Biomedical subjects

R Petryshyn

Publications and source records attributed to R Petryshyn.

At least 19 recordsLinked to original sources

PubMed2000

A cell-permeable peptide inhibits activation of PKR and enhances cell proliferation.

The double-stranded RNA dependent protein kinase (PKR) is a negative regulator of cell proliferation and thus itself a target for modulation. We show that a cell-permeable peptide (PRI), containing a conserved double-stranded RNA binding motif found in PKR, inhibits activation of the kinase and activity to phosphorylate its substrate. Further, the PRI-peptide localizes to the cytoplasm of murine embryonic fibroblasts and ablates cellular PKR activation. The PRI-peptide enhances cell proliferation compared to treatment with a variant control peptide, resulting in cultures with increased cell density. We conclude that peptides that interfere with PKR may be useful tools for regulating cell proliferation.

3T3 Cells↗

PubMed1999

Differential phosphorylation of PKR associates with deregulation of eIF-2alpha phosphorylation and altered growth characteristics in 3T3-F442A fibroblasts.

Murine embryonic 3T3-F442A fibroblasts contain elevated levels of a factor (dRF) inhibitory to the phosphorylation of PKR, when cultured under differentiation restrictive (10% cat serum) as compared to permissive conditions (10% fetal bovine serum). Experiments were conducted with the objective of understanding the effect of altered PKR activity on the growth characteristics of 3T3-F442A fibroblasts. Analysis of the phosphoprotein pattern confirmed that the phosphorylation of PKR was reduced in cells cultured in cat serum during specific stages of growth. In a similar manner, evaluation of eIF-2alpha phosphorylation by vertical slab gel iso-electric focusing indicated that inactivation of PKR correlated with reduction of eIF-2alpha phosphorylation. The expression of PKR was confirmed by western blotting ruling out the possibility of diminished protein as the cause of loss of activity. In addition, the expression of dRF coincided with the inactivation of PKR as shown by immunoblotting and phosphorylation studies. The reduction in PKR activity and subsequent deregulation of eIF-2alpha phosphorylation was related to appearance of tumor-like cellular morphology and increased cell density as shown by cell counts and [3H]-thymidine uptake. Taken together, these results support a hypothesis that PKR functions to regulate the growth of 3T3-F442A cells. Furthermore, our findings raise the possibility that deregulation of PKR by endogenous inhibitory molecules, such as dRF, may alter normal growth and differentiation. Such a deregulation of PKR may also contribute to the proliferation of tumor cells.

3T3 Cells↗

PubMed1998

Effect of laminin on the nuclear localization of nucleolin in rat intestinal epithelial IEC-6 cells.

Laminin is a major component of extracellular matrix. The mechanism of action of laminin on cell proliferation, differentiation, and migration is not fully understood. In this study, we investigated the role of extracellular matrix, especially laminin, on the cellular localization of the nuclear protein, nucleolin, and on cell proliferation. Immunofluorescent and western blot analysis indicated that nucleolin was translocated most efficiently to the nucleus in the small intestinal rat epithelial cell line (IEC-6) when cultured on laminin-coated plates. Specifically, nucleolin was observed predominantly in cytoplasm in the cells cultured without laminin. In contrast, nuclear localization was observed in the cells cultured on laminin. This effect of laminin on nucleolin translocation was time-dependent. Laminin was also observed to stimulate proliferation of IEC-6 cells in serum free medium. Our results suggest that laminin alters the distribution of nucleolin which may be an early signal for cell proliferation.

Animals↗

PubMed1996

Peptides derived from the interferon-induced PKR prevent activation by HIV-1 TAR RNA.

