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J. Biol. Chem., Vol. 277, Issue 21, 18365-18372, May 24, 2002

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Phosphorylation by Mitogen-activated Protein Kinase Mediates the Hypoxia-induced Turnover of the TAL1/SCL Transcription Factor in Endothelial Cells^*

Tong Tang, Jack L. Arbiser^§, and Stephen J. Brandt^¶**

From the Departments of  Medicine and ^¶ Cell Biology and  Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232, ^** Veterans Affairs Tennessee Valley Health Care System, Nashville, Tennessee 37232, and ^§ Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, October 10, 2001, and in revised form, February 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The basic helix-loop-helix transcription factor TAL1 (or SCL), originally identified from its involvement by a chromosomal rearrangement in T-cell acute lymphoblastic leukemia, is required for hematopoietic development. TAL1 also has a critical role in embryonic vascular remodeling and is expressed in endothelial cells postnatally, although little is known about its function or regulation in this cell type. We report here that the important proangiogenic stimulus hypoxia stimulates phosphorylation, ubiquitination, and proteasomal breakdown of TAL1 in endothelial cells. Tryptic phosphopeptide mapping and chemical inhibitor studies showed that hypoxia induced the mitogen-activated protein kinase-mediated phosphorylation of a single serine residue, Ser¹²², in the protein, and site-directed mutagenesis demonstrated that Ser¹²² phosphorylation was necessary for hypoxic acceleration of TAL1 turnover in an immortalized murine endothelial cell line. Finally, whereas TAL1 expression was detected in endothelial cells from both large and small vessels, hypoxia-induced TAL1 turnover was observed only in microvascular endothelial cells. Besides their implications for TAL1 function in angiogenic processes, these results demonstrate that a protein kinase(s) important for mitogenic signaling is also utilized in hypoxic endothelial cells to target a transcription factor for destruction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The TAL1 (or SCL) gene encodes a basic helix-loop-helix transcription factor originally identified from its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia (1, 2). TAL1 coding sequences have also been found fused to the promoter of an adjacent gene, SIL (for SCL interrupting locus), as the result of interstitial deletions (3, 4), whereas in other patients with T-cell acute lymphoblastic leukemia, the gene is expressed in the apparent absence of chromosomal rearrangement (5). Three related genes have also been found to be activated in T-cell acute lymphoblastic leukemia (6-8), although significantly less frequently than TAL1. In aggregate, overexpression of this subclass of basic helix-loop-helix genes is the most common gain of function mutation in this form of leukemia (reviewed in Ref. 9).

TAL1 binds DNA with any of the more widely expressed basic helix-loop-helix proteins known as E proteins, including the E12, E47, and E2-5 splice isoforms of the E2A gene, the related E2-2 protein, and the HEB/HTF4 gene products (10, 11). These TAL1-E protein heterodimers recognize a nucleotide motif, CANNTG, termed the E-box to activate or repress transcription (12-15). A variety of adaptor proteins or coregulators, including the LIM domain oncoproteins LMO1 and LMO2 (16, 17), histone acetyltransferases p300/CBP (14) and P/CAF (18), and nuclear corepressors mSin3A and mSin3B (15), also interact with TAL1 and modulate its transcriptional properties. With rare exceptions (19), however, TAL1's target genes remain unidentified.

TAL1 is a critical regulator of hematopoietic differentiation. Tal1 gene inactivation by homologous recombination resulted in midgestational lethality with the complete absence of yolk sac erythropoiesis (20, 21). From analysis of chimeric animals derived from mixtures of Tal1^/ and Tal1^+/+ embryonic stem cells, the gene also appears to be required for generation of lympho-hematopoietic cells of the adult (22, 23). In addition, TAL1 promotes the terminal differentiation of specific hematopoietic lineages (24-26).

Although less well characterized, TAL1 also has a role in blood vessel formation or maturation. Tal1 protein has been observed in endothelial progenitor cells, or angioblasts, and in endothelial and hematopoietic cells of the blood islands of the embryonic yolk sac (27, 28). Furthermore, enforced expression of TAL1 in zebrafish embryos resulted in overproduction of both hematopoietic and endothelial precursors (29), and studies of Tal1^/ embryos in which hematopoiesis was partially rescued by a Gata-1 promoter-driven Tal1 transgene revealed the gene is also required for vascular remodeling (30). Finally, Tal1 expression characterizes vascular and lymphatic endothelial cells of the adult (31).¹ However, its specific actions in this cell type and the extent to which its expression can be modulated by angiogenic molecules are not known.

