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Originally published In Press as doi:10.1074/jbc.M112153200 on February 27, 2002

J. Biol. Chem., Vol. 277, Issue 18, 15621-15628, May 3, 2002
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Rapid Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein C1/C2 in Response to Physiologic Levels of Hydrogen Peroxide in Human Endothelial Cells*

James R. StoneDagger and Tucker Collins§

From the Departments of Pathology, Children's Hospital and Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, December 19, 2001, and in revised form, February 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hydrogen peroxide (H2O2) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 µM) of H2O2 has been identified. Using a functional proteomic approach, nuclear extracts from human umbilical vein endothelial cells were analyzed by two-dimensional PAGE with or without prior treatment with a low concentration of H2O2. A protein doublet with a molecular mass of 39-41 kDa and a pI of ~5.0 was observed to be consistently altered by the treatment. Using proteolytic peptide mass fingerprinting, the protein was identified as heterogeneous nuclear ribonucleoprotein C1/C2, a nuclear restricted, pre-mRNA-binding protein. Upon two-dimensional PAGE, each heterogeneous nuclear ribonucleoprotein-C splice form was present as multiple spots because of differing levels of phosphorylation. Upon treatment with H2O2, there was an increase in phosphorylation at 10-20 min, which partially reversed by 30 min. Subsequently, at 60 min after treatment, a population of unphosphorylated protein was transiently present. The effects were observed with as little as 1 µM H2O2 and were maximal with 5-8 µM H2O2. The H2O2-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Partially reduced oxygen species, often referred to as reactive oxygen species, include superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>), hydrogen peroxide (H2O2), and hydroxyl radical (·OH). Of these species, H2O2 is the only one with the chemical stability required to establish significant steady-state concentrations in vivo (1). H2O2 has the added advantage of being small and uncharged, allowing it to freely diffuse across plasma membranes (2). These properties of relative stability, neutrality, and small size make H2O2 an ideal candidate for a signaling molecule analogous to nitric oxide (·NO).

It has been generally accepted that small quantities of H2O2 "leak" out of the mitochondria during oxidative phosphorylation. It has been estimated that 1-2% of all oxygen consumed in humans is transformed into H2O2 rather than water (3). It has also been known for some time that in leukocytes, NADPH oxidase enzymatically produces O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, which is then rapidly converted to H2O2 by both enzymatic and nonenzymatic routes (for review, see Ref. 4). The activity of this enzyme is responsible for the respiratory burst as part of the inflammatory response. The catalytic component of this multisubunit complex is a heme-containing glycoprotein (gp91phox). An important recent advance in biology has been the demonstration that there are multiple isoforms of this hemoprotein and that some of these isoforms are expressed in human endothelial cells and in human vascular smooth muscle (for reviews, see Refs. 5 and 6). The amount of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> and H2O2 produced by endothelial cells and vascular smooth muscle is considerably less than that produced by leukocytes, suggesting that the low-output enzymes in the vessel wall produce H2O2 for signaling rather than for cytostasis. This would be analogous to the ·NO systems in which an inducible high-output enzyme in macrophages generates ·NO for cytostasis and a constitutive low-output enzyme in endothelial cells generates ·NO for signaling (for review, see Ref. 7).

There is a large and growing list of growth factors, cytokines, and vasoactive modulators that stimulate the production of H2O2 and other reactive oxygen species within mammalian cells (8-17). In most of these cases, downstream effects of the agent can be inhibited by nonspecific antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate or by specific H2O2 scavengers such as catalase and glutathione peroxidase. For example, platelet-derived growth factor-induced DNA synthesis and migration in vascular smooth muscle are inhibited by catalase (17). Thus, the generation of H2O2 appears to be a fundamental aspect of receptor-mediated signaling events in mammalian cells. Furthermore, the presence of a low concentration of H2O2 appears to be required for the proper functioning of numerous signal transduction pathways.

