help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2005-0289
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
20/8/1786    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baan, B.
Right arrow Articles by Ouwens, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baan, B.
Right arrow Articles by Ouwens, D. M.
Molecular Endocrinology 20 (8): 1786-1795
Copyright © 2006 by The Endocrine Society

The Role of c-Jun N-Terminal Kinase, p38, and Extracellular Signal-Regulated Kinase in Insulin-Induced Thr69 and Thr71 Phosphorylation of Activating Transcription Factor 2

Bart Baan, Hans van Dam, Gerard C. M. van der Zon, J. Antonie Maassen and D. Margriet Ouwens

Department of Molecular Cell Biology, Section Signal Transduction and Ageing, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands

Address all correspondence and requests for reprints to: D. Margriet Ouwens, Ph.D., Department of Molecular Cell Biology, Section Signal Transduction and Ageing, Leiden University Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: d.m.ouwens{at}lumc.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The stimulation of cells with physiological concentrations of insulin induces a variety of responses, e.g. an increase in glucose uptake, induction of glycogen and protein synthesis, and gene expression. One of the determinants regulating insulin-mediated gene expression may be activating transcription factor 2 (ATF2). Insulin activates ATF2 by phosphorylation of Thr69 and Thr71 via a two-step mechanism, in which ATF2-Thr71 phosphorylation precedes the induction of ATF2-Thr69+71 phosphorylation by several minutes. We previously found that in c-Jun N-terminal kinase (JNK)–/– fibroblasts, cooperation of the ERK1/2 and p38 pathways is required for two-step ATF2-Thr69+71 phosphorylation in response to growth factors. Because JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, ERK1/2 and p38 in the insulin-induced two-step ATF2 phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1-adipocytes. The induction of ATF2-Thr71 phosphorylation was sensitive to MAPK kinase (MEK) 1/2-inhibition with U0126, and this phosphorylation coincided with nuclear translocation of phosphorylated ERK1/2. Use of the JNK inhibitor SP600125 or expression of dominant-negative JNK-activator SAPK kinase (SEK1) prevented the induction of ATF2-Thr69+71, but not ATF2-Thr71 phosphorylation by insulin. ATF2-dependent transcription was also sensitive to SP-treatment. Abrogation of p38 activation with SB203580 or expression of dominant-negative MKK6 had no inhibitory effect on these events. In agreement with this, the onset of ATF2-Thr69+71 phosphorylation coincided with the nuclear translocation of phosphorylated JNK. Finally, in vitro kinase assays using nuclear extracts indicated that ERK1/2 preceded JNK translocation. We conclude that sequential activation and nuclear appearance of ERK1/2 and JNK, rather than p38, underlies the two-step insulin-induced ATF2 phosphorylation in JNK-expressing cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN ELICITS COMPLEX responses in the body to maintain glucose and lipid homeostasis. Dependent on the target tissue, insulin regulates a variety of responses, including glucose uptake; glycogen, protein, and lipid synthesis; and gene expression. Next to the widely studied effects of insulin on the Forkhead/FOXO-transcription factors, phosphorylation of activating transcription factor 2 (ATF2), may also contribute to the effects of insulin on gene expression (1, 2).

ATF2 is a ubiquitously expressed member of the cAMP-responsive element-binding protein family of basic region-leucine zipper transcription factors also including cAMP response element-binding protein (3) and ATF3, ATF4, and ATF6 (4, 5). ATF2 can form homo- and heterodimers with other ATF family members (5), but also with the activating protein-1 family member cJun (6). The various dimer compositions confer a large repertoire of target genes on ATF2. Described ATF2 target-genes include cJun, cyclins A and D1, ATF3, TNF{alpha}, and peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (7, 8, 9, 10, 11, 12, 13).

In the absence of stimuli, ATF2 is prebound to DNA (13, 14) and held in an inactive conformation (15). Phosphorylation of residues within the activation domain leads to ATF2 transcriptional activation (16, 17, 18). In particular, phosphorylation of two threonine (Thr) residues, Thr69 and Thr71, seems to be required and sufficient for transcriptional activation of ATF2 (7, 19).

For mouse fibroblasts expressing the human insulin receptor, we previously reported that insulin induces ATF2-activation via phosphorylation of Thr69 and Thr71 via a two-step mechanism (2). Notably, this mechanism involves cooperation of two different pathways. Studies in c-Jun N-terminal kinase (JNK)1,2 –/– embryonic fibroblasts indicated that the ERK1/2 pathway mediates the induction of ATF2-Thr71 phosphorylation, whereas induction of ATF2-Thr69-phosphorylation involves the activity of the p38 pathway (2).

However, JNK is expressed in most cell types and is known to be capable of phosphorylating ATF2 (7, 19, 20). Purification of endogenous insulin-activated ATF2 kinases identified JNK, in addition to ERK and p38, as a putative candidate (2). These observations prompted us to further determine the contribution of JNK, ERK1/2 and p38, in insulin-induced ATF2-phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1 adipocytes. These cell types were used as representatives of the two major types of insulin responses: A14 cells respond primarily in a mitogenic manner, whereas 3T3L1 adipocytes respond more metabolically to insulin.

We studied in vivo and in vitro induction of ATF2-Thr71 and ATF2-Th69+71 phosphorylation at various time points after insulin stimulation using pharmacological inhibitors of the JNK, ERK1/2, and p38 pathways as well as overexpression of dominant-negative upstream kinases regulating the activity of JNK and p38. In addition, we examined the insulin-induced nuclear translocation of these kinases by immunofluorescence and assessed nuclear fractions for the presence of in vitro nuclear ATF2-kinase activity that could be ascribed to JNK, ERK1/2, or p38.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differential Onset of Insulin-Induced ATF2-Thr71 and ATF2-Thr69+71 Phosphorylation
Insulin treatment of A14 fibroblasts induced the phosphorylation of ATF2 on Thr71 within 2–4 min, whereas phosphorylation of ATF2-Thr69+71 was achieved only at 7–10 min after addition of insulin (Fig. 1AGo). A comparable situation was found in 3T3L1 adipocytes, in which insulin-induced ATF2-Thr71 phosphorylation was found after 4 min and preceded the induction of ATF2-Thr69+71 phosphorylation (Fig. 1BGo). In contrast, the induction of ATF2-Thr69+71 phosphorylation in response to osmotic shock (O.S.) was not preceded by ATF2-Thr71 phosphorylation (Fig. 1CGo).


