K protein) was associated with chromatin and ribosomes from transformed cells but not from untransformed cells. The 90K protein could be extracted from chromatin and ribosomes with 0.5 to 1.0 M NaCl or KCl. The 90K protein was still associated with the runoff ribosomes prepared by the puromycin reaction of the post-mitochondrial supernatant in the protein-synthesizing system. In vitro phosphorylation of chromatin and ribosomes from SV40 tsA-transformed mouse and rat cells indicated that the amounts of 90K protein associated with these organelles decreased greatly when the cells were cultivated at the restrictive temperature. A similar temperature-dependent decrease in the amount of (32)P-labeled 90K protein was observed in nonhistone chromosomal and ribosome-associated protein fractions prepared from SV40 tsA-transformed cells labeled with [(3)H]leucine and [(32)P]orthophosphate in vivo. In vitro phosphorylated 90K protein in nonhistone chromosomal and ribosome-associated proteins extracted with high salt was not immunoprecipitated with anti-SV40 T sera.     

Purification and properties of a non-histone chromatin protein with a molecular weight of 38,000 daltons specific for transformed rat cells

Non-histone chromatin proteins prepared from a normal rat cell line (No. 7) and the cells transformed with Rous sarcoma virus (RSV) (s7-1) were compared by means of reverse-phase high performance liquid chromatography (reverse-phase HPLC), followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that several proteins were specifically present in the transformed cell chromatin. A specific non-histone chromatin protein with a molecular weight of 38,000 daltons, 38K protein, was purified as a single species from s7-1 cells. This 38K protein was only detected in the transformed state of the cells transformed with a temperature-sensitive (ts) mutant of the src gene and the mutant cells which showed temperature sensitivity as to the transformation with wild type RSV.     

Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro.

Two species of polyoma virus middle T antigen were detected in both lytically infected and transformed cells by in vitro kinase assay of immunoprecipitates. A minor species with an apparent molecular weight of 58,000 (58K) represented less than 10% of the total middle T protein. This species was roughly 10 times more active as a phosphate acceptor than was the predominant 56K form. Partial proteolytic mapping experiments showed that the same site was phosphorylated in both species. Mapping of the middle T antigens from a series of deletion mutants suggested that the major site of phosphorylation is tyrosine residue 315. Phosphorylation occurred on both middle T species in vivo, involving sites predominantly other than the tyrosine labeled in vitro. The 56K and 58K middle T forms differed from each other in their in vivo phosphorylation patterns. Some phosphate was incorporated into the 58K species in a region of the molecule to which at least part of the apparent molecular weight different could be mapped. hr-t mutant NG-59, which codes for a slightly altered middle T, produced only a single species (56K) which was inactive in the in vitro kinase reaction. Moreover, no 58K species appeared in vivo with this mutant. hr-t mutants are therefore defective in both aspects of phosphorylation. Phenotypically normal revertant cells of a polyoma transformed line failed to express any middle T antigens or associated kinase activity.     

The C-terminal domain of the heavy chain of tetanus toxin rescues cerebellar granule neurones from apoptotic death: involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways

When cultured cerebellar granule neurones are transferred from a medium containing high extracellular potassium concentration ([K+]e) (25 mm) to one with lower [K+]e (5 mm), caspase-3 activity is induced and cells die apoptotically. In contrast, if cells in non-depolarizing conditions are treated with brain-derived neurotrophic factor (BDNF), caspase-3 activity, chromatin condensation and cell death are markedly diminished. In this study, we show that the C-terminal domain of the tetanus toxin heavy-chain (Hc-TeTx) is able to produce the same neuroprotective effect, as assessed by reduction of tetrazolium salts and by chromatin condensation. Hc-TeTx-conferred neuroprotection appears to depend on phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase, as is demonstrated by the selective inhibitors Wortmannin and PD98059, respectively. Hc-TeTx also induces phosphorylation of the tyrosine kinase BDNF receptor, activation of p21Ras in its GTP-bound form, and phosphorylation of the cascade including extracellular-signal-regulated kinases-1/2 (ERK-1/2), p90 ribosomal S6 kinase (p90rsk) and CREB (cAMP-response-element-binding protein). On the other hand, activation of the Akt pathway is also detected, as well as inhibition of the active form of caspase-3. These results point to an implication of both PI3K- and ERK-dependent pathways in the promotion of cerebellar granule cell survival by Hc-TeTx.     

Suppression of the mTOR-raptor signaling pathway by the inhibitor of heat shock protein 90 geldanamycin

Heat shock protein 90 (Hsp90) was co-immunoprecipitated with raptor, the binding partner of the mammalian target of rapamycin (mTOR) from HEK293 cells. Hsp90 was detected in the anti-raptor antibody immunoprecipitates prepared from the cell extract by immunoblot analysis using the anti-Hsp90 antibody, and the association of these two proteins was confirmed by immunoprecipitation from the cells co-expressing Hsp90 and raptor as epitope-tagged molecules. Geldanamycin, a potent inhibitor of Hsp90, disrupted the in vivo binding of Hsp90 to raptor without affecting the association of raptor and mTOR, and suppressed the phosphorylation by mTOR of the downstream translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The protein kinase activity of S6K as well as the phosphorylation of the substrate, 40S ribosomal protein S6, were lowered in the geldanamycin-treated cells. These results indicate that Hsp90 is involved in the regulation of protein translation by facilitating the phosphorylation reaction of 4E-BP1 and S6K catalyzed by the mTOR/raptor complex through the association with raptor, and that the mTOR signaling pathway is a novel target of geldanamycin.     

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.     

Mitogen-mediated protein phosphorylation in Werner's syndrome fibroblasts

DNA synthesis of WF-1 fibroblasts derived from a patient with Werner's syndrome was stimulated by fetal calf serum and adult human serum but not by various mitogens including epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). To clarify the cause of nonresponsiveness to these mitogens, we compared the rate of protein phosphorylation in normal fibroblasts HF-O and Werner's WF-1 cells. PDGF and TPA enhanced the phosphorylation of a Mr 80 K protein, which is known to be a substrate for protein kinase C, both in HF-O and WF-1 cells. This indicates that the pathway involving PDGF receptor, phosphatidylinositol turnover and protein kinase C activation is operational in WF-1 cells. Several species of phosphoproteins of Mr 250 K, 135 K, 110 K, 78 K and 42 K were detected in normal HF-O cells by immunoprecipitation using an anti-phosphotyrosine antibody. The same species of phosphoproteins were detected in Werner's WF-1 cells at passage 6, but only when treated with various mitogens and were not detected in WF-1 cells at passage 10 even after the PDGF- or TPA-treatment. These results suggest that the reduction of phosphorylation of these target proteins may be in part responsible for the diminished mitogenic responsiveness of Werner's fibroblasts.     

Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant

Centromeric H3-like histones, which replace histone H3 in the centromeric chromatin of animals and fungi, have not been reported in plants. We identified a histone H3 variant from Arabidopsis thaliana that encodes a centromere-identifying protein designated HTR12. By immunological detection, HTR12 localized at centromeres in both mitotic and meiotic cells. HTR12 signal revealed tissue- and stage-specific differences in centromere morphology, including a distended bead-like structure in interphase root tip cells. The anti-HTR12 antibody also detected spherical organelles in meiotic cells. Although the antibody does not label centromeres in the closely related species Arabidopsis arenosa, HTR12 signal was found on all centromeres in allopolyploids of these two species. Comparison of the HTR12 genes of A. thaliana and A. arenosa revealed striking adaptive evolution in the N-terminal tail of the protein, similar to the pattern seen in its counterpart in Drosophila. This finding suggests that the same evolutionary forces shape centromeric chromatin in both animals and plants.     

Transformed mammalian cells secrete specific proteins and phosphoproteins.

We have examined the proteins secreted into the growth medium by normal and transformed cells. Transformed cell lines from several mammalian species all secrete proteins in the 58,000 dalton molecular weight range. These proteins are all immunologically related and are secreted at low levels or not at all by the parental normal cell lines. Secretion of the 58K proteins occurs with either DNA or RNA virus transformation and with spontaneous transformation. The transformed cells also secrete phosphoproteins in the same size range, but these are immunologically distinct from the 58K proteins mentioned above. The sizes of the phosphoproteins are species-specific and unrelated to the transforming virus. Incubation of conditioned media from transformed cell cultures with gamma-32P-ATP labels phosphoproteins of the same sizes, indicating the presence in the media of both protein kinase and substrate. All three properties (58K protein, phosphoprotein, in vitro phosphorylation) are closely correlated with transformation in cells transformed by temperature-sensitive viruses. The biological implications of these results remain unknown, but the results may be relevant to recent data on the (phospho)proteins and protein kinase encoded by RNA tumor viruses and the molecular basis of the transformed phenotype.     

Correlation between phosphorylation of a 34,000-molecular-weight protein, pp60src-associated kinase activity, and tumorigenicity in transformed and revertant vole cells.

The phosphorylation of a 34,000-molecular-weight (34K) cell protein, purported to be a substrate of the avian retrovirus pp60src-associated protein kinase activity, was compared in three types of Rous sarcoma virus-infected vole cells: fully transformed cells, partial revertants which are morphologically normal in appearance but retain their tumorigenic potential, and full revertants which are similar to normal vole cells in all parameters including a lack of tumorigenicity. Although similar amounts of 34K protein are present in all three cell types, phosphorylation of the 34K protein was significantly reduced in the full revertant cell type. The reduced phosphorylation occurred at the tyrosine residue.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections.     

Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation

We have studied the phosphorylation of progesterone receptors (PR) in T47Dco human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A- (Mr approximately 94,000) and B- (Mr approximately 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [32P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [32P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [32P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A- and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A- and B-receptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020. Both agonists lead to hyperphosphorylation of nuclear PR before phosphorylation levels decrease, in parallel with the drop in protein levels as receptors down-regulate. Treatment of cells with RU 486, an antiprogestin, leads to PR transformation as determined by immunoblotting, but subsequent down-regulation does not occur. After transformation, chromatin-bound RU 486-occupied receptors become intensely phosphorylated however, with specific activities 15 times greater than those of untransformed PR. Since these receptors are phosphorylated but not processed, the hormone-induced nuclear phosphorylation of PR is unlikely to be a signal for receptor processing.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

NEURONAL PERIKARYA WITH DISPERSED, SINGLE RIBOSOMES IN THE VISUAL CORTEX OF MACACA MULATTA

Numerous small and medium-sized neuronal perikarya in layers III and IV of the visual cortex display an unusual pattern of ribosomal distribution. Instead of being aggregated in clusters, spirals, rows, and other regular polysomal configurations, the ribosomes, whether free or attached to the endoplasmic reticulum, are randomly dispersed, with no discernible pattern. The endoplasmic reticulum in such cells is reduced to a few (perhaps only one) meandering, broad cisternae, which delimit broad fields of cytoplasmic matrix occupied almost solely by scattered, single ribosomes. The Golgi apparatus is elaborate. Mitochondria are either small and numerous or large and infrequent. The other organelles, including the nucleus and nucleolus, are not remarkable. Axonal terminals synapse in the normal fashion on the surfaces of these cells and their dendrites. Associated with these cells are more numerous intermediate cells in which a few to many polysomal clusters can be found. It is proposed that the neurons with dispersed, single ribosomes are inactive in protein synthesis and that the suspension of such an important metabolic activity is probably temporary. Thus, these cells are considered to be part of a population undergoing cyclic fluctuations in the intensity of protein synthesis that should be correlated with their specific neural behavior.     

Inhibition of the cellular actions of nerve growth factor by staurosporine and K252A results from the attenuation of the activity of the trk tyrosine kinase.

The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.     

Phosphorylation of the ribosomal protein S6 is elevated in cells transformed by a variety of tumor viruses.

We have analyzed the phosphorylation of the ribosomal protein S6 in several cultures of normal cells and cells transformed with RNA or DNA tumor viruses. Serum deprivation of confluent, normal cells results in low levels of S6 phosphorylation. In contrast, under identical conditions, this protein is highly phosphorylated in virally transformed cells. No differences in the phosphopeptides generated by one-dimensional limited proteolysis were detected in S6 prepared from serum-stimulated or transformed cells. These results suggest that a common event in oncogenic transformation is the phosphorylation of S6.     

Membrane protein redistribution during differentiation of cultured human erythroleukemic cells.

Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium. In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin. However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation. Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membranes. A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 micrometers in diameter, is divided from the nucleus-containing part of the cell. With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin.     

VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis,p70, and p90 S6 kinase phosphorylation

FVIIa binding to tissue factor (TF) and subsequent signal transduction have now been implicated in a variety of pathophysiological processes, including cytokine production during sepsis, tumor angiogenesis and neoangiogenesis, and leukocyte diapedesis. The molecular details, however, by which FVIIa/TF affects gene expression and cellular physiology, remain obscure. Here we show that FVIIa induces a transient phosphorylation of p70/p85(S6K) and p90(RSK) in BHK cells stably transfected with either full-length TF or with a cytoplasmic domain-truncated TF but not in wild type BHK cells. Phosphorylation of these kinases was also observed in HaCaT cells, expressing endogenous TF. Phosphorylation of p70/p85(S6K) coincided with protein kinase B and GSK-3beta phosphorylation. Activation of p70/p85(S6K) was sensitive to inhibitors of phosphatidylinositol 3-kinase and to rapamycin, whereas phosphorylation of p90(RSK) was sensitive to PD98059. FVIIa stimulation of p70/p85(S6K) and p90(RSK) correlated with phosphorylation of the eukaryotic initiation factor eIF-4E, up-regulation of protein levels of eEF1alpha and eEF2, and enhanced [(35)S]methionine incorporation. These effects were not influenced by inhibitors of thrombin or FXa generation and were strictly dependent on the presence of the extracellular domain of TF, but they did not require the intracellular portion of TF. We propose that a TF cytoplasmic domain-independent stimulation of protein synthesis via activation of S6 kinase contributes to FVIIa effects in pathophysiology.