In the absence of additional factors, low concentrations of H2O2 appear to mediate a "pro-life" signal to mammalian cells in culture. Studies in which H2O2 was applied to mammalian cells have shown that low concentrations of H2O2 (<10 µM) stimulate mitogenesis and/or promote survival in a wide variety of cell types, including endothelial cells (18-23). Also, these low concentrations of H2O2 stimulate endothelial migration as well as tube formation in an in vitro model of angiogenesis (20). In addition, oxidized low density lipoprotein induces endothelial proliferation by stimulating the formation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> and H2O2 by activating the NADPH oxidase (24). Likewise, the overexpression of catalase inhibits proliferation of vascular smooth muscle (25). In contrast to the effects seen with low concentrations of H2O2, higher concentrations of H2O2 (50 µM to 1 mM) typically cause cells in culture to undergo growth arrest, apoptosis, and/or necrosis (for review, see Ref. 26).

Currently, the molecular mechanisms by which low concentrations of H2O2 function in mammalian cells are poorly understood. In Escherichia coli and Salmonella, there is a transcription factor named Oxy-R that serves as a sensor for H2O2 (27). Cysteines 199 and 208 of Oxy-R react with H2O2 to from a disulfide bond, resulting in the activation of the transcription factor. The reaction is fast, with a second order rate constant of 105 M-1 s-1, allowing the transcription factor to be activated by low concentrations of H2O2 (28). Once activated by H2O2, Oxy-R stimulates transcription of the bacterial catalase gene. To date, no definitive Oxy-R-like H2O2 sensor has been identified in humans. In fact, there is virtually no information on how human cells respond biochemically to concentrations of H2O2 below 10 µM. Several signal transduction pathways in cultured mammalian cells have been reported to be activated by the application of H2O2, including tyrosine kinases (29-31), mitogen-activated protein kinases (32-34), the epidermal growth factor receptor (35), and the transcription factors AP-1 (36, 37) and nuclear factor-kappa B (38). However, in these circumstances, the concentration of H2O2 required for activation typically ranged from 50 µM to 1 mM, well above the concentrations that would expected to be routinely generated for signaling physiologically. These higher concentrations are best considered as oxidative stress. Thus, although low concentrations of H2O2 (<10 µM) have been shown to be involved in numerous signal transduction pathways and to independently stimulate mitogenesis and survival, there is currently no information on precisely how human cells respond biochemically to these low concentrations of H2O2.

Here we have employed a functional proteomic approach to identify signaling pathways for sensing low physiologic levels of H2O2 in human endothelial cells. With this approach, it was determined that heterogeneous nuclear ribonucleoprotein (hnRNP)1 C1/C2, a nuclear restricted, pre-mRNA-binding protein (for review, see Ref. 39), is rapidly and reversibly phosphorylated in response to low concentrations of H2O2.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords and cultured as described previously (40). HUVECs were prepared confluent in 10-cm dishes at passage 3 in Medium 199 (BioWhittaker, Inc.) containing 20% fetal calf serum, 100 µg/ml heparin (Sigma), and 50 µg/ml endothelial cell growth supplement (Biomedical Technologies, Inc.).

Cell Treatment

Procedure 1-- Confluent endothelial cells were washed twice with Dulbecco's modified Eagle's medium (without phenol red; BioWhittaker, Inc.) and incubated in this medium in the absence of serum for 1 h at 37 °C. The cells were then incubated with the same medium in the absence or presence of varying concentrations of H2O2 (0-21 µM) for 60 min. The cells were washed twice with ice-cold phosphate-buffered saline (67 mM phosphate and 150 mM NaCl, pH 7.0) and harvested by scraping in Buffer A (10 mM Tris, 140 mM NaCl, and 1 mM EDTA, pH 8.0). The cells were then pelleted by spinning at 500 × g for 15 min. Nuclear extracts were prepared as described previously (41). Briefly, the cells were resuspended in Buffer B (10 mM HEPES, 10 mM KCl, 750 µM spermidine, 150 µM spermine, 0.2 mM EDTA, 1 mM dithiothreitol, and 20 µg/ml phenylmethylsulfonyl fluoride, pH 7.9) containing 0.1% Nonidet P-40 and incubated on ice for 10 min. The crude nuclei were isolated by centrifugation at 16,000 × g for 5 min. The crude nuclear pellet was resuspended in 600 µl of Buffer B and then underlaid with 300 µl of Buffer B containing 30% sucrose. The sample was centrifuged at 1300 × g for 10 min. The nuclear pellet was resuspended in 60 µl of Buffer C (20 mM Tris, 75 mM NaCl, 0.5 mM EDTA, and 50% glycerol, pH 8.0) to which was added an equal volume of cold 0.8 M ammonium sulfate. The nuclear slurry was incubated on ice for 20 min and then centrifuged at 16,000 × g for 5 min. The supernatant was applied to a Bio-Spin 6 chromatography column (Bio-Rad) equilibrated with isoelectric focusing (IEF) sample buffer (9 M urea, 65 mM dithiothreitol, 1% CHAPS, and 0.1% Bio-Lyte 3/10 ampholyte (Bio-Rad)).