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1. Differential Onset of Insulin-Induced ATF2-Thr71 and ATF2-Thr69+71 Phosphorylation

Serum-starved A14 cells (A) or 3T3L1 adipocytes (B) were treated with 10 nM insulin (INS). Total cell lysates were prepared at indicated time points and analyzed by Western blotting using phospho-specific ATF2-Thr71, ATF2-Thr69+71 antibodies. The faster migrating bands recognized by the phospho-specific ATF2-antibodies seem to represent shorter, alternatively spliced, ATF2 products (2 ). C, Cell lysates were prepared from A14 cells treated with 0.5 M NaCl (O.S.) and immunoblotted as described for panels A and B. D, Cell lysates prepared from insulin-treated A14 cells as described above were used for extended electrophoresis and subsequently blotted using ATF2-specific antibody.

 
The induction of ATF2-Thr69+71 phosphorylation was accompanied by a retarded mobility of the protein on ATF2 immunoblot, which was preceded by the induction of an intermediate form after 4 min of insulin treatment (Fig. 1DGo). The induction of this intermediate form coincided with the onset of ATF2-Thr71 phosphorylation and underscores the concept that in response to insulin treatment the ATF2 protein undergoes a transition from the nonphosphorylated form to the ATF2-Thr69+71-phosphorylated form via an intermediate ATF2-Thr71-phosphorylated form.

Differential Onset of Insulin-Induced MAPK Phosphorylation
Upon examining the time course of ERK1/2, p38, and JNK activation in A14 fibroblasts, we found that ERK1/2 was phosphorylated within 2 min of insulin treatment (Fig. 2AGo). This phosphorylation was accompanied by in vitro ATF2-directed kinase activity in ERK immunoprecipitates (Fig. 2BGo). Also, some phosphorylation of p38 and ATF2-directed kinase activity in p38 immunoprecipitates was observed within 2–4 min of insulin treatment of A14 fibroblasts (Fig. 2Go, A and B). The onset of JNK phosphorylation was delayed compared with ERK and p38 phosphorylation and was observed after 7 min of insulin treatment. The phosphorylation of JNK associated with the presence of ATF2 kinase activity in JNK immunoprecipitates (Fig. 2Go, A and B).


Figure 2
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 2. Time Course of Insulin-Induced MAPK Phosphorylation

A, Lysates from serum-starved A14 cells were prepared at the indicated time points after 10 nM insulin-treatment (INS) and were examined for phosphorylation of ERK1/2, p38, and JNK by immunoblotting with phospho-specific antibodies. B, ERK1/2, p38, and JNK immunoprecipitates were analyzed for ATF2-directed kinase activity in an in vitro kinase assay using GST-ATF2 as substrate for 1 h. The Coomassie-stained gel confirmed equal loading of GST-ATF2. IP, Immunoprecipitation; ERK1/2-PP, phosphorylated ERK1/2; p38-PP, phosphorylated p38; JNK-PP, phosphorylated JNK.

 
Early Insulin-Induced ATF2-Thr71 Phosphorylation Is Sensitive to Inhibition of the MAPK Kinase (MEK)-ERK Pathway
Because both ERK1/2 and p38 activation coincided with the onset of ATF2-Thr71 phosphorylation, we analyzed the involvement of these MAPKs using pharmacological inhibitors in A14 fibroblasts. Inhibition of p38 using SB203580 (21) had no effect on the induction of ATF2-Thr71 phosphorylation by insulin (Fig. 3AGo). However, prevention of ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126 (22) completely abrogated the induction of ATF2-Thr71 phosphorylation (Fig. 3AGo) and the appearance of the retarded (intermediate) form of ATF2 in response to 4-min insulin treatment (data not shown). Comparable results were obtained in 3T3L1 adipocytes: U0126 prevented insulin-induced increase in ATF2-Thr71 phosphorylation, and pretreatment with SB203580 had no inhibitory effect on insulin-induced ATF2-Thr71 phosphorylation (Fig. 3BGo).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3. Early Insulin-Induced ATF2-Thr71 Phosphorylation Is Sensitive to Inhibition of the MEK1/2-ERK1/2 Pathway

Serum-starved A14 cells (A) or 3T3L1 adipocytes (B) were incubated with 10 µM U0126 (U; 15 min) or 2.5 µM SB203580 (SB; 30 min) before insulin stimulation (INS; 10 nM) for 4 min. Total ATF2 and ATF2-Thr71-phosphorylation levels were determined by immunoblotting with specific antibodies. C, A14 cells were treated with inhibitors as described above, before stimulation with 10 nM of insulin for 4 min. Subsequently, cells were fixed and stained with phospho-specific ATF2-Thr71 antibodies followed by fluorescein isothiocyanate-conjugated secondary antibodies (green). DNA was stained with DAPI (blue). Neg, Negative; Veh, vehicle.

 
In line with these data, immunofluorescence experiments on A14 cells demonstrated nuclear phospho-ATF2-Thr71 immunoreactivity detected after 4 min of insulin treatment, which was abolished upon pretreatment with U0126, but not with SB203580 (Fig. 3CGo). The nuclear localization of ATF2 did not change during the insulin stimulation (data not shown). Collectively, these results suggest that ERK1/2, rather than p38, is responsible for the insulin-induced phosphorylation of nuclear ATF2 on Thr71 after 4 min of stimulation.

Cooperation of ERK1/2 with p38 or JNK Is Required for ATF2-Thr69+71 Phosphorylation in Vitro
When we analyzed lysates prepared from A14 cells treated for 4 min with insulin, predominantly ATF2-Thr71-directed kinase activity was found (Fig. 4AGo). This is in line with the observation that ERK1/2 can only phosphorylate ATF2 on Thr71 and not on Thr69 (2, 23). In lysates prepared after 15 min of insulin stimulation, ATF2-Thr71-directed kinase activity was similar to that found in 4-min lysates, but the level of ATF2-Thr69+71-directed kinase activity was markedly increased (Fig. 4AGo). To purify the kinase(s) responsible for the induction of ATF2-Thr69+71 phosphorylation, MonoQ fractionation was performed on lysates prepared after 15 min of insulin stimulation, and the obtained fractions were analyzed for the presence of ATF2-Thr71-directed vs. ATF2-Thr69+71-directed kinase activity (2). Approximately 90% of the input ATF2-Thr71-directed kinase activity was recovered after anion-exchange chromatography, and the majority of this fraction (80%) copurified with fractions containing ERK1/2 (2). The remaining ATF2-Thr71-directed kinase activity was recovered in fractions copurifying with JNK and p38, respectively (2). These fractions also contained ATF2-Thr69+71-directed kinase activity. However, in contrast to the almost complete recovery of ATF2-Thr71-directed kinase activity, only 5% of the input ATF2-Thr69+71-directed kinase activity was recovered in the fractions containing JNK and p38 (2). Comparable results were obtained for fractionation of lysates from 3T3L1 adipocytes (data not shown). Collectively, these findings raised the possibility that maximal induction of ATF2-Thr69+71 phosphorylation by insulin requires cooperation of ERK1/2 with p38 and/or JNK. To test this possibility, we added partially purified ERK1/2 to the kinase assays on fractions copurifying with JNK and p38. Indeed, the weak insulin-induced ATF2-Thr71-directed and Thr69+71-directed kinase activities copurifying with p38 and JNK were greatly enhanced by addition of ERK1/2 fraction to the assay (Fig. 4BGo). Similar results were obtained upon addition of recombinant ERK1/2 to the kinase assay (data not shown). Together, these findings suggest that phosphorylation of ATF2 on Thr69 by p38 and/or JNK is more efficient when ATF2 is already phosphorylated on Thr71, at least in vitro.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. Cooperation of ERK1/2 with p38 or JNK Is Required for Efficient Insulin-Induced ATF2-Thr69+71 Phosphorylation

A, Lysates from serum-starved A14 fibroblasts treated with 10 nM insulin (INS) for the indicated times were used in ATF2-directed in vitro kinase assays. Site-specific phosphorylation of GST-ATF2 was determined by Western blotting using ATF2-Thr71 and ATF2-Thr69+71 phospho-specific antibodies, respectively. B, MAPK-containing MonoQ fractions of lysates of 15-min insulin-treated serum-starved A14 cells were used in in vitro ATF2-directed kinase assays for 1 h. Site-specific phosphorylation of GST-ATF2 was determined as described above. Ponceau staining of the same blot is shown to verify equal loading of GST-ATF2.

 
Inhibition of JNK, But Not p38, Abrogates Insulin-Induced ATF2-Thr69+71 Phosphorylation and Activation
To determine the involvement of p38 and JNK in insulin-induced phosphorylation of ATF2 in intact cells, we used SB203580 and SP600125 (24) to inhibit p38 and JNK, respectively. As shown in Fig. 5Go, A and B, insulin-induced ATF2-Thr69+71 phosphorylation was abrogated by SP600125, but not SB203580, in both A14 fibroblasts and 3T3L1 adipocytes. It is noteworthy that we observed some inhibition of ATF2-Thr71 phosphorylation by SP600125 even under conditions when JNK is not activated, i.e. under basal conditions and at 4 min after insulin addition, indicating nonspecific side effects of this inhibitor (Fig. 5AGo). ATF2 band-shift analysis corroborated these results: the slowest migrating (Thr69+71 phosphorylated) form of ATF2 found after 10 min was not affected by SB203580 but shifted back to the intermediate form in the presence of SP600125 (Fig. 5AGo).


Figure 5
View larger version (50K):
[in this window]
[in a new window]
  		Fig. 5. Inhibition of JNK, But Not p38, Abrogates Insulin-Induced ATF2-Thr69+71 Phosphorylation

Serum-starved A14 fibroblasts (A) or 3T3L1 adipocytes (B) were incubated for 30 min with DMSO, 2.5 µM SB203580 (SB), 10 µM SP600125 (SP), or SB+SP before stimulation with 10 nM insulin (INS) for the indicated times. Phosphorylation of ATF2-Thr71, ATF2-Thr69+71, JNK, p38, and ERK1/2 was determined with phospho-specific antibodies. C, A14 fibroblasts were transfected with either an empty vector (e.v.) or expression vectors for epitope-tagged dominant-negative MKK6-KM or SEK1-KR together with pBabe-puro and pMT2-HA-ATF2. Puromycin-resistant cells were selected, serum-starved overnight, and subsequently stimulated with 10 nM insulin for 15 min. Phosphorylation of ATF2-Thr71, ATF2-Thr69+71 was determined with phospho-specific antibodies.

 
Similar results were found with the use of dominant-negative upstream kinases of p38 and JNK: MKK6-KM and SAPK kinase (SEK1) SEK1-KR, respectively (see Fig. 5CGo): Overexpression of SEK1-KR abrogated only the insulin-induced ATF2-Thr69+71 phosphorylation but not the Thr71-phosphorylation found after 15 min of insulin stimulation. Overexpression of MKK6-KM had no effect on insulin-induced ATF2-phosphorylation (Fig. 5CGo).

Immunofluorescence experiments showed that 10 min of insulin treatment increased nuclear ATF2-Thr69+71 immunoreactivity compared with 4 min and untreated A14 cells, and that this immunoreactivity was sensitive to SP600125 (Fig. 6AGo).


Figure 6
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 6. Inhibition of JNK, But Not p38, Abrogates the Insulin-Induced ATF2 Phosphorylation and Activation

A, Serum-starved A14 cells were treated with 10 µM SP600125 (SP) for 30 min before stimulation with 10 nM insulin (INS) for the indicated times. Cells were fixed and stained with antibodies for Thr69+71-phosphorylated ATF2 followed by fluorescein isothiocyanate-conjugated secondary antibodies (green). DNA was stained with DAPI (blue). B, Insulin-induced ATF2 transcriptional activity was examined in a GAL4-dependent luciferase reporter assay using the activation domain of ATF2 fused to the GAL4 DNA-binding domain (19 ). Cells were transiently transfected and grown for 8 h, subsequently serum-starved in DMEM containing 0.5% FBS overnight, and treated for 30 min with vehicle or inhibitors SB203580 or SP600125 before adding insulin (INS; 10 nM). Cells were lysed 16 h later, and luciferase activity was determined. The relative firefly luciferase activity is depicted as the mean enhancement of promoter activity in the absence or presence of insulin and/or inhibitors ± the SD of three independent experiments performed in triplicate. Note the different scaling of the left and right y-axis. **, P = 0.0097 (Student’s t test). neg, Negative; veh, vehicle.

 
To determine whether modulation of insulin-induced ATF2-Thr69+71 phosphorylation affected ATF2-mediated transcription accordingly, we examined the insulin-induced activation of a GAL4-ATF2-dependent luciferase reporter. Insulin-induced activation was significantly inhibited by SP600125 (P = 0.0097) but not by SB203580 (Fig. 6BGo).

The Onset of ATF2-Thr71 and ATF2-Thr69+71 Phosphorylation Associates with Nuclear Translocation of ERK1/2 and JNK
The experiments described above suggest that cooperation of ERK1/2 with JNK, rather than with p38, is required for insulin-induced two-step ATF2-Thr69+71 phosphorylation. To corroborate these findings we examined whether ERK1/2 and JNK translocate to the nucleus in response to insulin. We found that within 4 min, insulin induced the phosphorylation and nuclear translocation of ERK1/2 (ERK1/2-PP; see Fig. 7AGo). U0126 pretreatment strongly inhibited the ERK1/2-PP signal and reduced nuclear staining found with a pan-ERK1/2 antibody (Fig. 7AGo and data not shown).


Figure 7
View larger version (32K):
[in this window]
[in a new window]
  		Fig. 7. Time Course of Nuclear Translocation of ERK1/2 and JNK

Serum-starved A14 cells were incubated with DMSO, 10 µM U0126 (U) for 15 min, or 10 µM SP600125 (SP) for 30 min before stimulation with 10 nM insulin (INS) for the indicated times. Cells were fixed and stained with antibodies for (A) phosphorylated ERK (ERK-PP) or (B) JNK-PP and fluorescein isothiocyanate-conjugated secondary antibodies (green). DNA was stained with DAPI (blue). neg, Negative.

 
Despite the high aspecific background level of (nonnuclear) phosphorylated JNK (JNK-PP)-signal in the absence of insulin, no increase in JNK-PP staining could be detected after 4 min of insulin treatment (Fig. 7BGo). The amount of JNK-PP in the nucleus was substantially increased after 10 min of insulin stimulation, and this response was inhibited by SP600125 (Fig. 7BGo).

In line with the immunofluorescence data, we found that nuclear extracts prepared from cells after 4 min of insulin stimulation contained predominantly ATF2-Thr71-directed kinase activity (Fig. 8AGo). This ATF2-Thr71-directed activity could be abrogated by addition of ERK inhibitor 5-iodotubercidin [Itu (25)] and was absent in nuclear extracts from U0126-treated cells (data not shown). The ATF2-Thr71-directed kinase activity in these nuclear extracts was not affected by SP600125 or SB203580 (Fig. 8AGo).


Figure 8
View larger version (56K):
[in this window]
[in a new window]
  		Fig. 8. Sequential Appearance of ERK1/2- and JNK-Dependent ATF2-Directed Nuclear Kinase Activities

Serum-starved A14 cells stimulated with 10 nM insulin (INS) for 4 min (A) or 10 min (B). Nuclear proteins were extracted and used in in vitro ATF2-directed kinase assays for 2 h in the presence of DMSO or inhibitors 2.5 µM SB203580 (SB), 5 µM SP600125 (SP), or 5 µM Itu. Site-specific phosphorylation of GST-ATF2 was determined by Western blotting using ATF2-Thr71 and ATF2-Thr69+71 phospho-specific antibodies. Ponceau staining of the same blot is shown to verify equal loading of GST-ATF2.

 
Nuclear extracts obtained after 10 min of insulin stimulation contained both ATF2-Thr71- and ATF2-Thr69+71-directed kinase activity (Fig. 8BGo). Addition of the JNK inhibitor SP600125 abrogated only the ATF2-Thr69+71-phosphorylation, but not the ATF2-Thr71-directed activity (Fig. 8BGo). SB203580 did not affect the ATF2-Thr69+71 and ATF2-Thr71 activities, whereas Itu reduced both ATF2-Thr71 and ATF2-Thr69+71 kinase activities.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study addresses the activation of the transcription factor ATF2 by insulin. It extends our previous findings (2) by addressing the mechanism by which insulin accomplishes this activation in A14 cells and 3T3L1 adipocytes. The results of this study suggest the following model for the induction of ATF2-Thr69+71 phosphorylation in response to insulin (summarized in Fig. 9Go): within 2–4 min, insulin induces activation of ERK1/2, which translocates to the nucleus and mediates ATF2-Thr71 phosphorylation. Between 5 and 10 min after the addition of insulin, JNK is activated, translocates to the nucleus, and mediates Thr69 phosphorylation of phospho-Thr71-ATF2, thereby activating the transcription factor. We propose that the sequential activation and subsequent nuclear appearance of ERK1/2 and JNK are rate limiting for the two-step phosphorylation of ATF2-Thr69+71 in response to insulin.


Figure 9
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 9. Proposed Model for Insulin-Induced ATF2 Activation in A14 Cells and 3T3L1 Adipocytes

Insulin induces activation of ERK1/2 within 2–4 min. ERK1/2 subsequently translocates to the nucleus, where it mediates ATF2-Thr71 phosphorylation. Subsequently, JNK is activated, translocates to the nucleus, and efficiently phosphorylates Thr71-phosphorylated ATF2 on Thr69, thus inducing transcriptional activation of ATF2.

 
Previously, we reported that in JNK–/– cells, the ERK1/2 and the p38 pathways cooperate in the growth factor-induced phosphorylation of ATF2-Thr69+71 (2). Although this study indicates that JNK may be dispensable for insulin-mediated ATF2-phosphorylation, pharmacological inhibition of p38 in JNK-expressing cells had no inhibitory effect on the induction ATF2 phosphorylation, suggesting that JNK, rather than p38, regulates this response.

Accordingly, various studies have identified JNK as a bona fide ATF2 kinase (2, 7, 19, 20, 23). An important finding of the present study, however, is that JNK predominantly functions as ATF2-Thr69-kinase for ATF2 already phosphorylated on Thr71, in the proposed two-step ATF2-Thr69+71 phosphorylation mechanism induced by insulin. In contrast, in response to inducers of cellular stress such as UV, methyl methane sulfonate, O.S., and TNF{alpha}, the onset of ATF2-Thr69+71 phosphorylation coincides with the onset of ATF2-Thr71 phosphorylation, and this process seems completely JNK dependent in JNK-expressing cells (Refs. 2 , 7 , and 23 and data not shown). This raises the question whether activation of JNK alone may be sufficient for the induction of ATF2 phosphorylation in response to insulin treatment. However, the data shown here indicate that ERK1/2 activation also seems to be biologically relevant for insulin-mediated two-step ATF2-phosphorylation. Biochemical evidence for its involvement is provided by changes in electrophoretic mobility of the ATF2 protein during the course of insulin-mediated phosphorylation. After insulin stimulation, the ATF2 protein undergoes a transition via an ATF2-Thr71-phosphorylated form to an ATF2-Thr69+71-phosphorylated form. More importantly, pharmacological inhibition of ERK activation prevented early ATF2-Thr71 phosphorylation and the accompanying retarded mobility of the protein. In addition, ERK1/2 phosphorylation and ATF2-Thr71 phosphorylation are found several minutes before JNK activation and ATF2-Thr69+71 phosphorylation were detectable. Collectively, these findings highlight the importance of ERK1/2 for insulin-induced ATF2 phosphorylation and provide support for our model.

We and others have demonstrated that ERK preferentially phosphorylates ATF2 on Thr71, whereas the kinase seems unable to efficiently mediate ATF2-Thr69 phosphorylation (2, 23). Therefore, cooperation with another kinase seems required for induction of ATF2-Thr69+71 phosphorylation in response to insulin. Interestingly, whereas MonoQ fractionation of lysates from O.S.-treated cells yielded similar recoveries for ATF2-Thr69+71- and ATF2-Thr71-directed kinase activities, in cell extracts from insulin-treated cells, only about 5% of the ATF2-Thr69+71-directed in vitro kinase activity was recovered, in contrast to approximately 80% recovery of ATF2-Thr71-directed activity (for details see Ref. 2) Importantly, the weak insulin-induced ATF2-Thr69+71 kinase activities, which copurified with JNK and p38, respectively, were greatly enhanced by the addition of ERK1/2 to the kinase reaction. Collectively, these findings strongly suggest that insulin-activated MAPKs cooperate to induce efficient ATF2-Thr69+71 phosphorylation and provide strong support for our two-step model.

Previously, Waas et al. (26) found that recombinant active p38 phosphorylates glutathione-S-transferase (GST)-ATF2 via a two-step (double collision) mechanism, involving the dissociation of monophosphorylated Thr71-ATF2 or Thr69-ATF2 from the enzyme after the first phosphorylation step. Importantly, these authors found that monophosphorylation of ATF2-Thr69 strongly reduces the phosphorylation rate of Thr71, whereas monophosphorylation of Thr71 does not reduce the rate of Thr69 phosphorylation (26). Thus, efficient phosphorylation of ATF2 by recombinant active p38 only occurs in the order Thr71 -> Thr69+71. In our model, phosphorylation of ATF2-Thr71 by ERK1/2 might prime ATF2 for subsequent efficient ATF2-Thr69 phosphorylation. As described above, the kinase assays on MonoQ fractions indicate that both p38 and JNK can indeed enhance the phosphorylation of ATF2-Thr69+71 in the presence of activated ERK1/2 in vitro. In cultured cell lines expressing both kinases, however, JNK, rather than p38, seems to mediate this second phosphorylation. Several lines of evidence point in this direction. First, chemical inhibition of JNK activity with SP600125, or prevention of JNK activation by overexpression of dominant-negative SEK1, abrogated only the Thr69+71 phosphorylation in response to insulin. Second, prevention of p38 activation by overexpression of dominant-negative MKK6 or inhibition of p38 by SB203580 failed to affect insulin-mediated ATF2 phosphorylation in JNK-expressing cells. In part, the absence of the inhibitory effect of SB could be ascribed to an enhanced activation of JNK, which was observed in the presence of this inhibitor (Ref. 2 ; see also Fig. 5Go and supplemental Fig. S1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). However, simultaneous addition of SB203580 and SP600125 had the same effect as SP600125 alone, suggesting that this is not the case. In addition, we could not obtain evidence for nuclear translocation of activated p38 in insulin-treated cells neither in immunofluorescence assays nor in in vitro kinase assays on nuclear extracts.

To exclude the possibility that SB203580 was unable to inhibit p38, we analyzed inhibition of p38 activity by SB203580 in vitro and in vivo. In in vitro kinase assays, the SB compound completely abrogated ATF2-directed kinase activity found in p38-containing MonoQ-fractions of insulin-stimulated A14 fibroblasts (data not shown). Also, we found that phosphorylation of p38’s downstream nuclear target MAPKAPK2 [MK2 (27)] in response to insulin in JNK–/– cells was sensitive to SB203580-treatment (data not shown). Interestingly, although in A14 fibroblasts p38 activity was induced by insulin and reduced by SB203580 treatment, no MK2 phosphorylation could be detected in these cells (supplemental Fig. S1). In contrast, O.S. did induce robust phosphorylation of both p38 and MK2, which were reduced and abrogated, respectively, by SB203580 treatment (supplemental Fig. S1). These observations suggest that the SB203580 compound was functional in in vitro and in vivo assays. In addition, it seems that in JNK-containing A14 cells, insulin-induced phospho-p38 is confined to the cytosol or does not reach its nuclear target MK2, whereas it does reach MK2 after O.S. stimulation.

Collectively, these data support the idea that JNK, rather than p38, is responsible for the second phosphorylation event, and that p38 seems only capable of inducing ATF2 phosphorylation under conditions in which JNK is genetically absent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Cell Stimulation
A14 cells [NIH 3T3 fibroblasts overexpressing the human insulin receptor (28)] were cultured in DMEM containing 9% fetal bovine serum (FBS) and antibiotics. 3T3L1 fibroblasts were obtained from American Type Culture Collection (Manassas, VA) and differentiated to adipocytes as described previously (29). Briefly, 3T3L1 cells were grown to confluence. Differentiation was induced 2 d after confluence by culturing cells for 2 d on DMEM/FBS supplemented with 10 µg/ml of bovine insulin (Sigma Chemical Co., St. Louis, MO), 0.25 µM dexamethasone (Sigma), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), followed by DMEM/FBS containing only insulin (10 µg/ml). Differentiated adipocytes were maintained for another 6 d in DMEM/FBS before use. Before cell stimulations, the cells were serum starved (DMEM with 0.5% FBS) for 16 h. When inhibitors were used, cells were pretreated for 30 min with 10 µM SP600125 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) or 2.5 µM SB203580 (Promega Corp., Madison, WI) or for 15 min with 10 µM U0126 (Promega), before addition of bovine insulin to 10 nM or NaCl (osmotic shock; O.S.) to 500 mM.

Transient Transfection and Luciferase Assays
For overexpression experiments, A14 cells were cotransfected in six-well plates with a total of 1.5 µg of DNA per well; 0.875 µg of either a carrier vector or expression vectors encoding epitope-tagged dominant-negative MKK6-K82M or SEK1-K129R (kindly provided by Dr. J. Kyriakis), 0.5 µg pMT2-HA-ATF2 (2) and 0.125 µg pBabe-puro using FUGENE6 transfection reagent (Roche Biochemicals, Indianapolis, IN) according to the supplier’s protocol. Cells were selected with 3 µg/ml of puromycin (Sigma) for 3–5 d, after which cells were serum starved overnight, stimulated with 10 nM insulin for 15 min, and lysed and blotted as described above.

For GAL4-luciferase assays, A14 cells were transfected in six-well plates using the diethylaminoethyl-dextran method as described previously (30). For each well 0.25 µg pGl3-GAL4-E4-luciferase reporter (2) was cotransfected with 1 µg of carrier vector psp64 and 1 µg of either pC2-Gal4-ATF2-TAD (19) or the carrier vector. Briefly, DNA was mixed in 1 mg/ml diethylaminoethyl-dextran-supplemented Tris-buffered saline (TBS) [25 mM Tris (pH 7.4), 150 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, and 0.5 mM MgCl2] and added to cells. After a 30-min incubation, cells were washed and DMEM/FBS was added. After 8 h, cells were serum starved in DMEM containing 0.5% FBS and 24 h after transfection cells were pretreated with inhibitors before adding insulin (to 10 nM). Cells were lysed 16 h later in luciferase lysis buffer [25 mM Tris (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100], and luciferase activity was determined according to the manufacturer’s protocol (Promega).

Western Blot Analysis and Antibodies
Whole-cell lysates were prepared from 9-cm dishes that were rinsed twice with ice-cold PBS and lysed in 750 µl Laemmli sample buffer. Proteins were separated on polyacrylamide slab gels and transferred to Immobilon (Millipore Corp., Bedford, MA). Blots were stained with Ponceau S before blocking to verify equal loading and appropriate protein transfer. Filters were incubated with antibodies as described previously (2). The antibodies used were: lamin A and phospho-specific ATF2-Thr69+71, ATF2-Thr71, p38-Thr180/Tyr182, ERK1/2-Thr202/Tyr204 (all polyclonal), and JNK-Thr183/Tyr185 monoclonal (all from Cell Signaling Technology, Beverly, MA); p38 (N-20), ATF2 (C-19), and ERK1 (K-23) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and secondary antibodies: goat antirabbit and goat antimouse IgG-horseradish peroxidase conjugate (Promega). EF-1ß antibodies were described previously (31). For ATF2 band shift analysis, immunoblots were prepared after separation of proteins on large 7% polyacrylamide slab gels and incubated with ATF2 (C-19) antibody.

MonoQ/Anion-Exchange Chromatography
Anion-exchange chromatography was performed essentially as described previously (2). Briefly, stimulated cells were scraped in MonoQ lysis buffer [20 mM Tris (pH 7.0), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM sodium ß-glycerolphosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% (vol/vol) ß-mercaptoethanol and Complete protease inhibitors (Roche Biochemicals)]. Lysates of six 9-cm dishes (~8000 mg protein) were diluted 2-fold with MonoQ buffer [50 mM Tris·Cl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5% (vol/vol) glycerol, 0.03% (wt/vol) Brij-35, 1 mM benzamidine, 0.3 mM sodium orthovanadate, and 0.1% (vol/vol) ß-mercaptoethanol] and applied to a MonoQ HR 5/5 column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in MonoQ buffer. After washing, the column was developed with a linear salt gradient to 700 mM NaCl in MonoQ buffer, and fractions of 1 ml were collected. Aliquots of 10 µl from each fraction were used in in vitro ATF2 kinase assays.

Preparation of Nuclear Extracts
For extraction of nuclear proteins, cell lysates were prepared from 9-cm dishes that were rinsed twice with ice-cold PBS and scraped in 1 ml of cold RIPA buffer [30 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 0.5% Na-DOC, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and complete protease inhibitors (Roche)]. Nuclei were pelleted by centrifugation (10 min, 14,000 rpm, 4 C). Supernatants were collected and stored as cytosolic fractions. Nuclear pellets were washed twice with RIPA and were then incubated on ice for 1 h with 75 µl of extraction buffer [30 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM EDTA with Complete protease inhibitors (Roche)] and vortexed every 5 min. After centrifugation, supernatants were collected and used as nuclear protein extracts. Protein content was determined using the BCA-kit (Pierce Chemical Co., Madison, WI). Purity of the cell fractions was checked by Western blotting, using EF-1ß and lamin A antibodies as cytosolic and nuclear markers, respectively (see supplemental Fig. S2 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

ATF2 Kinase Assays
For in vitro ATF2 kinase assays, equal volumes of MonoQ fractions and equal amounts of protein from cytosolic or nuclear extracts were incubated at 30 C with 2 µg of purified GST-ATF2-N substrate (7) and 50 µM ATP in a total volume of 60 µl of kinase buffer [25 mM HEPES (pH 7.4), 25 mM MgCl2, 25 mM ß-glycerolphosphate, 5 mM ß-mercaptoethanol, and 100 µM sodium orthovanadate]. When indicated, kinase assays were performed in the presence of vehicle [dimethylsulfoxide (DMSO)] or inhibitors SB203580 (2.5 µM), SP600125 (5 µM), or 5 µM Itu (25). Reactions were terminated by the addition of 20 µl of 4x Laemmli buffer and subsequently analyzed by SDS-PAGE/immunoblotting with phospho-specific ATF2-Thr69+71 and ATF2-Thr71 antibodies. The specificity of these antibodies has been verified previously (2, 23).

For immunoprecipitation kinase assays, RIPA cell lysates were prepared as described above, and equal aliquots (750 µg) were rotated overnight with 10 µl of p38, JNK, or ERK antibodies coupled to protein A-sepharose beads (Pharmacia) at 4 C. The antibodies used for immunoprecipitation were: ERK 2199 (32), p38 N20 (Santa Cruz), and JNK1 FL (Santa Cruz). Beads were collected by centrifugation and were washed four times with RIPA buffer, and two times with kinase buffer. Subsequently, the beads were incubated with 2 µg of purified GST-ATF2-N substrate (7) and 50 µM ATP, containing 2 µCi [{gamma}-32P]ATP, in a total volume of 60 µl of kinase buffer for 1 h at 30 C.

Immunofluorescence
Cells were grown on coverslips. After stimulation, cells were washed twice with ice-cold PBS and subsequently fixed in 3.7% formaldehyde in PBS for 15 min at room temperature. Coverslips were washed with TBS (25 mM Tris, 100 mM NaCl, 5 mM KCl, 0.7 mM CaCl2·2 H2O, 0.5 MgCl2·6 H2O) and permeabilized for 5 min with 0.1% Triton X-100 in TBS, subsequently washed with 0.2% BSA/TBS, blocked for 30 min in 2% BSA/TBS at room temperature, and then incubated overnight at 4 C with primary antibodies diluted 1:250 in 0.2% BSA/TBS. After 0.2% BSA/TBS washes, coverslips were incubated with appropriate secondary antibodies diluted 1:100 in 0.2% BSA/TBS for 2 h at room temperature. Thereafter, coverslips were washed sequentially with 0.2% BSA/TBS and TBS, mounted in 4',6-diamidino-2-phenylindole (DAPI)-containing Vectashield solution (Vector Laboratories, Burlingame, CA), and fixed with nail polish. Fluorescence was detected using a Leica DM-RXA microscope (Leica Corp., Deerfield, IL). Pictures were acquired as color images (MERGE) and prepared using Photopaint and Coreldraw software (Corel Corp., Ottawa, Ontario, Canada).


   FOOTNOTES
 
B.B. was supported by a grant from the Dutch Diabetes Research Foundation (DFN 2001.00.46).

First Published Online April 6, 2006

Abbreviations: ATF, Activating transcription factor; DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; GST, glutathione-S-transferase; Itu, 5-iodotubercidin; JNK, c-Jun N-terminal kinase; JNK-PP, phosphorylated JNK; MEK, MAPK kinase; O.S., osmotic shock; SEK, SAPK kinase; TBS, Tris-buffered saline.

Received for publication July 14, 2005. Accepted for publication March 27, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Accili D, Arden KC 2004 FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117:421–426[CrossRef][Medline]
  2. Ouwens DM, De Ruiter ND, Van Der Zon GC, Carter AP, Schouten J, Van Der Burgt C, Kooistra K, Bos JL, Maassen JA, van Dam H 2002 Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38. EMBO J 21:3782–3793[CrossRef][Medline]
  3. Mayr B, Montminy M 2001 Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2:599–609[CrossRef][Medline]
  4. Hai T, Hartman MG 2001 The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 273:1–11[CrossRef][Medline]
  5. Hai TW, Liu F, Coukos WJ, Green MR 1989 Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev 3:2083–2090[Abstract/Free Full Text]
  6. Hai T, Curran T 1991 Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci USA 88:3720–3724[Abstract/Free Full Text]
  7. van Dam H, Wilhelm D, Herr I, Steffen A, Herrlich P, Angel P 1995 ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J 14:1798–1811[Medline]
  8. Shimizu M, Nomura Y, Suzuki H, Ichikawa E, Takeuchi A, Suzuki M, Nakamura T, Nakajima T, Oda K 1998 Activation of the rat cyclin A promoter by ATF2 and Jun family members and its suppression by ATF4. Exp Cell Res 239:93–103[CrossRef][Medline]
  9. Beier F, Lee RJ, Taylor AC, Pestell RG, LuValle P 1999 Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. Proc Natl Acad Sci USA 96:1433–1438[Abstract/Free Full Text]
  10. Kool J, Hamdi M, Cornelissen-Steijger P, van der Eb AJ, Terleth C, van Dam H 2003 Induction of ATF3 by ionizing radiation is mediated via a signaling pathway that includes ATM, Nibrin1, stress-induced MAPkinases and ATF-2. Oncogene 22:4235–4242[CrossRef][Medline]
  11. Tsai EY, Jain J, Pesavento PA, Rao A, Goldfeld AE 1996 Tumor necrosis factor {alpha} gene regulation in activated T cells involves ATF-2/Jun and NFATp. Mol Cell Biol 16:459–467[Abstract]
  12. Cao W, Daniel KW, Robidoux J, Puigserver P, Medvedev AV, Bai X, Floering LM, Spiegelman BM, Collins S 2004 p38 Mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene. Mol Cell Biol 24:3057–3067[Abstract/Free Full Text]
  13. Hayakawa J, Mittal S, Wang Y, Korkmaz KS, Adamson E, English C, Omichi M, McClelland M, Mercola D 2004 Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress. Mol Cell 16:521–535[CrossRef][Medline]
  14. Herr I, van Dam H, Angel P 1994 Binding of promoter-associated AP-1 is not altered during induction and subsequent repression of the c-jun promoter by TPA and UV irradiation. Carcinogenesis 15:1105–1113[Abstract/Free Full Text]
  15. Li XY, Green MR 1996 Intramolecular inhibition of activating transcription factor-2 function by its DNA-binding domain. Genes Dev 10:517–527[Abstract/Free Full Text]
  16. Sakurai A, Maekawa T, Sudo T, Ishii S, Kishimoto A 1991 Phosphorylation of cAMP response element-binding protein, CRE-BP1, by cAMP-dependent protein kinase and protein kinase C. Biochem Biophys Res Commun 181:629–635[CrossRef][Medline]
  17. Sevilla A, Santos CR, Vega FM, Lazo PA 2004 Human vaccinia-related kinase 1 (VRK1) activates the ATF2 transcriptional activity by novel phosphorylation on Thr-73 and Ser-62 and cooperates with JNK. J Biol Chem 279:27458–27465[Abstract/Free Full Text]
  18. Ban N, Yamada Y, Someya Y, Ihara Y, Adachi T, Kubota A, Watanabe R, Kuroe A, Inada A, Miyawaki K, Sunaga Y, Shen ZP, Iwakura T, Tsukiyama K, Toyokuni S, Tsuda K, Seino Y 2000 Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression. Diabetes 49:1142–1148[Abstract]
  19. Livingstone C, Patel G, Jones N 1995 ATF-2 contains a phosphorylation-dependent transcriptional activation domain. EMBO J 14:1785–1797[Medline]
  20. Gupta S, Campbell D, Derijard B, Davis RJ 1995 Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267:389–393[Abstract/Free Full Text]
  21. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC 1995 SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229–233[CrossRef][Medline]
  22. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM 1998 Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623–18632[Abstract/Free Full Text]
  23. Morton S, Davis RJ, Cohen P 2004 Signalling pathways involved in multisite phosphorylation of the transcription factor ATF-2. FEBS Lett 572:177–183[CrossRef][Medline]
  24. Bennett BL, Sasaki DT, Murray BW, O’Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, Anderson DW 2001 SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98:13681–13686[Abstract/Free Full Text]
  25. Fox T, Coll JT, Xie X, Ford PJ, Germann UA, Porter MD, Pazhanisamy S, Fleming MA, Galullo V, Su MS, Wilson KP 1998 A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase. Protein Sci 7:2249–2255[Abstract]
  26. Waas WF, Lo HH, Dalby KN 2001 The kinetic mechanism of the dual phosphorylation of the ATF2 transcription factor by p38 mitogen-activated protein (MAP) kinase {alpha}. Implications for signal/response profiles of MAP kinase pathways. J Biol Chem 276:5676–5684[Abstract/Free Full Text]
  27. Ben Levy R, Hooper S, Wilson R, Paterson HF, Marshall CJ 1998 Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol 8:1049–1057[CrossRef][Medline]
  28. Burgering BM, Medema RH, Maassen JA, van de Wetering ML, van der Eb AJ, McCormick F, Bos JL 1991 Insulin stimulation of gene expression mediated by p21ras activation. EMBO J 10:1103–1109[Medline]
  29. van den Berghe N, Ouwens DM, Maassen JA, van Mackelenbergh MG, Sips HC, Krans HM 1994 Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3–L1 adipocytes. Mol Cell Biol 14:2372–2377[Abstract/Free Full Text]
  30. Angel P, Hattori K, Smeal T, Karin M 1988 The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell 55:875–885[CrossRef][Medline]
  31. Sanders J, Brandsma M, Janssen GM, Dijk J, Moller W 1996 Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1 ß {gamma} {delta} in the endoplasmic reticulum. J Cell Sci 109:1113–1117[Abstract]
  32. Pronk GJ, Vries-Smits AM, Ellis C, Bos JL 1993 Complex formation between the p21ras GTPase-activating protein and phosphoproteins p62 and p190 is independent of p21ras signalling. Oncogene 8:2773–2780[Medline]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
C. M. Kusminski, N. F. da Silva, S. J. Creely, F. M. Fisher, A. L. Harte, A. R. Baker, S. Kumar, and P. G. McTernan
The in Vitro Effects of Resistin on the Innate Immune Signaling Pathway in Isolated Human Subcutaneous Adipocytes
J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 270 - 276.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. A. Bogoyevitch and B. Kobe
Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases
Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 1061 - 1095.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
20/8/1786    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baan, B.
Right arrow Articles by Ouwens, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baan, B.
Right arrow Articles by Ouwens, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals