The NGFI-B Protein, an Inducible Member of the Thyroid/Steroid Receptor Family, Is Rapidly Modified Posttranslationally TIMOTHY J

Home , Thyroid hormone receptor

The NGFI-B Protein, an Inducible Member of the Thyroid/Steroid Receptor Family, Is Rapidly Modified Posttranslationally TIMOTHY J. FAHRNER, STEVEN L. CARROLL, AND JEFFREY MILBRANDT* Departments ofPathology and Internal Medicine, Division of Laboratory Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110 Received 23 July 1990/Accepted 12 September 1990

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of ==61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate- polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

The cellular response to growth factors includes the acti- chromocytoma cells (21); its mouse homolog (nur77) was vation of a set of genes termed early-response genes (15). identified similarly in serum-stimulated fibroblasts (11). In These genes are characterized by several features: their PC12 cells, the 2.4-kb NGFI-B mRNA is normally present at transcription is activated within minutes of growth factor very low levels, but upon administration of NGF, the gene is administration, their induction is not dependent on de novo transcriptionally activated (37). The amount of NGFI-B protein synthesis, and their corresponding mRNAs and the mRNA increases dramatically within 30 min, but this in- proteins they encode are short-lived. Most early-response crease is very transient, with levels returning to base line genes have been isolated by differential hybridization tech- within 4 h. The NGFI-B gene is expressed at relatively high niques, and the functions of these genes are therefore largely levels in the adult rat brain, adrenal gland, and muscle (M. unknown. However, some (e.g., c-fos and c-jun) are known Watson and J. Milbrandt, Development, in press). As has to regulate transcription, and others are inferred to have been observed for a number of early-response genes, the similar functions because they share sequence homology level of NGFI-B mRNA rapidly and dramatically increases with other transcriptional regulators. These include the in the brain following Metrazol-induced seizures (37). genes encoding the zinc finger proteins NGFI-A (20) (also By virtue of its sequence similarity to other ligand-depen- called and Egrl [35], ztf7268 [5], Krox 24 [16]), Krox 20 dent transcriptional regulators, NGFI-B probably plays an (4)/Egr2 (13), and NGFI-B (21)Inur77 (11). Like many early- important role in regulating the cellular response to growth response genes, the NGFI-B gene is activated in a variety of factors. At present, little is known about the protein encoded cell types by multiple, diverse stimuli, including growth by the NGFI-B cDNA. In this we describe seizure calcium and study, the factors, induction, ionophores, phorbol production of antibodies to the NGFI-B protein and the esters. Thus, NGFI-B may function as a nuclear messenger subsequent characterization of NGFI-B biosynthesis, post- of the signal transduction process and represent, along with translational modification, and cellular location. NGFI-B the products of other early-response genes, the primary was detected as a heterogeneous collection of protein spe- effectors of the cellular response to environmental changes. cies ranging in apparent molecular mass from 63 to 88 kDa. The nucleotide sequence of the NGFI-B cDNA predicts a Pulse-chase analysis revealed that NGFI-B is a short-lived protein of 61 kDa that is closely related to members of the protein that undergoes rapid and extensive modification via thyroid/steroid receptor gene family. Each of these receptor phosphorylation in NGF-stimulated and, to a lesser degree, proteins functions as a ligand-dependent, transcriptional basic fibroblast that is growth factor (bFGF)-stimulated PC12 cells. regulator activated by binding to its respective hor- Other agents, including epidermal growth factor (EGF), mone ligand. The NGFI-B gene was identified by virtue of its phorbol ester, and the calcium ionophore A23187 induce nerve factor in rat induction by growth (NGF) PC12 pheo- NGFI-B synthesis but do not promote its phosphorylation. Cellular localization studies demonstrated that NGFI-B is found in approximately equal amounts in the cytoplasm and * Corresponding author. nucleus of NGF-stimulated PC12 cells. 6454 VOL. 10, 1990 POSTTRANSLATIONAL MODEL OF NGFI-B 6455

MATERIALS AND METHODS intestinal alkaline phosphatase in phosphatase buffer (12.5 mM Tris [pH 7.91, 6 mM MgCl2, 5% glycerol, 0.05% Nonidet Materials. NGF was the gift of Eugene Johnson (Depart- P-40, 50 mM KCI) for 30 min at 30°C, followed by a second ment of Pharmacology, Washington University School of incubation with 20 U of bacterial alkaline phosphatase for 30 Medicine). The pATH plasmids were the gift of A. Tza- min at 68°C in the same buffer. When indicated, phosphatase goloff, Columbia University. [35S]methionine (Translabel) inhibitors (100 mM NaF, 10 mM Na4P207, and 1 mM and 32Pi were obtained from ICN Biomedicals (Irvine, NaVO4) were added. Calif.). Leupeptin, 0-12-tetradecanoylphorbol-13-acetate Cell fractionation. Nuclear and cytoplasmic fractionation (TPA), A23187, and Pansorbin were obtained from Calbio- was done essentially as described by Challberg and Kelly chem (San Diego, Calif.). Calf intestinal alkaline phos- (3). Cell lysis was confirmed by examination under a phase- phatase was purchased from Boehringer-Mannheim (India- contrast microscope. After cell lysis, the nuclei were pel- napolis, Ind.). Bacterial alkaline phosphatase and restriction leted by centrifugation at 2,000 x g for 5 min. The cytosolic enzymes were obtained from New England BioLabs (Bev- fraction was decanted and brought to 0.1% sodium dodecyl erly, Mass.). All reagents, unless otherwise indicated, were sulfate (SDS). The nuclear pellet was homogenized in RIPA purchased from Sigma (St. Louis, Mo.). buffer equal in volume to the cytosolic fraction. Cell culture. PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and RESULTS 5% horse serum (19). The DG44 CHO cell line was grown in F12 medium supplemented with 10% fetal calf serum (36). Production and characterization of anti-NGFI-B antibodies. Production of bacterial fusion proteins. A fusion protein To characterize the NGFI-B protein, we produced two containing the bacterial TrpE protein and a portion of MAbs (1A8 and 2E1) to a bacterial fusion protein (B1440) NGFI-B was produced by using the pATH expression vector containing a portion of NGFI-B that encodes part of the (10). This construct (pB1440) was made by ligating a 353- putative ligand-binding domain of this receptor. These anti- nucleotide Sacl fragment (nucleotides 1440 to 1793 of the NGFI-B MAbs were shown to immunoprecipitate NGFI-B NGFI-B cDNA) into pATH 11. Escherichia coli DHSa cells produced in a reticulocyte lysate system primed with bearing this plasmid produced a protein of the predicted NGFI-B RNA and by CHO cells transfected with an molecular mass when induced by tryptophan depletion and NGFI-B expression vector (data not shown). The specificity addition of 25 ,ug of indole acrylic acid per ml. The B1440 of these MAbs was confirmed by blocking experiments with fusion protein, which contains NGFI-B residues from Glu- the B1440 fusion protein (see Materials and Methods). 410 to Leu-527, was purified by preparing an insoluble NGFI-B is rapidly synthesized and modified in PC12 cells fraction from the bacterial lysate (39). after NGF administration. In the initial characterization of Production of MAbs. Female BALb/cJ mice (Jackson the MAbs, we noticed that NGFI-B produced in vitro Laboratory) were immunized with 30 ,ug of B1440 fusion migrated more rapidly on SDS-polyacrylamide gels than did protein. The mice were injected subcutaneously once each that synthesized by the transfected CHO cells. This obser- week for 3 weeks and then injected intraperitoneally every vation suggested that NGFI-B was posttranslationally mod- month for 4 months. Three days prior to fusion, 20 ,ug of ified and led us to examine the initial induction of NGFI-B in B1440 fusion protein was injected intravenously. The ani- PC12 cells treated with NGF. To examine the synthesis of mals were sacrificed, spleen cells were harvested, and hy- NGFI-B protein after NGF stimulation, PC12 cells were bridomas were generated by using the nonsecreting my- incubated in the presence of [35S]methionine and treated eloma cell line P3X63AG8.6.5.3 (34). Hybridoma culture with NGF for the indicated lengths of time. Cells were also supernatants were screened for reactivity against NGFI-B labeled in the absence of NGF for 3 h. Lysates were translated in the presence of [35S]methionine in a reticulo- prepared from cells harvested at each time point, and cyte lysate system (Promega, Madison, Wis.), using a stan- NGFI-B levels were assayed by immunoprecipitation with dard immunoprecipitation assay. Supernatants from the anti-NGFI-B MAbs, followed by electrophoresis on SDS- strongest positive clones, designated monoclonal antibody polyacrylamide gels and autoradiography. A low level of (MAb) 1A8 and MAb 2E1, were harvested for further use. NGFI-B was present in uninduced PC12 cells, and NGFI-B Immunoprecipitation of labeled NGFI-B proteins. Twenty- levels rose rapidly after NGF treatment, with peak levels four hours prior to labeling, 5 x 106 PC12 cells were seeded occurring 70 min after NGF addition (Fig. 1A). Also, onto 10-cm plates. PC12 cells were incubated in medium NGFI-B from uninduced PC12 cells migrated more rapidly lacking methionine for 1 h and labeled by addition of than did that present in NGF-treated cells (compare lane 1 [35S]methionine (150 ,Ci/ml) for 70 min in either the absence with lanes 2 to 8), an observation consistent with the or the presence of NGF (50 ng/ml) or other agents as presence of a rapid posttranslational modification. indicated. Cells were harvested, and immunoprecipitation To determine whether posttranslational modifications of assays were performed as previously described (8). Blocking NGFI-B could account for the heterogeneous nature of the experiments were performed by incubating the antibodies detected species and the altered mobility of NGFI-B after with either 10 pVg of B1440 or nonrecombinant TrpE protein NGF stimulation, a pulse-chase experiment was performed. for 1 h on ice before adding them to the cell lysate. PC12 cells were incubated in the absence of methionine for For phosphorylation studies, PC12 cells were seeded onto 1 h before the addition of NGF. The cells were then plates as described above and incubated in phosphate-free incubated in the presence of NGF for 45 min, pulse-labeled medium containing dialyzed serum for 14 h. This medium with [35S]methionine for 15 min, and chased with an excess was replaced with identical medium for an additional hour, of cold methionine for increasing lengths of time. NGFI-B and cells were labeled by addition of 32pi (500 mCi/ml). After expression was analyzed by immunoprecipitation with anti- 3 h of incubation, NGF (50 ng/ml) was added for 70 min and NGFI-B MAbs, followed by electrophoresis on SDS-poly- cells were lysed in RIPA buffer containing phosphatase acrylamide gels and autoradiography. Immediately after the inhibitors. Phosphate was removed from immunoprecipi- 15-min pulse, a heterogeneous mixture of NGFI-B molecules tated proteins by incubating the proteins with 20 U of calf ranging in apparent size from 63 to 88 kDa was present (Fig. 6456 FAHRNER ET AL. MOL. CELL. BIOL.

A min: 0 15 30 45 60 75 90 120 180 116

84 58 4I

B min: 0 15 30 45 60 75 90 120 180 C 0 45 116

IS #w 84 58

FIG. 1. Posttranslational modification of NGFI-B in NGF-stimulated PC12 cells. (A) Time course of NGFI-B induction in NGF-treated PC12 cells. PC12 cells were grown in medium containing [35S]methionine for 1 h before the addition of NGF (50 ng/ml). Cells were harvested at the indicated times, and NGFI-B was analyzed by immunoprecipitation with anti-NGFI-B MAbs. Positions of size standards are indicated in kilodaltons on the left. (B and C) Pulse-chase analysis of NGFI-B in NGF-stimulated PC12 cells. (B) PC12 cells were grown in medium lacking methionine for 1 h, treated with NGF for 45 min, pulsed with [35S]methionine (150 p.Ci/ml) for 15 min, and chased with medium containing 1 mM methionine for increasing lengths of time. Cell lysates were incubated with anti-NGFI-B MAbs, and immunoprecipitates were electrophoresed on an SDS-polyacrylamide gel. The lanes are labeled with the length of the chase period at the time the cells were harvested (i.e., time 0 represents cells harvested at the end of the pulse without a chase). (C) As for panel B except that the length of the [35S]methionine pulse was 2 min.

1B). Within 30 min after the pulse, essentially all NGFI-B The immunoprecipitated proteins were incubated with both was present in the most slowly migrating fraction. This calf intestinal and bacterial alkaline phosphatases in either suggested that NGFI-B was undergoing a rapid posttransla- the absence or the presence of the phosphatase inhibitors tional modification that resulted in the decreased mobility sodium pyrophosphate, sodium fluoride, and sodium vana- and heterogeneous nature of the detected species. As a date. A dramatic shift in the mobility of NGFI-B was 15-min pulse was not adequate to clearly demonstrate the observed after treatment with alkaline phosphatases (Fig. extent of the modification, a similar experiment was per- 2A). The results demonstrated that NGFI-B from both formed in which the pulse length was decreased to 2 min. uninduced and NGF-treated PC12 cells had an equivalent, Cell lysates were prepared immediately after the pulse and increased mobility after treatment with alkaline phos- after 45 min of chase. NGFI-B was synthesized as a protein phatases. The presence of phosphatase inhibitors during the with an apparent molecular mass of 63 kDa that was rapidly incubation with alkaline phosphatases prevented the shift in modified to more slowly migrating forms with apparent mobility, thereby demonstrating that it was due to the molecular masses ranging up to 88 kDa (Fig. 1C). The removal of phosphate from NGFI-B. heterogeneity observed is presumably a reflection of inter- To confirm these results, we directly labeled NGFI-B with mediate forms in this modification process. These experi- phosphate. PC12 cells were grown in the presence of 32p; for ments also demonstrated that NGFI-B is a relatively short- 3 h before the addition of NGF for 70 min. Lysates were lived protein with a half-life of approximately 20 to 30 min, prepared and incubated with anti-NGFI-B MAbs to immu- consistent with another report (31) and similar to that of noprecipitate 32P-labeled NGFI-B. A broad band extending other early-response genes such as c-fos (22) and the from apparent molecular mass 63 to 88 kDa was detected in NGFI-A gene (8). cells treated with NGF, and a smaller band of much weaker The NGFI-B protein is phosphorylated. Many members of intensity was observed in uninduced cells (Fig. 2B). Treat- the thyroid/steroid receptor family are phosphorylated both ment of the immunoprecipitated protein with alkaline phos- on serine/threonine (32) and tyrosine residues (18). The phatases eliminated the observed signal. To ensure that the functional consequences of these phosphorylation events are labeling proceeded as expected, the rat NGF receptor, a not fully understood but are under intense investigation. The protein known to be heavily phosphorylated, was also NGFI-B protein is very rich in serines and threonines and immunoprecipitated from these lysates with an anti-NGF contains numerous possible phosphorylation sites. The het- receptor MAb and found to be strongly labeled. These erogeneous nature of the detected NGFI-B band suggested results demonstrate that NGFI-B is heavily phosphorylated. that multiple species slightly different in mobility are present Presumably, as with other members of this hormone recep- in the cell. We therefore explored the possibility that tor family, this modification plays an important role in its NGFI-B was phosphorylated in PC12 cells by treating it with function. phosphatases. Cells were cultured in the presence of Induction of NGFI-B in PC12 cells by other agents. Many [35S]methionine for 70 min either with or without NGF, and early-response genes, including NGFI-B, are activated by a NGFI-B was immunoprecipitated from these cell lysates. variety of agents, including phorbol esters, calcium iono- VOL. 10, 1990 POSTTRANSLATIONAL MODEL OF NGFI-B 6457 A. B.

1 2 3 4 5 6 1 2 3 4 116 116 -

84 - 84 --- * 3E 58 58

FIG. 2. Demonstration that NGFI-B is a phosphorylated protein. (A) [35S]methionine-labeled NGFI-B was immunoprecipitated from PC12 cells grown in the absence (lanes 1 to 3) or presence (lanes 4 to 6) of NGF and treated as follows: no treatment (lanes 1 and 4), incubation with alkaline phosphatase (lanes 2 and 5), or incubation with alkaline phosphatase in the presence of phosphatase inhibitors (100 mM NaF, 10 mM Na4P207, and 1 mM NaVO4) (lanes 3 and 6). (B) PC12 cells were grown in medium containing 32p; in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of NGF. Cell lysates were immunoprecipitated with anti-NGF receptor (lane 1) or anti-NGFI B (lanes 2 to 4) MAbs. Lane 4 contains immunoprecipitated lysate that was also incubated with alkaline phosphatase. Positions of size standards are indicated in kilodaltons on the left. phores, carbachol, and Metrazol. This is true for the onco- upon it is translocated to the nucleus (27). To examine the gene c-fos, which undergoes dramatic posttranslational mod- cellular location of NGFI-B, cell fractionation studies were ification. The extent of posttranslational modification of performed on PC12 cells labeled with [35S]methionine in the c-fos, however, is dependent on the type of stimulus used to absence or presence of NGF. Nuclear and cytoplasmic activate the gene (1, 7). To explore whether this was also fractions were obtained, and NGFI-B levels were assayed by true for NGFI-B, we treated PC12 cells grown in the immunoprecipitation with anti-NGFI-B MAbs (Fig. 4). The presence of [35S]methionine with NGF, bFGF, EGF, phor- small amount of NGFI-B detected in uninduced PC12 cells bol ester (TPA), or A23187 for 70 min. Cell lysates were was present solely in the nucleus; no signal was detected in prepared, and NGFI-B was immunoprecipitated. The slowly the cytoplasm from these cells. However, in PC12 cells migrating NGFI-B species were detected in cells stimulated treated with NGF for 70 min, NGFI-B was present in both with NGF and, to a lesser degree, by bFGF, both of which the nucleus and cytoplasm in approximately equal quanti- promote the differentiation of PC12 cells (Fig. 3). EGF, ties. It is also apparent that the modal size of NGFI-B in the phorbol ester, and calcium inophore, agents that do not nucleus was smaller than that of the NGFI-B species found promote differentiation, were also capable of stimulating in the cytoplasm, suggesting that nuclear NGFI-B species NGFI-B synthesis. However, only the more rapidly migrat- contain less phosphate than do those present in the cyto- ing, underphosphorylated species were observed in cells plasm. This observation is consistent with results from treated with these agents, suggesting that the signals neces- uninduced cells, in which only the rapidly migrating, under- sary for NGFI-B phosphorylation were not activated by phosphorylated form of NGFI-B was present (Fig. 4; com- these agents. pare lanes 1 and 4). In addition, treatment of both the Cellular location of NGFI-B. Most members of this recep- cytoplasmic and nuclear forms with phosphatases reduced tor family have a consistently nuclear location (14, 26). An them to the same apparent size, demonstrating that the exception is the glucocorticoid receptor, which is found in differences in modal size are not due to other modifications the cytoplasm until it is complexed with its ligand, where- (data not shown).

1 2 3 4 5 6 1 2 3 4 5 6 116 84 84 58 II,,4 Is 58 sb-e

FIG. 3. Induction of NGFI-B by NGF, EGF, bFGF, phorbol ester (TPA), and calcium ionophore (A23187). Immunoprecipita- tions were performed on [35S]methionine-labeled lysates from cells induced for 70 min with the indicated reagents: no treatment (lane FIG. 4. Subcellular localization of NGFI-B in PC12 cell,. Shown 1), NGF, 50 ng/ml (lane 2), bFGF, 1.5 ng/ml (lane 3), TPA, 50 ng/ml is an immunoprecipitation analysis of [35S]methionino -labeled (lane 4), A23187, 10 ,uM (lane 5), and EGF, 100 ng/ml (lane 6). NGFI-B from PC12 cells grown in the absence (lanes I to 3) or Lysates were incubated with anti-NGFI-B MAbs, and immunopre- presence (lanes 4 to 6) of NGF. Lanes: 1 and 4, total cellular lysate; cipitates were analyzed by SDS-polyacrylamide gel electrophoresis. 2 and 5, nuclear fraction; 3 and 6, cytoplasmic fraction. Positions of Positions of size markers are indicated in kilodaltons on the left. size markers are indicated in kilodaltons on the left. 6458 FAHRNER ET AL. MOL. CELL. BIOL.

DISCUSSION ing NGFI-B, effect changes in their activity, stability, or subcellular location. The NGFI-B cDNA was identified as a member of a class The subcellular location of the thyroid/steroid receptors of genes known as the early-response or immediate-early has been extensively examined. Early work demonstrated genes. These genes are rapidly activated in many cell types that the glucocorticoid receptor is stationed in the cytoplasm in response to a variety of environmental cues, including until it is complexed with cortisol, whereupon it is translo- growth factors, membrane depolarization, tumor promoters, cated to the nucleus (38). However, it is now known that and antigenic stimulation. Many early-response genes en- many other receptors, including those for thyroid hormone, code transcriptional regulatory proteins, including c-Fos vitamin D, estrogen, and progesterone, are constitutively (28), Fral (6), Fra2 (24), FosB (40), c-Jun (2), JunB (30), and intranuclear and are capable of interacting with their cognate JunD (29), and zinc finger proteins such as NGFI-A, Krox- DNA recognition sequences in the absence of hormone (9). 20, and NGFI-B. These gene products are presumably A cell fractionation study using NGF-stimulated PC12 cells important in regulating the cell's response to environmental revealed that NGFI-B is found at approximately equal levels changes and can be thought of as the nuclear messengers of in the nucleus and cytoplasm. The distribution is further this response. The NGFI-B gene is a unique member of this distinguished by the fact that the larger, more heavily subset because it is homologous to members of the thyroid/ phosphorylated species are predominantly in the cytoplasm, steroid receptor gene family, a group of ligand-dependent whereas species migrating more rapidly are overrepresented transcriptional regulators. Thus, its activity is potentially in the nucleus. In contrast, NGFI-B was found only in the under dual control: its expression is controlled by environ- nucleus in Cos (31) and CHO (data not shown) cells trans- mental cues, and its transcriptional regulatory activities are fected with an NGFI-B expression vector. This difference in presumably governed by a hormone ligand. Because of these NGFI-B localization is not due to factors present in serum, unique features, we set out to characterize the protein since NGFI-B synthesized in cells cultured without serum encoded by the NGFI-B cDNA. had a similar distribution (data not shown). One possibility We used anti-NGFI-B antibodies raised against a TrpE- for this discrepancy is that the hormone ligand for NGFI-B is NGFI-B bacterial fusion protein to establish that a low level an intracellular mediator which is present at different levels of NGFI-B exists in uninduced PC12 cells and that NGFI-B in CHO and PC12 cells. Alternatively, it may reflect other levels increase rapidly but transiently after NGF treatment. cell-type-specific differences. On SDS-polyacrylamide gels, NGFI-B was detected as a NGFI-B, like many early-response genes, can be induced broad band ranging in apparent molecular mass from 63 to 88 by a variety of growth factors and other agents. However, kDa, suggesting that it is composed of a heterogeneous the ability of these inducing agents to activate processes mixture of molecules. The heterogeneous nature of the which posttranslationally modify these proteins varies NGFI-B proteins observed in NGF-stimulated PC12 cells widely. NGF and, to a lesser extent, bFGF stimulate the and the demonstration of posttranslational modification of synthesis of highly phosphorylated NGFI-B species, NGFI-B in the pulse-chase experiments prompted us to whereas other agents such as EGF, phorbol ester, or A23187 determine whether NGFI-B was glycosylated. However, the do not. Similar observations have been reported in studies of results of several experiments designed to detect the pres- the c-Fos protein (7), which is also induced in PC12 cells by ence of carbohydrates on this protein were negative (unpub- a variety of agents. When c-fos is induced by NGF or FGF, lished data). it is extensively phosphorylated, but when barium ions, Transcription factors, including members of the thyroid/ calcium ionophores, or potassium ions are used to activate steroid receptor family, are commonly modified via phos- this gene, the c-Fos protein is modified to a lesser extent. As phorylation. We found that NGFI-B was also phosphory- we have observed for NGFI-B, the extent of c-Fos phos- lated and that the heterogeneous collection of NGFI-B phorylation was manifested as changes in its mobility on species coalesced to a single species after treatment with SDS-polyacrylamide gels (7). The functional consequences alkaline phosphatases. NGFI-B present in uninduced cells of producing NGFI-B that differs in phosphate content are was also reduced in apparent molecular weight by phos- not known but may be critical, considering the important phatase treatment such that NGFI-B derived from unin- role of phosphorylation in regulating these receptors and duced and NGF-stimulated cells comigrated. This result transcriptional regulators in general. demonstrates that some sites on this protein are phosphor- Establishing the function of NGFI-B and determining the ylated even in the basal state. The estrogen, glucocorticoid, relative contribution of phosphorylation state to its activity, and progesterone receptors are also phosphorylated in the localization, and hormone-binding capability must await the basal state; however, the extent of phosphorylation is in- discovery of both the hormone with which it interacts and creased by interaction with the cognate ligand (12, 32). genes that it regulates. However, by virtue of its similarity to Indeed, hormone binding itself is dependent on the state of other thyroid/steroid hormone receptors, its activation by receptor phosphorylation in the case of glucocorticoid and growth factors and depolarizing agents, and its differential estrogen receptors (17, 23). Hormone-dependent phosphor- expression during postnatal development (Watson and Mil- ylation of the progesterone receptor results in a decrease in brandt, in press), it is reasonable to assume that it plays an its relative mobility on SDS-polyacrylamide gels (33) much important role in modulating the cell's response to environ- like that observed with NGFI-B. This change in phosphor- mental change. ylation state has also been correlated with a decreased ability to interact with HSP90 and an increased affinity to bind DNA (9). It has also been reported that glucocorticoid ACKNOWLEDGMENTS receptors that are tightly bound to the nuclear matrix contain We thank Eugene Johnson for supplying us with the anti-NGF a decreased number of phosphorylated residues (25), an receptor MAb and NGF. observation reminiscent of that seen with NGFI-B in PC12 This work was supported by Public Health Service grants cells (Fig. 4). Collectively, these observations suggest that NS01018 from the National Institute of Neurological and Commu- alterations in the phosphorylation of these receptors, includ- nicative Diseases and Stroke and P01 CA49712 from the National VOL. 10, 1990 POSTTRANSLATIONAL MODEL OF NGFI-B 6459

Cancer Institute and by grant RG1779B2 from the Multiple Sclerosis 20. Milbrandt, J. 1987. A nerve growth factor gene encodes a Foundation. possible transcriptional regulatory factor. Science 238:797-799. 21. Milbrandt, J. 1988. Nerve growth factor induces a gene homol- LITERATURE CITED ogous to the glucocorticoid receptor gene. Neuron 1:183-188. 1. Barber, J. R., and I. M. Verma. 1987. Modifications of fos 22. Muller, R., R. Bravo, J. Burckhardt, and T. Curran. 1984. proteins: phosphorylation of c-fos, but not v-fos, is stimulated Induction of c-fos gene and protein by growth factors precedes by 12-tetradecanoylphorbol-13-acetate and serum. Mol. Cell. activation of c-myc. Nature (London) 312:716-720. Biol. 7:2201-2211. 23. Nielsen, C., J. Sando, and W. Pratt. 1977. Evidence that 2. Bohmann, D., T. J. Bos, A. Admon, T. Nishimura, P. K. Vogt, dephosphorylation inactivates glucocorticoid receptors. Proc. and R. Tjian. 1987. Human proto-oncogene c-jun encodes a Natl. Acad. Sci. USA 74:1398-1402. DNA binding protein with structural and functional properties 24. Nishina, H., H. Sato, T. Suzuki, M. Sato, and H. Iba. 1990. of transcription factor AP-1. Science 238:1386-1392. Isolation and characterization of fra-2, an additional member of 3. ChaUberg, M., and T. Kelly. 1979. Adenovirus DNA replication the fos gene family. Proc. Natl. Acad. Sci. USA 87:3619-3623. in vitro. Proc. Natl. Acad. Sci. USA 76:655-659. 25. Orti, E., D. B. Mendel, L. I. Smith, and A. Munck. 1989. 4. Chavrier, P., M. Zerial, P. Lemaire, J. Almendral, R. Bravo, Agonist-dependent phosphorylation and nuclear dephosphory- and P. Charnay. 1988. A gene encoding a protein with zinc lation of glucocorticoid receptors in intact cells. J. Biol. Chem. fingers is activated during GO/Gl transition in cultured cells. 264:9728-9731. EMBO J. 7:29-35. 26. Perrot-Applanat, M., F. Logeat, M. T. Groyer-Picard, and E. 5. Christy, B. A., L. F. Lau, and D. Nathans. 1988. A gene Milgrom. 1985. Immunocytochemical study of mammalian pro- activated in mouse 3T3 cells by serum growth factors encodes a gesterone receptor using monoclonal antibodies. Endocrinology protein with "zinc finger" sequences. Proc. Natl. Acad. Sci. 116:1473-1484. USA 85:7857-7861. 27. Picard, D., and K. R. Yamamoto. 1987. Two signals mediate 6. Cohen, D., P. Ferreira, R. Gentz, B. Franza, and T. Curran. hormone-dependent nuclear localization of the glucocorticoid 1989. The product of a fos-related gene, fra-1, binds coopera- receptor. EMBO J. 6:3333-3340. tively to the AP-1 site with Jun: transcription factor AP-1 is 28. Rauscher, F. J., L. C. Sambucetti, T. Curran, R. J. Distel, and comprised of multiple protein complexes. Genes Dev. 3:173- B. M. Spiegelman. 1988. Common DNA binding site for fos 184. protein complexes and transcription factor AP-1. Cell 52:471- 7. Curran, T., and J. Morgan. 1986. Barium modulates c-fos 480. expression and post-translational modification. Proc. Natl. 29. Ryder, K., A. Lanahan, E. Perez-Albuerne, and D. Nathans. Acad. Sci. USA 83:8521-8524. 1989. Jun-D: a third member of the Jun gene family. Proc. Natl. 8. Day, M. L., T. J. Fahrner, S. Ayken, and J. Milbrandt. 1990. Acad. Sci. USA 86:1500-1503. The zinc finger protein NGFI-A exists in both nuclear and 30. Ryder, K., L. Lau, and D. Nathans. 1988. A gene activated by cytoplasmic forms in nerve growth factor-stimulated PC12 cells. growth factors is related to the oncogene v-jun. Proc. Natl. J. Biol. Chem. 265:15253-15260. Acad. Sci. USA 85:1487-1491. 9. Denner, L. A., N. I. Weigel, W. T. Schrader, and B. W. 31. Ryseck, R.-P., H. Macdonald-Bravo, M.-G. Mattei, S. Ruppert, O'MaUley. 1989. Hormone-dependent regulation of chicken pro- and R. Bravo. 1989. Structure, mapping and expression of a gesterone receptor deoxyribonucleic acid binding and phos- growth factor inducible gene encoding a putative nuclear hor- phorylation. Endocrinology 125:3051-3058. EMBO J. 8:3327-3335. 10. Dieckmann, C., and A. Tzagoloff. 1985. Assembly of the mito- monal binding receptor. chondrial membrane system. J. Biol. Chem. 260:1513-1520. 32. Sheridan, P. L., R. M. Evans, and K. B. Horwitz. 1989. 11. Hazel, T. G., D. Nathans, and L. F. Lau. 1988. A gene inducible Phosphotryptic peptide analysis of human progesterone recep- by serum growth factors encodes a member of the steroid and tors. J. Biol. Chem. 264:6520-6528. thyroid hormone receptor superfamily. Proc. Natl. Acad. Sci. 33. Sheridan, P. L., M. D. Francis, and K. B. Horwitz. 1989. USA 85:8444 8448. Synthesis of human progesterone receptors in T47D cells. J. 12. Hoeck, W., S. Rusconi, and B. Groner. 1989. Down-regulation Biol. Chem. 264:7054-7058. and phosphorylation of glucocorticoid receptors in cultured 34. Springer, T. 1980. Cell surface differentiation in the mouse, p. cells. J. Biol. Chem. 264:14396-14402. 185-217. In R. Kennett, T. McKearn, and K. Bechtol (ed.), 13. Joseph, L. J., M. M. Le Beau, G. A. Jamieson, Jr., S. Acharya, Monoclonal antibodies. Plenum Publishing Corp., New York. T. B. Shows, J. D. Rowley, and V. P. Sukhatme. 1988. Molecular 35. Sukhatme, V. P., X. Cao, L. C. Chang, C. H. Tsai-Morris, D. cloning, sequencing, and mapping of EGR2, a human early Stamenkovich, P. C. P. Ferreira, D. R. Cohen, S. A. Edwards, growth response gene encoding a protein with "zinc-binding T. B. Shows, T. Curran, M. Le Beau, and E. D. Adamson. 1988. finger" structure. Proc. Natl. Acad. Sci. USA 85:7164-7168. A zinc finger-encoding gene coregulated with c-fos during 14. King, W. J., and G. L. Greene. 1984. Monoclonal antibodies growth and differentiation, and after cellular depolarization. localize estrogen receptor in the nuclei of target cells. Nature Cell 53:37-43. (London) 307:745-749. 36. Urlaub, G., E. Kas, A. Carothers, and L. Chasin. 1983. Deletion 15. Lau, L. F., and D. Nathans. 1985. Identification of a set of genes of the diploid dihydrofolate reductase locus from cultured expressed during the GO/Gl transition of cultured mouse cells. mammalian cells. Cell 33:405-412. EMBO J. 4:3145-3151. 37. Watson, M. A., and J. Milbrandt. 1989. The NGFI-B gene, a 16. Lemaire, P., 0. Revelant, R. Bravo, and P. Charnay. 1988. Two transcriptionally inducible member of the steroid receptor gene mouse genes encoding potential transcription factors with iden- superfamily: genomic structure and expression in rat brain after tical DNA-binding domains are activated by growth factors in seizure induction. Mol. Cell. Biol. 9:4213-4219. cultured cells. Proc. Natl. Acad. Sci. USA 85:4691-4695. 38. Wikstrom, A. C., 0. Bakke, S. Okret, M. Bronnegard, and J. A. 17. Migliaccio, A., and F. Auricchio. 1981. Hormone binding of Gustafson. 1987. Intracellular localization of the glucocorticoid estradiol-17P receptor: evidence for its regulation by cytoplas- receptor: evidence for cytoplasmic and nuclear localization. mic phosphorylation and nuclear dephosphorylation by anties- Endocrinology 120:1232-1242. trogens. J. Steroid Biochem. 15:369-381. 39. Williams, D., R. Van Frank, W. Muth, and J. Burnett. 1982. 18. Migliaccio, A., A. Rotondi, and F. Auricchio. 1986. Estradiol Cytoplasmic inclusion bodies in Escherichia coli producing receptor: phosphorylation on tyrosine in uterus and interaction biosynthetic human insulin proteins. Science 215:687-689. with antiphosphotyrosine antibody. EMBO J. 5:2867-2872. 40. Zerial, M., L. Toschi, R. Ryseck, M. Schuermann, R. Muller, 19. Milbrandt, J. 1986. Nerve growth factor rapidly induces c-fos and R. Bravo. 1989. The product of a novel growth factor mRNA in PC12 rat pheochromocytoma cells. Proc. Natl. Acad. activated gene, fos B, interacts with jun proteins enhancing their Sci. USA 83:4789-4793. DNA binding activity. EMBO J. 8:805-813.