Procedure 2-- Confluent HUVECs in Medium 199 containing fetal calf serum, heparin, and endothelial cell growth supplement were incubated in the absence or presence of varying concentrations of H2O2 (0-9 µM) for 0-90 min at 37 °C. In some experiments, the cells were treated with catalase (1000 units/ml) or actinomycin D (5 µg/ml), either without H2O2 or added 1 min prior to H2O2. The nuclear pellet was obtained as described for cell treatment Procedure 1. The nuclear pellet was resuspended in 100 µl of IEF sample buffer and then centrifuged at 16,000 × g for 15 min. The supernatant was applied to a Bio-Spin 6 chromatography column equilibrated with IEF sample buffer.

Two-dimensional Electrophoresis

HUVEC nuclear extracts were subjected to IEF using a PROTEAN IEF cell and 17-cm IPG Ready-Strips (pH 3-10 and 4-7; Bio-Rad). Upon completion of IEF, the IPG Ready-Strips were incubated first with equilibration buffer (375 mM Tris, 6 M urea, 2% SDS, and 20% glycerol, pH 8.8) containing 130 mM dithiothreitol for 10 min and then with equilibration buffer containing 135 mM iodoacetamide for 10 min. The strips were placed on 10% polyacrylamide gels and electrophoresed with a constant current of 20 mA/gel. In some experiments, the gels were then fixed and stained using Silver-Stain Plus (Bio-Rad) following the procedure supplied by the manufacturer. Stained gels were analyzed using the program PD-Quest (Bio-Rad) to detect changes in the protein spots upon treatment.

Protein Identification

The portion of the gel containing a protein spot of interest was excised using a razor blade, cutting as close to the spot as possible. The silver-stained spot was then destained by washing the gel spot with 15 mM potassium ferricyanide and 50 mM sodium thiosulfate until clear. The gel portion was washed three times with H2O, followed by two washes with 200 mM ammonium bicarbonate and 50% acetonitrile. The gel portion was then dehydrated in a SpeedVac concentrator (Savant Instruments, Inc.) and rehydrated using 200 mM ammonium bicarbonate containing 0.5 µg of trypsin (Promega). The gel portion was incubated at 37 °C overnight. The reaction was quenched with 1% trifluoroacetic acid, and then the gel spot was washed twice with 60% acetonitrile in 0.1% trifluoroacetic acid. The quench and the two washes were pooled and concentrated to 30% of the initial volume in the SpeedVac concentrator. The sample was then absorbed onto a reverse-phase Zip-Tip (Millipore Corp.). The resin was washed with 0.1% trifluoroacetic acid, and the peptides were eluted with 60% acetonitrile in 0.1% trifluoroacetic acid. The eluate was then concentrated to 30% of the initial volume in the SpeedVac concentrator. The masses of the tryptic peptides were determined at the University of Michigan Protein Core Facility by matrix-assisted laser desorption ionization (MALDI) mass spectrometry using a VESTEC-2000 mass spectrometer. The peptide masses together with the apparent molecular mass and pI of the protein were used to search the NCBI Non-redundant Human Protein Database using the program PROFOUND (42).2