The double-stranded RNA-dependent protein kinase (PKR) is believed to mediate cellular antiviral responses, function as a tumor suppressor, and regulate cell growth and differentiation. Its activation is dependent on double-stranded RNA (dsRNA) structures but these interactions are not fully understood. The possibility of direct interaction between dsRNA and the arginine and lysine-rich region of PKR (residues 54-74) was examined using synthetic peptides. We found that addition of a synthetic peptide corresponding to residues 54-74 of murine PKR or residues 60-80 of human PKR inhibited the autophosphorylation and activation of the kinase by either poly(I)-poly(C) or the 82-nucleotide-long TAR RNA. Gel-shift analysis indicated that the peptides disrupted the kinase-TAR complex by binding directly to TAR RNA. These findings delineate at least one dsRNA-binding domain in PKR which may be important for its cellular activation.

Amino Acid Sequence↗

PubMed1996

Effect of interferon on protein translation during growth stages of 3T3 cells.

Interferons (IFNs) elicit a spectrum of biological responses from target cells, including inhibition of proliferation in several types of cells in vivo and in culture. The mechanism of action of IFN is complex and not fully understood. Previous evidence has indicated that part of the antiproliferative effect of IFN is due to modulation of protein translation. Here we report that there is a transient autocrine production of beta-interferon during specific periods of growth of mouse 3T3-F442A and 3T3-C2 cells. Treatment of preconfluent mouse 3T3-C2 cells with interferon reduced protein synthesis in these cells. This reduction began after 3 h of interferon treatment and was correlated with the appearance of phosphorylated double-stranded RNA dependent eIF-2 alpha kinase (PKR) measured in vitro. This inhibition of protein synthesis was associated with diminished exchange of GTP for GDP in the eLF-2.GDP complex. This diminished guanine nucleotide exchange activity was due to the inhibition of eukaryotic initiation factor eIF-2B, the factor required for the dissociation of GDP from eIF-2, and the formation of the functional eIF-2.GTP complex. The autocrine effect of IFN resulted in elevated PKR activity, increased phosphorylation of eIF-2 alpha, and diminished eIF-2B activity. These results suggest that interferon regulates the initiation of protein synthesis by a mechanism involving PKR, eIF-2 alpha phosphorylation, and eIF-2B activity. Since 3T3-F442A cells produce and secrete interferon in a transient fashion during growth, this regulatory mechanism may be significant in the normal growth and differentiation of these cells.

3T3 Cells↗

PubMed1995

A 110-kD nuclear shuttling protein, nucleolin, binds to the neurite-promoting IKVAV site of laminin-1.

The basement membrane protein laminin and the IKVAV-containing sequence from the laminin alpha 1 chain have been found to promote the differentiation of primary neurons and a variety of neural cell lines. We previously reported that a 110-kd IKVAV-binding protein (LBP110) isolated from brain appears to be a member of the beta-amyloid precursor protein (APP) family by immunologic and functional studies, which showed that LBP110/APP is also important in neurite outgrowth (Kibbey et al.: Proc Natl Acad Sci USA 90:10150-10153, 1993). In the preparation of this binding protein, a contaminating IKVAV-binding protein of identical molecular weight, nucleolin, was also identified. Here we have studied the relationship between these binding proteins. We find that nucleolin binds specifically to the IKVAV sequence independently of LBP110/ApP. We have also demonstrated significant levels of nucleolin in mature brain and in differentiating neural cells, suggesting that nucleolin functions not only in cell proliferation and in ribosome biogenesis as was previously reported, but also in the differentiation and maintenance of neural tissue. Our identification of cytoplasmic and cell-surface nucleolin, an IKVAV-binding protein, suggests that this protein may function in signalling by extra-cellular matrix.

Amino Acid Sequence↗

PubMed1993

Inhibition of the dsRNA-dependent protein kinase by a peptide derived from the human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus (HIV) is the etiologic agent leading to the development of acquired immunodeficiency syndrome (AIDS). Interferons (IFNs) are known for eliciting antiviral responses from cells, and studies have indicated that infection with HIV induces the production of IFN. Previous studies have shown that the trans-acting response element (TAR) sequence of HIV-1 mRNA can activate the IFN-induced double-stranded (ds) RNA-dependent protein kinase (DAI). DAI, when activated, is a potent inhibitor of protein synthesis and has been implicated in mediating part of IFN's antiviral activity. Here, we report that a synthetic peptide containing the basic region of HIV Tat protein is effective in preventing the activation of DAI. Evidence is presented that indicates that the Tat peptide exerts its effect by binding to the TAR RNA sequence and thus preventing this RNA from binding to and activating DAI. It appears that in addition to its role in trans-activation, the tat protein may also function to overcome the antiviral activity of IFN by regulating DAI activity. Thus, inhibition of DAI by the Tat protein early in the life cycle of HIV may provide a mechanism by which the virus can escape a translational block imposed by the kinase.

Enzyme Activation↗

PubMed1992

Mechanism of action of a cellular inhibitor of the dsRNA-dependent protein kinase from 3T3-F442A cells.

When mouse 3T3-F442A preadipocyte fibroblasts reach confluence in the appropriate culture medium, their growth is arrested, and the cells undergo terminal differentiation to adipocytes. Two proteins that may be involved in this process are interferon and the interferon-induced double-stranded RNA (dsRNA)-dependent protein kinase (DAI). In 3T3-F442A cells, interferon and DAI are transiently expressed with a maximum level of active kinase appearing at confluence. Interestingly, the level of active DAI was found to be low when cells were maintained under conditions nonpermissive for differentiation. This reduction in DAI was at least partly because of the presence of elevated levels of a specific inhibitor of DAI, termed dRF, which appeared to be a reversible inhibitor of the autophosphorylation (activation) of DAI. In the present study, the mechanism of action of dRF was investigated. Photocross-linking experiments indicated that dRF prevented the binding of ATP to DAI. Since the binding of ATP to DAI is dsRNA-dependent, we examined the effect of dRF on the binding of dsRNA to the kinase using RNA mobility shift assays. dRF was found to prevent the formation of DAI-dsRNA complexes without a direct effect on the dsRNA. This suggests that dRF exerts its effect through an interaction with DAI.

3T3 Cells↗

PubMed1992

Immunolocalization and expression of functional and nonfunctional cell-to-cell channels from wild-type and mutant rat heart connexin43 cDNA.

The carboxyl terminal cytoplasmic domain of distinct gap junction proteins may play an important role in assembly of functional channels as well as differential responsiveness to pH, voltage, and intracellular second messengers. Oligonucleotide-directed site-specific mutagenesis in a paired Xenopus laevis oocyte expression system was used to examine the expression of mRNAs encoding wild-type and carboxyl terminal mutant connexin43 (Cx43) proteins. Oocytes were stripped, injected with mRNA or distilled water (dH2O), preincubated for 16-20 hours, and then paired for 5-10 hours; this process was followed by electrophysiological recording using the dual voltage-clamp technique. Initial experiments compared the relative junctional conductances (Gjs) in oocyte pairs expressing Cx43 (382 amino acid residues) and two truncated mutants lacking most or a portion of the cytoplasmic carboxyl terminal. The shortest mutant (M241) contained 240 amino acid residues and was devoid of all phosphorylatable serine residues in the cytoplasmic tail; its length approximated the length of liver connexin26. The longest mutant (M257) tested contained 256 amino acid residues, including two serine residues. Oocyte pairs expressing M241 yielded a Gj similar to that of oocytes injected with dH2O, whereas M257 yielded a Gj similar to that of oocytes injected with Cx43. Immunoprecipitation studies showed that Cx43, M257, and M241 proteins were readily detectable in oocytes injected with their respective mRNAs, indicating that the lack of Gj observed with the M241 mRNA was not due to reduced translation. Immunocytochemical studies revealed that wild-type and both truncated mutants were localized to the area of cell-to-cell contact between the paired oocytes, indicating that protein targeting to the membrane was not inhibited in oocytes injected with M241 mRNA. Oocyte pairs expressing mutants in which serine residues were replaced with nonphosphorylatable amino acids (serine codon No. 255 AGC was converted to GCC, alanine, designated as M255S----A, and serine codon No. 244 AGC was converted to GGC, glycine, designated as M244S----G) showed Gjs similar to M257, indicating that these serine residues and, by inference, their phosphorylation state are not critical for expression of functional channels. The importance of the length of the carboxyl terminus was assessed by comparing the Gjs in a series of mutants that were intermediate in length between M257 and M241. Gradual shortening of the carboxyl terminus produced a gradual reduction of Gj relative to M257. However, simple deletion of amino acid residues 241-257 from the wild-type Cx43 did not affect Gj relative to M257.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Phosphorylation and proteolytic degradation of nucleolin from 3T3-F442A cells.

The effect of phosphorylation on the proteolysis of nucleolin has been investigated. Nucleolin is readily phosphorylated both in vitro and in vivo. Utilizing phosphorylation assays and immunoblotting with anti-nucleolin serum, we have observed that phosphorylation enhances nucleolin as a substrate for a protease. This protease activity cleaves the protein into a highly phosphorylated 30 kDa peptide and a 72 kDa peptide. The involvement of casein kinase II is suggested since this cleavage is promoted by spermine and inhibited by heparin, which are, respectively, a stimulator and an inhibitor of casein kinase II activity. The molecular identity of the protease and the physiologic significance of the proteolytic cleavage of nucleolin remain to be studied.

3T3 Cells↗

PubMed1991

Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases.

Adenosine Triphosphate↗

PubMed1991

Partial characterization of a cellular factor that regulates the double-stranded RNA-dependent eIF-2 alpha kinase in 3T3-F442A fibroblasts.

The interferon-induced double-stranded RNA-dependent eIF-2 alpha kinase (dsI) has an established role in mediating part of interferon's antiviral effects. Numerous studies have suggested that dsI also has regulatory functions in cells not infected with virus. Our previous results have indicated that the activation of this kinase may be an important regulatory signal in controlling growth arrest of mouse 3T3-F442A fibroblasts prior to their subsequent differentiation to adipocytes. Here, we report that extracts from 3T3-F442A cells cultured under conditions nonpermissive for differentiation exhibit significantly reduced dsI activity and that this reduction is due, at least in part, to the presence of elevated levels of a novel inhibitor of dsI activation (dRF). This inhibitor is also detected in reduced amounts in extracts from cells cultured under conditions which are permissive for differentiation. We have achieved a 1,000-fold purification of dRF activity, and highly purified dRF preparations were found to be greatly enriched for a 15-kDa protein that was greater than 90% pure. Our results indicate that dRF is not a protein phosphatase or protease but a reversible inhibitor of dsI autophosphorylation. In addition, our results imply that dRF is a physiologic regulator of dsI, since dRF activity correlates with the ability of 3T3-F442A cells to undergo adipose conversion.

Adipose Tissue↗

PubMed1989

Disulfide bond formation in the regulation of eIF-2 alpha kinase by heme.

The inhibition of the autophosphorylation of the heme-regulated eukaryotic initiation factor (eIF)-2 alpha kinase (HRI) by hemin is very similar to that produced by thiol oxidation by diamide. The results obtained from the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unphosphorylated and phosphorylated HRI under reducing and nonreducing conditions indicate that hemin promotes disulfide formation in HRI. Hemin-promoted disulfide formation in HRI occurs under quasi-physiological conditions, i.e. 30 degrees C, 10 min at hemin concentrations of 5-10 microM. Under nondenaturing conditions, unphosphorylated HRI, phosphorylated HRI, hemin-treated unphosphorylated HRI, and hemin-treated prephosphorylated HRI are all eluted identically on Sephacryl S-300 column chromatography with an apparent molecular mass of 290,000 daltons. It appears, therefore, that the disulfide formation promoted by hemin occurs within the unit of 290,000 daltons. In addition, hemin treatment of phosphorylated HRI results in the appearance of a disulfide-linked form of higher molecular mass when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. A similar high molecular mass form is observed when HRI is treated with 1,6-bismaleimidohexane, a double sulfhydryl cross-linker agent, and the autophosphorylation of HRI and the phosphorylation of eIF-2 alpha by HRI are greatly diminished; these effects are similar to the effects of hemin on HRI. We conclude that disulfide formation by hemin provides a likely mechanism by which hemin prevents the activation and inhibits the activity of HRI.

Animals↗

PubMed1989

Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism.

All mRNAs of human immunodeficiency virus 1 (HIV-1) contain in their 5' untranslated region a sequence termed TAR that responds to trans-activation by the tat (trans-activating) protein. This RNA sequence assumes a stable secondary structure, and its cap structure is relatively inaccessible. Here we report that these structural properties of the TAR sequence underlie the ability of TAR to inhibit in trans the translation of other mRNAs. This mechanism of translation inhibition involves the activation of the double-stranded RNA-dependent kinase (dsl), which in turn phosphorylates the protein synthesis initiation factor 2 (eIF-2). Mutations in the TAR region that diminish the stability of the secondary structure cause a significant reduction in the trans-inhibition. A similar reduction in the dsl activation occurs when TAR is placed further downstream of the cap structure. This is a clear demonstration of a specific naturally occurring mRNA sequence that can activate dsl. We suggest a novel translational regulatory mechanism that interdigitates the activities of eIF-2 and eIF-4F.

Animals↗

PubMed1988

Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells.

We have previously reported that cultured mouse 3T3-F442A cells exhibit a transient, double-stranded RNA (dsRNA)-dependent phosphorylation of the dsRNA-dependent eIF-2 alpha kinase (eIF-2 alpha, alpha-subunit of the eukaryotic initiation factor 2) (dsI). When dsI is activated by low levels of dsRNA, it is a potent inhibitor of protein synthesis. The transient expression of dsI is due to an autocrine effect of interferon at specific stages of growth and differentiation, and it may represent a mechanism for regulating cell growth and differentiation in 3T3-F442A cells. In this report, the purification of dsI from 3T3-F442A cell cultures by a two-step procedure is described. A specific immune serum to dsI was prepared by immunizing a rabbit with highly pure preparations. Immune precipitation studies demonstrate that the serum reacts with phosphorylated dsI both in vitro and in vivo and with de novo synthesized dsI after induction with interferon. We find that dsI of 3T3 cells can undergo phosphorylation in vitro without the addition of dsRNA and in vivo in the absence of viral infection. These results are consistent with a physiologic role for dsI in the growth and differentiation of these cells.

Animals↗

PubMed1986

The effects of N-ethylmaleimide on the phosphorylation and aggregation of insulin receptors in the isolated plasma membranes of 3T3-F442A adipocytes.

We have examined the insulin-dependent phosphorylation of the insulin receptor in the isolated plasma membranes of 3T3-F442A adipocytes. Phosphorylation of the insulin receptor is detected readily in the plasma membrane of these cells by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulfate and under nonreducing conditions, the predominant species of phosphorylated insulin receptor has an apparent molecular mass of 350,000 daltons with the beta-subunit (92,000 daltons) being phosphorylated. The phosphorylation of the insulin receptor in the isolated plasma membrane is absolutely dependent on insulin; tyrosine residues and, to a lesser extent, serine residues of the receptor are phosphorylated. Treatment of the plasma membrane with N-ethylmaleimide (NEM) has two effects; 1) NEM prevents the formation of a larger form (greater than 350,000 daltons) of the phosphorylated insulin receptor. The formation of this larger form of the receptor involves sulfhydryl oxidation which occurs at 37 degrees C under nondenaturing conditions, but does not occur at 0 degrees C or at room temperature even in the presence of sodium dodecyl sulfate. These results indicate that the larger form of the phosphorylated receptor can occur under physiological conditions and suggest that this process may be relevant to aggregation of the receptor-ligand complex. 2) Prior to treatment with insulin, NEM enhances the phosphorylation of the insulin receptor. Phosphopeptide analysis indicates that the site(s) of phosphorylation of the receptor is identical in the presence or absence of NEM.

Adipose Tissue↗

PubMed1984

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells.

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.

Animals↗

PubMed1984

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases.

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.

Blood Proteins↗