We used an immortalized murine endothelial cell line to investigate the effects of an important proangiogenic stimulus on Tal1 protein dynamics. We found that hypoxia greatly accelerated Tal1 turnover in these cells through mitogen-activated protein kinase (MAPK)²-mediated phosphorylation, ubiquitination, and proteasomal degradation. In addition to their implications for TAL1's actions in angiogenesis, these studies show that a protein kinase(s) important for mitogenic signaling can also be employed in targeting a transcription factor for destruction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- The MS1 cell line was generated by transduction of pancreatic microvascular endothelial cells with a temperature-sensitive simian virus 40 large T antigen (32). Human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were purchased from Clonetics (San Diego, CA), and COS-7 and HeLa cells were obtained from the American Type Culture Collection (Manassas, VA). The immortalized human bone marrow microvascular endothelial cell line BMEC-1 (33) was provided by Francisco Candal (Centers for Disease Control and Prevention, Atlanta, GA).

MS1 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) with 5% heat-inactivated fetal bovine serum (FBS) in a humidified 10% CO₂ atmosphere at 37 °C. HUVECs were cultured in endothelial growth medium (EGM; Clonetics), and HMVECs were grown in microvascular endothelial growth medium (EGM-MV; Clonetics). COS-7, HeLa, and murine erythroleukemia cells were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated FBS, and BMEC-1 cells were grown in Medium 199 with 15% FBS, 40 µg/ml endothelial cell growth supplement (Sigma), 1 mM L-glutamine, 10 mM HEPES, 16 units/ml heparin, and 25 mM sodium bicarbonate. To induce hypoxia, tissue culture dishes were transferred to a Billups-Rothenberg chamber, which was flushed with a defined gas mixture, sealed, and returned to a 37 °C incubator (34). These mixtures contained 10% CO₂ and either 0%, 0.5%, or 1.0% O₂.

Plasmids-- An expression plasmid (pT123) for influenza virus hemagglutinin (HA) epitope-tagged ubiquitin (35) was provided by Dirk Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany). Construction of pcDNA1-Tal1 and pcDNA1-Tal1^S122A plasmids has been described previously (36). To express Myc epitope-tagged Tal1 and Tal1^S122A proteins, the appropriate cDNA was subcloned into vector pcDNA3.1(+) (Invitrogen), and a nucleotide sequence encoding a human c-Myc epitope was inserted by site-directed mutagenesis immediately before the stop codon (GeneEditor In Vitro Site-Directed Mutagenesis System, Promega, Madison, WI).

Protease Inhibitors-- Protease inhibitors MG132 (N-carbobenzyloxy-L-leucinyl-L-leucinyl-L-leucinal; Sigma), LLM (N-acetyl-L-leucinyl-L-leucinyl-methioninal; Sigma), LLnL (N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; Sigma), and E64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; Sigma) were dissolved in Me₂SO before addition to culture.

Antibodies-- Affinity-purified rabbit polyclonal antibody to mouse Tal1 has been described previously (28). Rabbit polyclonal antibody to human TAL1 was provided by Richard Baer (Columbia University, New York, NY). Purified mouse monoclonal antibody to the HA epitope (16B12) was purchased from BAbCO (now Covance Research Products, Richmond, CA), mouse monoclonal antibody to HIF-1 (NB 100-123) was purchased from Novus Biologicals (Littleton, CO), and mouse monoclonal antibody 9E10 to the human c-Myc epitope was purchased from Sigma.

Western Blot Analysis-- Nuclear extracts were prepared by the method of Dignam et al. (37) and denatured by heating at 95 °C for 3 min in Laemmli buffer (38). Samples were subjected to SDS-PAGE in 10% polyacrylamide gels. After electrophoresis, proteins were electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad), which was incubated in blocking buffer (5% milk, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) at 4 °C for 4 h and then incubated with the indicated antibodies overnight. Binding of the primary antibody was detected by an enhanced chemiluminescence method (ECL Plus; Amersham Biosciences) using horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) or sheep anti-mouse IgG (Amersham Biosciences).

Protein Stability Analysis-- For pulse-chase analysis, MS1 cells were grown in 10-cm-diameter dishes, washed once with methionine- and cysteine-free Dulbecco's modified Eagle's medium (Invitrogen), and incubated at 37 °C for 1 h in this medium plus 5% dialyzed FBS. After labeling with 200 µCi/ml [³⁵S]methionine/cysteine (Tran³⁵S-Label; ICN, Irvine, CA) at 37 °C for 1 h, cells were washed with phosphate-buffered saline and chased with Dulbecco's modified Eagle's medium with 5% FBS under hypoxic or normoxic conditions at 37 °C. Cells were then lysed in 1 ml/dish radioimmune precipitation assay (RIPA) buffer (9.1 mM Na₂HPO₄, 1.7 mM NaH₂PO₄, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1.0% Nonidet P-40, and 1 mM dithiothreitol) containing 50 µM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml antipain, 10 µg/ml leupeptin, and 50 µg/ml aprotinin. The resulting lysates were clarified by centrifugation, and radiolabeled Tal1 was immunoprecipitated with a specific antibody. Immune precipitates were fractionated by SDS-PAGE, and band intensities were quantitated from phosphorimaging of dried gels (Molecular Dynamics, Sunnyvale, CA).

Protein half-life was also determined by Western blot analysis after treatment of cells with the protein synthesis inhibitor cycloheximide. Following a medium change, cycloheximide was added at a concentration of 100 µM, and the cells were incubated under hypoxic or normoxic conditions at 37 °C. Nuclear extracts were subjected to Western blot analysis as described above.

Cell Transfections-- COS-7 and HeLa cell cultures of ~80% confluence were transfected with 10 µg of plasmid DNA by calcium phosphate coprecipitation (39), whereas subconfluent MS1 cells were transfected with 8 µg of liposome-complexed DNA (LipofectAMINE; Invitrogen). Using a -galactosidase expression vector under identical conditions, a transfection efficiency of 60% was achieved for COS-7 cells, 80% for HeLa cells, and 10% for MS1 cells.

In Vivo Ubiquitination Assay-- HeLa cells cultured in 10-cm-diameter dishes were transiently transfected with 4 µg of the HA-tagged ubiquitin expression vector, 4 µg of Tal1 expression vector, or both. MS1 cells were transfected with 8 µg of the HA-ubiquitin expression vector and split 24 h after transfection. After an additional 48 h, cells were treated with 10 µM MG132 for 4 h and lysed with ice-cold RIPA buffer containing 50 µM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml antipain, 10 µg/ml leupeptin, and 50 µg/ml aprotinin. Cell lysates were clarified by centrifugation and incubated with Tal1 antibody at 4 °C overnight. The resulting immune complexes were precipitated with protein A-Sepharose (Pierce) at 4 °C for 2 h. These complexes were washed four times with ice-cold RIPA buffer, heated in Laemmli sample buffer to 95 °C for 3 min, and subjected to Western blot analysis as described above using a mouse monoclonal antibody to the HA epitope. Horseradish peroxidase-conjugated anti-mouse IgG was used to detect binding of the HA antibody.

Metabolic Labeling of Cells and Radioimmunoprecipitation Analysis-- MS1 cells were metabolically labeled as described by Tang et al. (36). Briefly, cells were incubated in phosphate-free minimum essential medium with 5% dialyzed FBS at 37 °C for 1 h and radiolabeled with 0.9 mCi/ml [³²P]orthophosphate (ICN) for 1.5 h in the presence of 10 µM MG132. After treatment with 50 µM PD98059 or vehicle for 30 min, cells were subjected to severe hypoxia (0% O₂) or continued normoxia for 2 h. Nuclei were extracted with ice-cold RIPA buffer containing 1 mM sodium vanadate, 20 mM -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml antipain, and 10 µg/ml pepstatin A. Radiolabeled Tal1 was then immunoprecipitated from the resulting extracts with Tal1 antibody at 4 °C overnight. Immune precipitates were collected with protein A-Sepharose, washed four times with RIPA buffer, boiled for 5 min in Laemmli buffer, and resolved by SDS-PAGE. Dried gels were subjected to autoradiographic analysis.

Two-dimensional Tryptic Phosphopeptide Analysis-- After immunoprecipitation and fractionation by SDS-PAGE, radiolabeled Tal1 protein was transferred to a polyvinylidene difluoride membrane, eluted, and digested exhaustively with trypsin. Proteolytic fragments were then separated by two-dimensional phosphopeptide analysis by the method of Boyle et al. (40). Briefly, tryptic digests were applied to a cellulose thin layer electrophoresis plate (EM Science, Gibbstown, NJ) and fractionated by electrophoresis for 20 min in pH 1.9 buffer at 1000 V and by ascending thin layer chromatography in the second dimension in a solvent containing 39% butanol, 30% pyridine, and 6% glacial acetic acid. Phosphorimaging was used to visualize radioactive digestion products and assess phosphorylation of specific fragments.

MAPK Assay-- MS1 cells were transferred to serum-free medium for 12 h and lysed as described above. MAPK protein was immunoprecipitated from nuclear extracts by the method of Chen and Blenis (41) using an antibody reactive with both ERK1 and ERK2 (Upstate Biotechnology, Lake Placid, NY), and protein kinase activity was assayed as described by Robbins et al. (42) with myelin basic protein as substrate. Briefly, immunoprecipitated protein was incubated with 0.2 mg/ml myelin basic protein and 100 µM [-³²P]ATP (2 µCi/reaction) in 75 mM Tris-HCl, pH 8.0, 10 mM MgCl₂ at 30 °C for 30 min. Reactions were terminated by the addition of electrophoresis sample buffer and fractionated by SDS-PAGE in a 15% gel. Myelin basic protein phosphorylation was detected by autoradiography and quantitated from phosphorimaging of the dried gel.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hypoxia Reduces Tal1 Protein Abundance in Microvascular Endothelial Cells-- Hypoxia's effect on TAL1 expression was investigated using an immortalized murine pancreatic endothelial cell line, MS1 (32). Western blot analysis revealed that Tal1 protein abundance declined significantly in MS1 cells exposed to 1.0% (Fig. 1A), 0.5% (Fig. 1B), or 0% O₂ (Fig. 1C) or incubated with 100 µM CoCl₂ (Fig. 1D), a mimetic of hypoxia. This was not the result of cytotoxicity, however, because no reduction in cellular viability was observed by trypan blue analysis even after 24 h of severe hypoxia (data not shown), and no evidence of apoptosis was noted in flow cytometric analysis of propidium iodide-labeled cells (data not shown). Moreover, Tal1 levels recovered in hypoxic cells with restoration of a normal ambient oxygen concentration (Fig. 1C). The decline in Tal1 expression was also associated with accumulation of the HIF-1 transcription factor (Fig. 1E), excluding a general increase in protein turnover.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Hypoxia decreases Tal1 protein abundance in microvascular endothelial cells. MS1 cells were exposed to 1% O2 (A), 0.5% O2 (B), 0% O2 (C), or 100 µM CoCl2 (D) for the times indicated, and Tal1 protein was detected by Western blot analysis as described under "Experimental Procedures." In one experiment, cells were returned to a normal ambient oxygen concentration (C). As a control, HIF-1 protein expression was quantitated by Western blot analysis in MS1 cells that were made hypoxic (E).

Hypoxia Accelerates Tal1 Protein Turnover in Microvascular Endothelial Cells-- Pulse-chase analysis showed that the decline in Tal1 protein in hypoxic MS1 cells was the result of its increased turnover (Fig. 2). Whereas Tal1 had a half-life (t_1/2) of 8 h under ambient oxygen tensions, a biphasic decay curve with a t_1/2(initial) of 2 h was observed after cells were exposed to severe hypoxia. Similar results were obtained from Western blot analysis of cycloheximide-treated cells (data not shown). In contrast to its effects on protein stability, hypoxia effected a slight stabilization of Tal1 mRNA, increasing its t_1/2 from 3 to 4 h (data not shown). Thus, hypoxia decreased Tal1 expression in MS1 cells by accelerating its turnover.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Hypoxia accelerates Tal1 protein turnover in microvascular endothelial cells. MS1 cells were labeled with [35S]methionine/cysteine for 2 h, cultured under normoxic (closed circles) or hypoxic (0% O2) (open circles) conditions in the presence of cycloheximide, and lysed in RIPA buffer after 2, 4, 6, 8, 10, or 12 h. Radiolabeled Tal1 was immunoprecipitated and fractionated on a 10% SDS-polyacrylamide gel and then analyzed by autoradiography. The mean percentage ± S.E. of Tal1 remaining is expressed relative to time following the addition of the protein synthesis inhibitor. Data represent the results of five independent experiments.

Proteasomal Proteolysis Mediates Hypoxia-induced Tal1 Turnover-- To identify the pathway responsible for Tal1 destruction, cells were treated with different protease inhibitors before being made hypoxic, and Tal1 protein levels were then quantitated by Western blot analysis. The potent proteasomal inhibitor MG132 was tested first in Tal1-transfected HeLa cells, and, whereas hypoxia was without effect on its expression in these cells (Fig. 3A), MG132 did increase Tal1 steady-state levels. In contrast, hypoxia effected a decline in Tal1 expression in MS1 cells that was prevented by MG132 (Figs. 3B and 4) and attenuated by the less potent proteasomal inhibitor ALLN (Fig. 3B) but unaltered by the lysosomal protease inhibitor E64 and the calpain inhibitor LLM (Fig. 3B). These results thus implicate the 26 S proteasome in the increased turnover of Tal1 in hypoxic microvascular endothelial cells.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Tal1 degradation is mediated by the ubiquitin/proteasome pathway. A, Tal1-transfected HeLa cells were pretreated with the specific proteasomal inhibitor MG132 and cultured under normoxic or hypoxic (0% O2) conditions for 16 h. Tal1 expression was then measured by Western blot analysis. B, Tal1 protein abundance was determined by Western blot analysis in MS1 cells pretreated with MG132 (potent proteasome inhibitor), E64 (lysosomal protease inhibitor), LLnL (weak proteasome inhibitor), or LLM (calcium-dependent protease inhibitor) and then subjected to hypoxia (0% O2) for 16 h. C, extracts of MG132-treated HeLa cells transfected with expression vectors for Tal1, HA-ubiquitin, or both were immunoprecipitated with Tal1 antibody, and the immune precipitates were subjected to Western blot analysis with HA antibody. D, MG132-treated MS1 cells transfected with an expression vector for HA-ubiquitin were exposed to severe hypoxia (0% O2) or continued normoxia for 4 h and then extracted with RIPA buffer. Extracts were immunoprecipitated with Tal1 antibody, and immune precipitates were subjected to Western blot analysis with HA antibody.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   MG132 inhibits Tal1 protein turnover in hypoxic MS1 cells. MS1 cells were incubated for 16 h with 10 µM MG132 or vehicle, treated with 100 µM cycloheximide to inhibit further protein synthesis, and subjected to severe hypoxia (0% O2) or continued normoxia for 3, 6, or 9 h. Nuclear extracts were then fractionated on a 10% SDS-polyacrylamide gel and electrotransferred to a membrane. Tal1 protein was detected by Western blot analysis and quantitated from densitometry of the x-ray film. The mean percentage ± S.E. of Tal1 remaining is expressed relative to the time following the addition of the protein synthesis inhibitor. Data represent the results of three independent experiments.

Hypoxia Stimulates Tal1 Ubiquitination in Vivo-- With rare exception (43), proteins destined for proteasomal degradation are first marked by conjugation with ubiquitin. To investigate whether TAL1 undergoes ubiquitination, HeLa cells were cotransfected with expression vectors for Tal1 and HA-tagged ubiquitin, Tal1 protein was immunoprecipitated with a specific antibody, and the presence of ubiquitin-conjugated Tal1 was determined by Western blot analysis using antibody to the HA epitope. As shown in Fig. 3C, a smear of HA-labeled Tal1 was noted only in cells transfected with HA-ubiquitin and Tal1, thus demonstrating that this transcription factor is subject to polyubiquitination in vivo.

To determine whether endogenously expressed Tal1 also was ubiquitinated and the effect hypoxia had on this process, analogous immunoprecipitation/immunoblot studies were carried out 2 (data not shown) and 4 h (Fig. 3D) after exposure of HA-ubiquitin-expressing MS1 cells to severe hypoxia. Whereas Tal1 ubiquitination was detectable even under normal oxygen tensions, the abundance of ubiquitinated Tal1, and in particular the highest molecular weight forms (Fig. 3D), increased in cells made hypoxic. Thus, TAL1 ubiquitination is stimulated by hypoxia in microvascular endothelial cells.

Hypoxia Stimulates the MAPK-catalyzed Phosphorylation of Tal1 Ser¹²²-- Phosphorylation induces the ubiquitin-mediated proteolysis of a number of proteins, including transcription factors (reviewed in Ref. 44 and discussed below). To investigate whether TAL1 phosphorylation was affected by hypoxia, MS1 cells were metabolically labeled with [³²P]orthophosphate, and nuclear extracts were subjected to radioimmunoprecipitation analysis. Incorporation of ³²P into immunoprecipitated Tal1, in fact, was strongly stimulated by hypoxia (Fig. 5A). Because MAPKs are activated by hypoxia in a number of cell types (45-50), including microvascular endothelial cells (51, 52), and mediate growth factor-stimulated phosphorylation of TAL1 (36, 53), we examined the effect of the MAPK kinase 1 (MEK) inhibitor PD98059 (54, 55) on TAL1 phosphorylation in MS1 cells made hypoxic. As shown in Fig. 5A, phosphorylation of Tal1 was virtually eliminated in cells pretreated with PD98059, providing strong evidence for the involvement of a MAPK in this process. PD98059 can inhibit MAPK kinase 5 (56, 57) in addition to MEK, so its effect on Tal1 phosphorylation could have resulted from inhibition of ERK1, ERK2, or ERK5 (56, 57).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Hypoxia elicits MAPK-mediated phosphorylation of Tal1 on Ser122. MS1 cells metabolically labeled with [32P]orthophosphate were preincubated with 50 µM of MEK inhibitor PD98059 or vehicle and then exposed to severe hypoxia (0% O2) or continued normoxia for 2 h. A, radiolabeled Tal1 protein was immunoprecipitated from nuclear extracts with Tal1 antibody, fractionated on a 10% SDS-polyacrylamide gel, and analyzed by autoradiography. B, radiolabeled Tal1 protein was immunoprecipitated from nuclear extracts, fractionated on a 10% SDS-polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane. Eluates were exhaustively digested with trypsin, and the resulting proteolytic products were subjected to two-dimensional electrophoresis. Extracts from EGF-treated, Tal1-transfected COS-7 cells were processed in parallel as a positive control for Ser122 phosphorylation.

