Ubiquitin in stressed chicken embryo fibroblasts

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.     

A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein

Heat shock proteins of chick embryo fibroblasts were analyzed on SDS polyacrylamide gradient gels and were found to include not only three previously well-characterized proteins of 25,000, 73,000, and 89,000 D, but also a 47,000-D protein. Two-dimensional gel electrophoresis revealed that this protein was unusually basic (pI = 9.0) and corresponded to a recently characterized, major gelatin- and collagen-binding protein. The induction of synthesis of this 47,000-D membrane glycoprotein after heat stress of fibroblasts was particularly apparent in preparations isolated by gelatin-affinity chromatography. Regulation of this 47,000-D phosphoprotein was more sensitive than that of three major heat shock proteins in that a substantial stimulation of synthesis occurred at even 42 degrees C, as well as at higher temperature. Phosphorylation of the 47,000-D protein was not altered after heat shock. These studies establish this phosphorylated membrane glycoprotein as a member of the heat shock/stress protein family, and they add collagen binding to the unexpectedly diverse spectrum of biochemical activities induced by exposure of cells to stress.     

The dynamic state of heat shock proteins in chicken embryo fibroblasts

Subcellular fractionation and immunofluorescence microscopy have been used to study the intracellular distributions of the major heat shock proteins, hsp 89, hsp 70, and hsp 24, in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed. Hsp 89 was localized primarily to the cytoplasm except during the restress when a portion of this protein concentrated in the nuclear region. Under all conditions, hsp 89 was readily extracted from cells by detergent. During stress and restress, significant amounts of hsp 70 moved to the nucleus and became resistant to detergent extraction. Some of this hsp 70 was released from the insoluble form in an ATP-dependent reaction. Hsp 24 was confined to the cytoplasm and, during restress, aggregated to detergent-insoluble perinuclear phase-dense granules. These granules dissociated during recovery and hsp 24 could be solubilized by detergent. The nuclear hsps reappeared in the cytoplasm in cells allowed to recover at normal temperatures. Sodium arsenite also induces hsps and their distributions were similar to that observed after a heat shock, except for hsp 89, which remained cytoplasmic. We also examined by immunofluorescence the cytoskeletal systems of chicken embryo fibroblasts subjected to heat shock and found no gross morphological changes in cytoplasmic microfilaments or microtubules. However, the intermediate filament network was very sensitive and collapsed around the nucleus very shortly after a heat shock. The normal intermediate filament morphology reformed when cells were allowed to recover from the stress. Inclusion of actinomycin D during the heat shock--a condition that prevents synthesis of the hsps--did not affect the intermediate filament collapse, but recovery of the normal morphology did not occur. We suggest that an hsp(s) may aid in the formation of the intermediate filament network after stress.     

Arsenite-induced changes in methylation of the 70,000 dalton heat shock proteins in chicken embryo fibroblasts

Methylated lysyl and arginyl residues are present in the two major heat shock proteins, hsp70A and hsp70B, of chicken embryo fibroblasts. Here, we demonstrate that this methylation can be modulated by sodium arsenite, a chemical that increases the synthesis of hsp70. In particular, in hsp70A the amount of epsilon-N-trimethyl-lysine significantly decreases and the amount of epsilon-N-dimethyl-lysine and epsilon-N-monomethyl-lysine increases, while in hsp70B, the quantity of NG-monomethyl-arginine is reduced fivefold after arsenite treatment. To determine the specificity of these changes in methylation the pool size of S-adenosyl-L-methionine (AdoMet) and the total cellular level of methylated protein was measured. After arsenite treatment, no significant change in AdoMet pool size and the level of protein methylation was observed with the exception of an apparent increase in NG-monomethyl-arginine in total cellular protein. Thus, the arsenite-induced changes in methylation of hsp70 polypeptides are not a generalized phenomenon and may reflect a modulation in the structure or function of these two polypeptides after their induced synthesis by this chemical.     

Alterations in protein kinase C type III-alpha during heat shock of rat embryo fibroblasts.

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-alpha isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.     

The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts

The addition of certain amino acid analogues (canavanine, hydroxynorvaline, o-methylthreonine) or a mild heat shock at 45 degrees C caused chicken embryo fibroblasts to increase rapidly the synthesis of three proteins (molecular weights 22,000, 76,000 and 95,000 daltons) to levels which dominate the cells biosynthetic capacity and exceed the level of synthesis of the major cell structural proteins. Actinomycin D blocked the increased synthesis of p22, p76 and p95 in both analogue and heat shock-treated cells, while cycloheximide addition during the "induction" period blocked formation of these proteins only in analoguetreated cells. The elevated levels of synthesis for this set of proteins began to decrease shortly after restoration of the normal amino acid or normal temperature, and the normal pattern of cell protein synthesis was found 8 hr later. Induction of a similar set of proteins was detected in mouse L cells and baby hamster kidney cells after treatment with amino acid analogues or heat shock. Several laboratories have reported synthesis of proteins with similar molecular weights in cells subjected to conditions that alter glucose metabolism, and we speculate that these proteins may be associated with a hexose transport system.     

Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts

Utilizing video-enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and—in some cases—fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial-matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug. The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.     

A rearranged junD transforms chicken embryo fibroblasts.

A complementary DNA clone synthesized from the chicken junD mRNA, containing 5'- and 3'-untranslated sequences, was inserted in the retroviral expression vector RCAS to yield the construct JD. A second RCAS construct (DDDD) contained only the coding domains of JunD. DDDD did not transform upon primary transfection, but JD produced small numbers of transformed cell foci in chicken embryo fibroblast cultures. The virus recovered from these foci, JDV, was moderately transforming for chicken fibroblasts and weakly oncogenic in the animal. Its genome was rearranged, showing evidence for two recombination events. The first crossover was located between 5'-untranslated and coding sequences of junD and incorporated part of the 5'-untranslated region into an open reading frame. The second crossover occurred between junD and gag. The two crossovers generate a single open reading frame of 2064 nucleotides that encodes an 85 kilodalton protein in which sequences in the amino-terminal region of JunD are duplicated. This gag junD reading frame was recloned and then reconstituted into a replication-defective but transformation-competent retrovirus, indicating that the Gag-JunD fusion protein is the effector of transformation. A construct containing this rearranged coding sequence of JunD in Rc/RSV transactivated the collagenase promoter in chicken cells. Southern blot analysis of several independently isolated JunD transformants and deletion analysis of JDV indicated that duplication of a domain in the amino-terminal region of JunD is crucial for transformation and transactivation.     

Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.     

The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.     

Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.     

Lamin B is a prompt heat shock protein

The cellular response to hyperthermia involves the increased synthesis of heat shock proteins (HSPs) within several hours after treatment. In addition, a subset of proteins has been shown to be increased immediately after heating. These “prompt” HSPs are predominantly found in the nuclear matrix–intermediate filament fraction and are not present or detectable in unheated cells. Since the nuclear matrix has been suggested to be a target for heat-induced cell killing, prompt HSPs may play a prominent role in the heat shock response. Using Western blotting and flow cytometry, we found that an increase in the synthesis of lamin B, one of the major proteins of the nuclear lamina, is induced during heating at 45.5°C but not during heating at 42°C. Since it is an abundant protein which is constitutively expressed in mammalian cells, lamin B appears to be a unique member of the prompt HSP family. The kinetics of induction of lamin B during 45.5°C heating did not correlate with the dose-dependent reduction in cell survival. While increased levels of lamin B during 45.5°C heating do not appear to confer a survival advantage directly, a possible role for lamin B in cellular recovery after heat shock cannot be discounted. J Cell Physiol 178:28–34, 1999. © 1999 Wiley-Liss, Inc.     

The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts.

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Increased collagen synthesis in myeloblastosis-associated virus-infected chicken embryo fibroblasts.

Chicken embryo fibroblasts infected with a non-transforming derivative of avian myeloblastosis virus [MAV-2(0)] showed a threefold increase in the biosynthesis of collagen compared to values in normal counterparts. In contrast, non-collagen protein synthesis was unchanged.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.