Two-dimensional Immunoblots

Two-dimensional polyacrylamide gels were electroblotted onto polyvinylidene difluoride membrane at 0.2 A for 16 h at 4 °C. Membranes were blocked with 5% nonfat dry milk (Santa Cruz Biotechnology) in Tris-buffered saline/Tween buffer (7 mM Tris, 150 mM NaCl, and 1% Tween 20, pH 7.5) and then incubated with goat anti-hnRNP-C1/C2 polyclonal antibody (Santa Cruz Biotechnology) at 1:100 dilution. After primary incubation and washing, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) at 1:5000 dilution. In some experiments, the membranes were blocked with 5% bovine serum albumin in Tris-buffered saline/Tween buffer, and anti-phospho-casein kinase II substrate monoclonal antibody (Calbiochem) at 1:100 dilution was used as the primary antibody, followed by horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Blots were imaged using an ECL Plus detection kit (Amersham Biosciences). Film was developed using an Eastman Kodak M35A X-Omat processor.

Alkaline Phosphatase Treatment

Nuclear extracts from 107 endothelial cells (prepared by cell treatment Procedure 2) were applied to spin columns (Bio-Rad) equilibrated with 50 mM Tris, 100 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol, pH 7.9. The extracts were then incubated for 2 h at 37 °C in the presence of either alkaline phosphatase (100 units; New England Biolabs Inc.) or the phosphatase inhibitors sodium fluoride (20 mM) and sodium orthovanadate (1 mM). After incubation, the samples were applied to spin columns equilibrated with IEF sample buffer and then subjected to two-dimensional PAGE and immunoblotting.

Miscellaneous Methods

H2O2 concentrations of stock solutions were determined using an epsilon  of 81 M-1 at 230 nm (43). UV-visible absorption spectra were recorded on a Cary 50 Bio UV-Visible spectrophotometer. If not otherwise specified, chemicals were obtained from Sigma.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

H2O2-induced Protein Alteration in HUVEC Nuclear Extracts-- A functional proteomic approach was used to screen for nuclear proteins that are altered by the application of low concentrations of H2O2 to HUVECs. In the screening procedure (cell treatment Procedure 1), confluent HUVECs were serum-starved for 1 h and then treated with a low concentration of H2O2 for an additional hour. Nuclear extracts were prepared from isolated nuclei by ammonium sulfate extraction. With this procedure, nuclear extract from ~107 HUVECs typically yielded ~300 protein spots upon two-dimensional PAGE at pH 3-10 (Fig. 1A). The vast majority of these spots were unaltered by the addition of low concentrations of H2O2. However, one of these protein spots was consistently altered by the application of H2O2 (Fig. 1, A (arrow) and B). This spot appeared as a doublet with molecular masses of 39 and 41 kDa and a pI of ~5.0. Based on silver staining, the lower protein (39 kDa) was present at approximately three times the concentration of the upper protein (41 kDa). Initially, the protein doublet appeared as single broad spots with a pI of 5.00-5.05. One hour after the application of H2O2, each protein composing the doublet appeared as four spots with apparent pI values ranging from 5.00 to 5.15. As shown in Fig. 1B, there was significant alteration of the spot(s) with just 1 µM H2O2, and the alteration appeared to be complete with 8 µM H2O2. The alteration was consistent with a portion of the protein present in the control sample being altered to a higher pI 60 min after treatment with low concentrations of H2O2.


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  		Fig. 1.   Two-dimensional electrophoresis of HUVEC nuclear extract. A, the soluble nuclear extract from 107 HUVECs (after treatment with 8 µM H2O2 for 60 min) was subjected to two-dimensional PAGE, first with isoelectric focusing at pH 3-10, followed by SDS-PAGE on a 10% gel. The arrow indicates the protein further characterized here. The numbers at the top refer to the pH from the first-dimension IEF, and the numbers on the left refer to the apparent molecular mass in kilodaltons. B, shown is the identification of an H2O2-altered protein. The cells were first treated with the indicated concentrations of H2O2 for 1 h. First-dimension IEF was performed at pH 4-7, followed by SDS-PAGE on 10% gels. Displayed are gel portions at pH 4.9-5.2 and containing the 39-41-kDa protein indicated by the arrow in A. After treatment, each of the mass forms was present as four spots with apparent pI values of 5.00, 5.05, 5.10, and 5.15.

Identification of the Altered Protein as hnRNP-C1/C2-- Proteolytic peptide mass fingerprinting was performed on the lower portion of the middle two spots (pI 5.05 and 5.10) present after treating cells with 8 µM H2O2 for 1 h (Fig. 1B). Spots were pooled from a total of 12 gels, utilizing a total of ~3 × 108 endothelial cells. Representative MALDI mass spectra are depicted in Fig. 2. Using the program PROFOUND, the peptide masses were used to search the NCBI Non-redundant Human Protein Database (Table I). Both spots matched hnRNP-C1/C2 with significant Z scores of 2.43 (44). hnRNP-C1/C2 is a nuclear restricted, RNA-binding protein that is present as a heterotetramer (C13C2) in which C1 and C2 are splice variants differing by the presence of an additional 13 amino acids in C2 (45, 46). This heterotetrameric structure explains the doublet pattern observed upon two-dimensional PAGE. Significant regions of the N-terminal RNA-binding portion of the protein could be accounted for by the observed tryptic peptides (Fig. 2C). However, much of the acidic C-terminal regulatory portion of the protein was not identified, suggesting that this portion of the protein may be the site of potential post-translational modifications.


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  		Fig. 2.   Tryptic peptide mass fingerprinting. HUVECs were treated with 8 µM H2O2 for 60 min, followed by two-dimensional PAGE of nuclear extracts and silver staining. The lower spots with apparent pI values of 5.05 and 5.10 in Fig. 1B were subjected to tryptic in-gel digestion and peptide mass fingerprinting. A, a representative MALDI mass spectrum. Asterisks indicate peaks present in the no-protein control spectra. B, MALDI mass spectrum showing two sets of peaks, with each set arising from natural isotopic abundances. C, amino acid sequence for hnRNP-C1 (44). The boldface underlined residues indicate regions with matching peptides from either the spot at pI 5.05 or the spot at pI 5.10.

                              
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Table I
Tryptic peptide assignments
All masses are monoisotopic.

Characterization of the H2O2 Effect on hnRNP-C1/C2-- To more fully explore the effect of H2O2 on hnRNP-C1/C2, the time course was examined. In initial studies, it was observed that serum starvation, as used in the screening procedure, caused subtle alterations in the hnRNP-C1/C2 spot positions (data not shown). Therefore, all subsequent analyses were performed in complete medium (see "Experimental Procedures"). In addition, because hnRNP-C1/C2 has been reported to be resistant to salt extraction (47), the protein was extracted from the nuclei with urea. As depicted by the two-dimensional immunoblots in Fig. 3A, prior to treatment, hnRNP-C1/C2 existed as up to three spots with pI values of 5.00, 5.05, and 5.10. The presence of the form at pI 5.10 was variable; and frequently, only the spots at pI 5.00 and 5.05 were present under resting conditions. At 10-20 min after treatment with 8 µM H2O2, the more acidic spots were enhanced, and a spot at pI 4.95 appeared. This partially reversed by 30 min; and by 45-60 min, a fifth hnRNP-C1/C2 protein spot appeared at pI 5.15. This latter alteration reversed by 90 min. Thus, the apparent post-translational modification status of hnRNP-C1/C2 is complex, with at least five major forms present in human endothelial cells. Low concentrations of H2O2 stimulate the appearance of a more acidic form at 10-20 min, followed by the transient appearance of a more basic form at 45-60 min.


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  		Fig. 3.   Two-dimensional immunoblots of hnRNP-C1/C2. Nuclear extracts from HUVECs were subjected to two-dimensional immunoblotting for hnRNP-C1/C2. Shown are portions of the two-dimensional immunoblots at pH 4.9-5.2. A, HUVECs were treated with 8 µM H2O2 for the indicated times. B, shown is the concentration dependence for the alteration at 20 min. HUVECs were treated for 20 min with the indicated concentrations of H2O2.

Because the appearance of the acidic form at pI 4.95 (10-20 min after treatment) was more proximal in the H2O2 signaling pathway, the concentration dependence of this alteration was determined by immunoblotting. As depicted in Fig. 3B, there was significant formation of the form at pI 4.95 with just 2 µM H2O2, and the alteration appeared to be complete with 5 µM H2O2. Thus, the concentration dependence for the appearance of the form at pI 4.95 (10-20 min after treatment) is in good agreement with that observed for the appearance of the more basic form at pI 5.15 (60 min after treatment) (Fig. 1B).

To verify that the observed effect at 20 min was due to H2O2 and not to the presence of any contaminants, the effect of catalase on the H2O2-induced alteration was determined. Interestingly, the addition of catalase to the cells in the absence of H2O2 caused a modest decrease in the quantity of the more acidic forms of hnRNP-C1/C2 (Fig. 4A). This implies that endogenous levels of H2O2 present during cell culture are sufficient to alter the pI of some of the hnRNP-C1/C2. When added together with H2O2, catalase substantially inhibited the H2O2-induced shift to lower pI values (4.95 and 5.00) at 20 min. Thus, the effect of H2O2 on the pI of hnRNP-C1/C2 is inhibited by a specific H2O2 scavenger.


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  		Fig. 4.   Effect of catalase and actinomycin D. Nuclear extracts from HUVECs were subjected to two-dimensional immunoblotting for hnRNP-C1/C2. Shown are portions of the two-dimensional immunoblots at pH 4.9-5.2. A, HUVECs were treated for 20 min with or without 8 µM H2O2 in the presence or absence of catalase (Cat; 1000 units/ml). B, HUVECs were treated as indicated for 20 min with or without 8 µM H2O2 in the presence or absence of actinomycin D (Act; 5 µg/ml).

The biological role of hnRNP-C1/C2 is to bind pre-mRNA as it is generated in the nucleus (39). Thus, the alteration of this protein seen 20 min after treatment could be a direct effect or could be the result of rapidly synthesized pre-mRNA. To determine whether transcription is required for the H2O2-induced alteration 20 min after treatment, the effect of the transcriptional inhibitor actinomycin D was investigated. As shown in Fig. 4B, the appearance of the more acidic form at pI 4.95 (20 min after H2O2 treatment) was not inhibited by actinomycin D. This observation suggests that the production of new pre-mRNA is not required for the effect and that H2O2 may stimulate the alteration of hnRNP-C1/C2 more directly.

H2O2 Alters the Phosphorylation Status of hnRNP-C1/C2-- It has been demonstrated that hnRNP-C is phosphorylated "in vivo" in HeLa cells (48). The number and sites of phosphorylation have not been determined. The pattern of five spots present on the two-dimensional gels would be consistent with hnRNP-C containing zero to four phosphate groups. It would be expected that the spots on the left (lower pI) would contain more phosphates than the spots on the right. To verify that the pattern of four spots was due to differences in phosphorylation, the two-dimensional electroblot from H2O2-treated HUVECs (60 min after treatment) was probed with a monoclonal antibody specific for the phosphorylated form of casein kinase II substrates. This antibody has cross-reactivity to a wide range of phosphoserine-containing sequences, with the strongest reactivity for phosphoserine residues flanked by acidic residues, as found in the C-terminal portion of hnRNP-C1/C2. At 60 min after treatment, the two hnRNP-C-reactive spots on the left (pI 5.00 and 5.05) showed strong reactivity for this phospho-specific antibody, whereas the third spot from the left (pI 5.10) showed less reactivity (Fig. 5A). The spot on the right at pI 5.15 showed no significant reactivity for the phospho-specific monoclonal antibody. Thus, phosphorylation status does account at least in part for the heterogeneity of hnRNP-C1/C2 in human endothelial cells. Furthermore, this suggests that the basic form at pI 5.15 present transiently 60 min after treatment is the completely unphosphorylated form of the protein.


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  		Fig. 5.   Phosphorylation status of hnRNP-C1/C2. Shown are portions of the two-dimensional immunoblots at pH 4.9-5.2. A, HUVECs were treated for 60 min with 8 µM H2O2. Two-dimensional immunoblots were then probed with either anti-hnRNP-C1/C2 polyclonal antibody (upper panel) or anti-phospho-casein kinase II (CKII) substrate monoclonal antibody (lower panel). B, HUVECs were treated for 20 min with or without 8 µM H2O2. Nuclear extracts were then incubated for 120 min in the presence or absence of alkaline phosphatase (AP), followed by two-dimensional immunoblotting for hnRNP-C1/C2.

To demonstrate that the acidic form of hnRNP-C1/C2 present 20 min after the application of H2O2 resulted from phosphorylation, nuclear extracts were treated with alkaline phosphatase to remove phosphate groups prior to two-dimensional immunoblotting. After treatment with alkaline phosphatase, most of the hnRNP-C1/C2 was present at pI 5.15, consistent with this form being the dephosphorylated form of the protein (Fig. 5B). Importantly, alkaline phosphatase treatment abolished the difference between the H2O2-treated and control samples. The presence of other post-translational modifications could not be completely ruled out. However, these findings indicate that in confluent human endothelial cells, hnRNP-C1/C2 is present predominantly as the diphosphorylated and triphosphorylated forms. Upon treatment with low concentrations of H2O2, there is increased phosphorylation, such that a population of quatramodified protein rapidly forms and is mostly absent by 30 min. At 45-60 min after treatment, there is additional dephosphorylation, with the transient formation of a population of completely unphosphorylated hnRNP-C1/C2.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is considerable evidence that H2O2 functions as a signaling agent in mammalian cells. The compound is small, uncharged, and freely diffusible and thus is analogous to nitric oxide (·NO). Most cells appear to contain a plasma membrane-bound NADPH oxidase that produces O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, which is then converted to H2O2 by both enzymatic and nonenzymatic routes (4-6). In addition, significant H2O2 is believed to diffuse out of the mitochondria (3). Numerous growth factors and cytokines have been shown to stimulate the formation of H2O2 by mammalian cells, and specific H2O2 scavengers such as catalase and glutathione peroxidase have been shown to inhibit several signal transduction pathways (8-17). Typically, low concentrations of H2O2 (<10 µM) appear to be a pro-life signal to mammalian cells in culture, enhancing survival and/or stimulating proliferation (18-23). Higher concentrations of H2O2 (50 µM to 1 mM) typically cause cells in culture to undergo growth arrest, apoptosis, and/or necrosis (26). This latter situation is best described as oxidative stress, and many signaling pathways have been shown to be affected by these higher concentrations of H2O2. In contrast, the pro-life stimulus from low concentrations of H2O2 is best thought of as H2O2 signaling, and there is currently no information on how mammalian cells sense and respond biochemically to these low concentrations of H2O2.

In this study, a functional proteomic approach revealed that hnRNP-C1/C2 is structurally altered by very low concentrations of H2O2 in human endothelial cells. In confluent human endothelial cells, hnRNP-C1/C2 is present predominantly as the diphosphorylated and triphosphorylated forms. Upon treatment with low concentrations of H2O2, there is increased phosphorylation, such that a population of quatramodified protein rapidly forms and is mostly absent by 30 min. At 45-60 min, there is additional dephosphorylation of the protein, with the transient formation of the completely unphosphorylated form. This is the first demonstration of an agonist-stimulated phosphorylation of hnRNP-C1/C2 in cells in culture.

It has been previously demonstrated that hnRNP-C1/C2 is phosphorylated in vivo in HeLa cells (48) and in vitro in HeLa cell nuclear extracts (49-52). The number and locations of phosphorylations present and the kinases involved are not well understood. The protein also appears to undergo a process termed "hyperphosphorylation," which is associated not only with phosphorylation, but also with an increase in the apparent mass of the protein. So-called hyperphosphorylated protein has been observed during mitosis in HeLa cells and upon the addition of naked mRNA or okadaic acid to HeLa cell nuclear extracts (50-52). It seems unlikely that the process being reported here is the same process occurring during hyperphosphorylation because there is no apparent mass change associated with the increase in phosphorylation induced by H2O2. In addition, the failure of actinomycin D to inhibit the phosphorylation argues against the process being the result of newly transcribed pre-mRNA.

Based on in vitro phosphorylation studies, a model has been proposed for how phosphorylation may regulate the binding of pre-mRNA by hnRNP-C1/C2 (49). In this model, it was suggested that the protein is bound to pre-mRNA in a basally phosphorylated state. Additional phosphorylation was proposed to promote the release of the protein from the RNA. Once released, it was proposed that the protein was dephosphorylated, possibly all the way to the unphosphorylated state before binding another pre-mRNA. If this model holds, then it appears that H2O2 may be stimulating the release of a subset of hnRNP-C1/C2 from pre-mRNA as evidenced by the increased phosphorylation at 20 min and the subsequent dephosphorylation. In addition, the model would be supported by noting that the basal protein contains two to three phosphates. The addition of a fourth phosphate may promote the release of the protein from the RNA. Knowledge of the sites of phosphorylation and of the relative binding affinities of the differentially phosphorylated forms will greatly aid in elucidating the mechanisms that regulate pre-mRNA binding by hnRNP-C1/C2.

Thus, the rapid phosphorylation of hnRNP-C1/C2 in response to low concentrations of H2O2 may result in the mobilization or removal of a population of hnRNP-C1/C2 from the nuclear mRNA pool. There are several potential consequences of such an event. Recently, it was reported that the application of an osmotic shock to NIH-3T3 cells stimulated the phosphorylation of hnRNP-A1 and the cytoplasmic accumulation of the protein, resulting in a change in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter (53). Presumably, hnRNP-A1 was acting as a negative effector, and its removal from the pre-mRNA allowed for alternative splicing. Likewise, phosphorylation and removal of a subset of hnRNP-C1/C2 from the pre-mRNA may allow for alternative splicing of a select group of pre-mRNAs. Alternatively, because hnRNP-C1/C2 is a nuclear restricted protein with a nuclear retention sequence (54), the protein must be removed from the mRNA before the mRNA can be exported. It is possible that phosphorylation of hnRNP-C1/C2 regulates a post-transcriptional response to H2O2 in which a select population of existing mRNAs are mobilized for processing and/or export. The mechanism by which H2O2 alters the phosphorylation of hnRNP-C1/C2 and the effect of this phosphorylation on the endothelial nuclear transcriptome are being actively pursued.

The observation that physiologic levels of H2O2 regulate the phosphorylation status of a pre-mRNA-binding protein in endothelial cells has potentially important implications for vascular biology and for the mechanisms of vascular diseases. For example, atherosclerosis is typically more severe at sites in the vasculature where non-laminar shear stress is imparted on the endothelium (for review, see Ref. 55). Such stress stimulates endothelial cells to produce more H2O2 than when exposed to laminar shear stress (56). Thus, in these atherosclerosis-prone sites, hnRNP-C1/C2 may have an altered phosphorylation status, and alternative (and possibly detrimental) post-transcriptional processes may be favored. For example, hnRNP-C1 has been shown to bind selectively to an internal ribosomal entry site in platelet-derived growth factor B chain mRNA and by so doing may regulate the production of this protein (57). The signaling events and potential post-translational modifications that allow hnRNP-C1 to accomplish this are unknown. Additionally, vascular endothelial growth factor stimulates the production of H2O2 by endothelial cells, and at least some of the effects of this factor are inhibit by catalase (58). Vascular endothelial growth factor plays an important role in both angiogenesis and the endothelial response to injury (for review, see Ref. 59). Both of these processes may rely in part on alternative post-transcriptional processes mediated by the H2O2-dependent phosphorylation of hnRNP-C1/C2.

    ACKNOWLEDGEMENTS

We thank Kay Case, Vannessa Davis, and Deanna Lamont for excellent technical assistance in HUVEC isolation and culture and Dr. Tara L. Sander, Dr. Woei-Jong Robert Liu, and Jenny L. Maki for critically reviewing the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant R37 HL35716.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Grant T32 HL07627.

§ To whom correspondence should be addressed: Dept. of Pathology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-5806; Fax: 617-734-4721; E-mail: tcollins@rics.bwh.harvard.edu.

Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M112153200

2 Available at prowl.rockefeller.edu/cgi-bin/ProFound.

    ABBREVIATIONS

The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein; HUVEC, human umbilical vein endothelial cell; IEF, isoelectric focusing; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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