Phosphorylation of TAL1 has been shown previously to be restricted to serine residues (58-60). Furthermore, a specific serine in the putative transactivation domain of the protein, Ser¹²², is preferentially phosphorylated by ERK1 in vitro (53) and is the target in cells of epidermal growth factor (EGF)- and erythropoietin-stimulated MAPK phosphorylation (36, 53). To determine which residues were phosphorylated in response to hypoxia, two-dimensional tryptic phosphopeptide analysis was carried out on radiolabeled Tal1 immunoprecipitated from hypoxic MS1 cells that had been pretreated with PD98059. As a positive control for Ser¹²² phosphorylation, immune precipitates were also prepared from Tal1-transfected COS-7 cells treated with EGF. Although more than one radiolabeled tryptic phosphopeptide was obtained, presumably the result of incomplete enzyme digestion, an identical fragment pattern was apparent for hypoxic MS1 cells and EGF-stimulated COS-7 cells (Fig. 5B). Because mutagenesis has shown this serine to be the only site in TAL1 of EGF-induced phosphorylation (36, 53), these results demonstrate that Ser¹²² is also the target of hypoxia-induced phosphorylation. Finally, nuclear MAPK activity was found to increase within 30 min after transferring MS1 cells to a hypoxic environment (data not shown), confirming previously published results (51, 52).

MAPK-stimulated Phosphorylation of Ser¹²² Is Required for Hypoxic Destabilization of Tal1 in Microvascular Endothelial Cells-- To assess the importance of MAPK-mediated phosphorylation in hypoxic stimulation of Tal1 protein turnover, Tal1 protein expression was analyzed by Western blot analysis after exposing PD98059-pretreated MS1 cells to severe hypoxia. This MEK inhibitor increased the protein's t_1/2(initial) from 2 h to more than 6 h (Fig. 6A), consistent with the involvement of a MAPK in hypoxic acceleration of Tal1 turnover. To investigate the requirement of Ser¹²² phosphorylation specifically, cDNAs for Myc epitope-tagged wild-type protein and a Ser¹²² to Ala (S122A) mutant were transfected into MS1 cells, which were then made hypoxic. Whereas the level of Myc-tagged wild-type Tal1 declined with kinetics similar to that of the endogenously expressed protein, the S122A mutant was stable even in the face of severe hypoxia (Fig. 6B). Western blot and radioimmunoprecipitation analyses confirmed that the transfected proteins were expressed at comparable levels before hypoxic exposure and that phosphorylation of the S122A mutant was reduced in cells made hypoxic compared with tagged wild-type protein (Fig. 6C). Together, these results demonstrate that MAPK-mediated phosphorylation of Ser¹²² is required for hypoxic destabilization of Tal1 in microvascular endothelial cells.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Phosphorylation of Ser122 is required for Tal1 degradation in hypoxic MS1 cells. A, MS1 cells pretreated with the MEK inhibitor PD98059 or vehicle were subjected to severe hypoxia (0% O2) for the indicated times, and Tal1 protein levels were determined by Western blot analysis. B, MS1 cells transfected with cDNAs for Myc-tagged wild-type Tal1 or S122A mutant were treated with cycloheximide and made hypoxic (0% O2) for 2, 4, or 6 h. Levels of epitope-tagged Tal1 proteins were then quantitated by Western blot analysis with Myc antibody. C, nuclear extracts from MS1 cells transfected with cDNAs for Myc-tagged wild-type Tal1 or S122A mutant but not yet exposed to hypoxia were subjected to Western blot analysis with Myc antibody (Protein). Extracts of transfected cells metabolically labeled with [32P]orthophosphate and rendered hypoxic for 2 h were subjected to radioimmunoprecipitation analysis with Myc antibody (Phosphorylation).

Hypoxic Stimulation of Tal1 Degradation Is Characteristic of Small but Not Large Vessel Endothelial Cells-- HUVECs were the only cultured endothelial cells known to express TAL1 before initiation of this work (31). Because they derive from a large vessel and most of these studies were carried out in a microvascular endothelial line, we investigated whether TAL1 protein expression was similarly affected by hypoxia in endothelial cells from different sources. HMVECs, the immortalized human bone marrow microvascular endothelial cell line BMEC-1 (33), HUVECs, and, as a control, murine erythroleukemia cells were exposed to either severe hypoxia (0% O₂) or the hypoxia mimetic cobalt for 24 h, and TAL1 levels were measured by Western blot analysis with species-specific TAL1 antibodies. Although all of these lines expressed this transcription factor, TAL1 was only reduced by hypoxia in HMVECs and BMEC-1 cells and actually increased in response to this stimulus in murine erythroleukemia cells (Fig. 7, A-D). Similar results were noted when cells were exposed to 1% O₂ and 100 µM CoCl₂ (Fig. 7E). Thus, in addition to extending the number of endothelial cell lines found to express the protein, these findings suggest that hypoxic acceleration of Tal1 turnover may be restricted to microvascular endothelial cells.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Hypoxic acceleration of TAL1 protein turnover is restricted to microvascular endothelial cells. AD, cells depicted were cultured under normoxic or hypoxic conditions and extracted for Western blot analysis with murine or human TAL1 antibody. A, HMVECs treated with 100 µM CoCl2, a hypoxia mimetic, or subjected to severe hypoxia (0% O2) for 24 h. B, human bone marrow microvascular endothelial cell line (BMEC-1) subjected to hypoxia (0% O2) for 24 h. C, HUVECs subjected to hypoxia (0% O2) for 24 h. D, murine erythroleukemia (MEL) cell line subjected to hypoxia (0% O2) for 8 and 16 h. E, results of studies of cells exposed to 1% O2 or 100 µM CoCl2 for 24 h. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 relative to HUVECs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hypoxia poses a significant challenge to cellular and organismal function and is an important stimulus of angiogenesis in developmental, physiological, and pathological contexts. As for other environmental stimuli (61), hypoxia induces the expression of a large number of genes that ultimately mediate the cell's adaptations (62). This genomic response has increasingly been recognized to result from posttranslational modification (ubiquitination, most prominently) of specific transcription factors. Indeed, the levels of the most important transcriptional mediator of hypoxia-induced angiogenesis, HIF-1, have been shown to be tightly controlled by this process (reviewed in Ref. 63). Although the consequence (proteasomal degradation) is identical, the oxygen tensions under which HIF-1 and TAL1 ubiquitination are elicited differ considerably.

Hypoxia also stimulates the phosphorylation of transcription factors. There is evidence of hypoxic activation of MAPKs for diverse cell types (45, 46, 48), including microvascular endothelial cells (51, 52), and MAPK-mediated phosphorylation of Elk-1 was shown to be important for hypoxic stimulation of c-fos and Egr-1 transcription (46, 48). HIF-1 has also been reported to be phosphorylated by MAPK, with an increase in its transcriptional potency but, interestingly, not its stability (51, 64). The ubiquitination and proteolysis of other transcription factors, including IB (65), p53 (66), MyoD (67), c-Myb (68), progesterone receptor (69), microphthalmia (Mi) (70), BCL-6 (71), and Spi-B (72), are elicited by phosphorylation, although examples to the contrary have also been described (73, 74). Although ubiquitin-mediated destruction of BCL-6 (71) and Mi (70) by MAPK or MAPK-activated ribosomal S6 kinase 1 has recently been described, to our knowledge, this work establishes the first connection between hypoxic activation of MAPK and degradation of a transcription factor.

A separate mechanism has been described for the hypoxia-stimulated destruction of the transcription factor cAMP-response element-binding protein involving its decreased dephosphorylation by the serine/threonine phosphatase PP1 (75). Although a contribution of reduced dephosphorylation to hypoxic acceleration of TAL1 turnover cannot be completely excluded, the amino acid sequence encompassing Ser¹²² shows no homology to the proteasomal targeting motif in cAMP-response element-binding protein, PP1 at least would not be expected to act as a MAPK phosphatase, and, most directly, studies using a protein kinase inhibitor suggest that TAL1 stability is regulated at the level of phosphorylation.

The model most compatible with our results, then, has phosphorylation of Ser¹²² by a MAPK(s) acting to stimulate TAL1 ubiquitination and, ultimately, its trafficking to the 26 S proteasome. A critical step in this process must be in the conjugation of ubiquitin to TAL1, and we surmise that phosphorylation of this serine, which is sufficient to alter the protein's mobility in denaturing polyacrylamide gels (53), must make it a more favorable substrate for an E2 ubiquitin-conjugating enzyme and/or E3 ubiquitin ligase. The observation that the N-terminal half of TAL1 encompassing Ser¹²² otherwise acts to stabilize the protein is demonstrated by the much shorter half-life in cells of the smaller pp24^TAL1 isoform (76, 77) as compared with the full-length protein (data not shown).

Whereas MAPK-stimulated Ser¹²² phosphorylation is necessary for induced degradation of TAL1 in microvascular endothelial cells, in contrast to BCL-6 (71) and Mi (70), it is not sufficient to accelerate the protein's turnover in heterologous cell types. Specifically, TAL1 protein stability was unaltered in hypoxic HeLa cells, in which MAPK can be presumed to have been activated (46) (Fig. 3B), and in EGF-treated Tal1-transfected COS-7 cells, in which MAPK-mediated Ser¹²² phosphorylation has been demonstrated directly (53). Thus, an additional factor(s), potentially a unique E2 and/or E3 enzyme, must be present in hypoxic endothelial cells for Ser¹²² phosphorylation to signal for increased ubiquitination. Proximity to a PEST domain, a proline-, glutamine-, serine-, and threonine-rich motif that acts to target proteins for degradation, is likely not involved, however, because no such regions are predicted to be present in either TAL1 or Mi (data not shown).

Activation from phosphorylation or, in the case of certain nuclear steroid receptors, ligand binding has recently been recognized to hasten transcription factor destruction (reviewed in Ref. 78). Furthermore, tumor-specific mutations were found to reduce the ubiquitination and subsequent proteasomal turnover of viral and cellular Myc proteins by disrupting critical phosphorylation sites (79-81). These and other studies (82, 83) have led to the concept that transcriptional activation domains also function as degradation motifs ("degrons"). In contrast to Mi, however, in which recruitment of the coactivator p300 (84) and destruction by the ubiquitin-proteasome pathway are both increased by MAPK-mediated phosphorylation, the region encompassing Ser¹²² in TAL1 exhibits little transactivating activity as linked to the DNA-binding domain of yeast transcription factor GAL4 without concomitant p300 overexpression (14). The sequences mediating TAL1's association with p300 are also located some distance from its MAPK phosphorylation site (14), and mutagenesis of this serine has no effect on p300 coactivation.³ MAPK-mediated degradation of TAL1 (data herein) and, in particular, the dedicated repressor BCL-6 (71) thus indicate that transcriptional activation and degradation are not invariably linked.

Endothelial cells from different vascular beds manifest distinct ultrastructural characteristics, growth properties, responses to cytokines, and repertoires of gene expression (85, 86). In this context, it may be significant that hypoxia reduced TAL1 protein abundance only in endothelial cells from small vessels. Hypoxic down-regulation of this transcription factor was observed in microvascular endothelial cells from two species (human and mouse), three organs (pancreas, skin, and bone marrow), and primary as well as immortalized cells, so this difference cannot reflect adaptation to culture or tissue or species of origin. Because hypoxia induced TAL1 phosphorylation in both HUVECs and MS1 cells (data not shown), recognition of phosphorylated protein by the ubiquitination machinery likely underlies this differential processing. Although the involvement of TAL1 in endothelial cell responsiveness to hypoxia remains to be determined, these studies have, at a minimum, revealed a novel difference in the biochemistry of large and small vessel endothelial cells.

The specific functions of TAL1 in this cell type are unknown, although preliminary studies revealed that, in contrast to its growth-promoting effects in erythroid progenitors (25, 26),⁴ TAL1 overexpression inhibited the proliferation of MS1 cells without inducing their death.⁵ Given its transient expression in angiogenic processes in vivo¹ and its down-regulation by the proangiogenic stimulus of hypoxia, TAL1 may act to inhibit or at least limit blood vessel formation. Whatever its actions, this basic helix-loop-helix transcription factor is subject to posttranslational modification in endothelial cells, with consequences for its level of expression.

	ACKNOWLEDGEMENTS

We thank Drs. Dirk Bohmann for ubiquitin expression vectors, Richard Baer for human TAL1 antibody, Francisco Candal for the BMEC-1 cell line, and Mark Koury for useful discussions.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant R01 HL49118 (to S. J. B.) and a Merit Review Award from the Department of Veterans Affairs (to S. J. B.).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.

To whom correspondence should be addressed: Division of Hematology-Oncology, Rm. 777, Preston Research Bldg., Vanderbilt University Medical Center, Nashville, TN 37232. Tel.: 615-936-1809; Fax: 615-936-3853; E-mail: stephen.brandt@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, March 19, 2002, DOI 10.1074/jbc.M109812200

¹ T. Tang, S. R. Opalenik, J. M. Davidson, D. M. Brantley, J. Chen, and S. J. Brandt, manuscript in preparation.

³ S. Huang, Y. Qiu, R. W. Stein, and S. J. Brandt, unpublished observations.

⁴ S. Huang, Y. Shi, and S. J. Brandt, unpublished observations.

⁵ T. Tang and S. J. Brandt, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; HUVEC, human umbilical vein endothelial cell; HMVEC, human dermal microvascular endothelial cell; FBS, fetal bovine serum; HA, hemagglutinin; RIPA, radioimmune precipitation assay; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; EGF, epidermal growth factor; Mi, microphthalmia; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES