©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Activating Transcription Factor-1 and the cAMP Response Element-binding Protein by Ca/Calmodulin-dependent Protein Kinases Type I, II, and IV (*)

(Received for publication, August 10, 1995; and in revised form, November 15, 1995)

Peiqing Sun (1) (2) Liming Lou (1) Richard A. Maurer (1)(§)

From the  (1)Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201 and the (2)Genetics Ph.D. Program, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ability of activating transcription factor-1 (ATF1) or the cAMP response element-binding protein (CREB) to enhance transcription can be stimulated by increases in intracellular Ca concentrations. To identify protein kinases which may mediate the ability of Ca to activate these transcription factors, we compared the ability of constitutively active forms of several Ca/calmodulin-dependent protein kinases (CaM kinases) to activate ATF1 or CREB. We find that constitutively active CaM kinase I and IV can activate both ATF1 and CREB. In addition, expression vectors for full-length CaM kinase I and IV were able to augment the ability of Ca influx to activate ATF1 or CREB consistent with a role for these kinases in mediating transcriptional responses to Ca signaling. In contrast, CaM kinase II was unable to activate either ATF1 or CREB. These findings provide a potential mechanism that may permit variation in the ability of ATF1 and CREB to respond to changes in intracellular Ca concentrations depending on differences in the relative concentrations of specific CaM kinases.


INTRODUCTION

The cAMP and Ca signaling pathways are used widely to regulate the transcription of specific genes. In at least some cases, cAMP and Ca converge to regulate the activity of a single transcription factor(1, 2) . A key study which led to this view of convergent regulation by cAMP and Ca involved analysis of the DNA sequences required for Ca-regulated transcription of the c-fos proto-oncogene(1) . It was found that the regions of the fos gene that are required for responses to Ca mapped to a previously identified cAMP response element. Other studies provided evidence that cAMP response element-binding protein (CREB) (^1)can bind to the fos cAMP response element and that elevations in intracellular Ca result in phosphorylation of CREB at sites similar to those that were phosphorylated in response to elevated cAMP levels. The use of a GAL4-CREB fusion provided strong evidence that the transcription stimulating activity of CREB can be regulated by increases in intracellular Ca(2) . It seems likely that convergent regulation of CREB activity by cAMP and Ca permits integration of multiple extracellular signals in the regulation of specific genes.

The ability of cAMP and Ca to regulate CREB activity involves phosphorylation of CREB by the cAMP-dependent protein kinase (PKA) or Ca/calmodulin-dependent protein kinases (CaM kinases). PKA phosphorylates CREB on a single major site at Ser and phosphorylation of this residue is crucial for activation of CREB by PKA(3) . CaM kinases I, II, and IV have been shown to phosphorylate CREB in vitro, providing evidence that each of these protein kinases is a candidate for mediating the effects of Ca on CREB activity(2, 4, 5, 6) . Interestingly, recent studies have demonstrated that specific CaM kinases can have very different effects on the activation of CREB(7, 8) . In particular, CaM kinase II can inhibit activation of CREB while CaM kinase IV can activate CREB. The inhibitory effects of CaM kinase II are due to phosphorylation of a negative regulatory site located at Ser of CREB. These observations provide a mechanism that would permit the Ca signaling pathway to be either antagonistic or additive with the cAMP pathway for activation of CREB depending on the relative activity of specific CaM kinases.

Although CaM kinase I has been shown to phosphorylate CREB in vitro, the ability of CaM kinase I to activate CREB has not been tested. CaM kinase I was originally identified in brain extracts as an activity that could phosphorylate a specific site of synapsin I, a protein associated with synaptic vesicles. The purified kinase was found to effectively phosphorylate only synapsin I and II(9) . This led to the view that CaM kinase I had a very restricted substrate specificity and was probably functionally restricted to the modulation of neurotransmitter release. However, CaM kinase I can be found in tissues other than the brain and it has been shown that CREB appears to be good substrate for CaM kinase I in vitro(2) .

In addition to CREB, at least one other member of the CREB/ATF family has been shown to be responsive to both the cAMP and Ca pathways. Transfection studies have shown that ATF1 can be activated by increases in either cAMP or Ca(10, 11) . Although it is clear that the activity of ATF1 can be regulated by Ca influx, the role of specific CaM kinases in regulating ATF1 activity has not been explored. It is possible that specific CaM kinases might have differential effects on the activity of CREB and ATF1. For instance, although previous studies have shown that CaM kinase II can block activation of CREB(7, 8) , it is possible that ATF1 can be activated by CaM kinase II. If this were the case, changes in the relative concentration of CREB and ATF1 would permit changes in the interaction between the cAMP and Ca pathways. Thus it is important to examine the ability of specific CaM kinases to modulate ATF1 activity.

In the present study we have compared the ability of CaM kinase I, II, and IV to alter the transcription stimulating activity of ATF1 and CREB. The results indicate that CaM kinase I and IV can activate ATF1 and CREB. Phosphorylation of Ser of CREB or Ser of ATF1 was found to be essential for transcriptional activation by CaM kinase I and IV. Thus, both CaM kinase I and IV may participate in mediating Ca-stimulated activation of ATF1 and CREB in specific tissues. In contrast, we find that CaM kinase II cannot activate either ATF1 or CREB. As observed previously with CREB, CaM kinase II phosphorylates two sites in ATF1. Thus the negative regulatory effects of CaM kinase II are observed for both ATF1 and CREB.


MATERIALS AND METHODS

Plasmid Constructions

To prepare expression vectors encoding full-length and constitutively active CaM kinase I, cDNAs were isolated by polymerase chain amplification based on the published sequence of rat CaM kinase I(12) . Appropriate primers were used to isolate the complete coding sequence (residues 1-332) or a truncated coding sequence (residues 1-295) using single-stranded cDNA from PC12 cells as a template. The correct sequences of the amplified and cloned coding sequences were confirmed by nucleotide sequence analysis (13) and comparison to the published sequence of rat CaM kinase I. The full-length or truncated CaM kinase I coding sequences were used to replace the globin coding sequence in the RSV-globin expression vector (14) . To prepare a GAL4-ATF1Deltab-zip expression vector, the coding sequence for the transcriptional activation domain of ATF1 (amino acids 1-213) was isolated by polymerase chain reaction amplification from RSV-ATF1 (10) and fused in frame to the 3` end of the coding sequence for the DNA binding domain of GAL4 (amino acids 1-147) downstream of the viral cytomegalovirus promoter in plasmid pcDNAI-amp (Invitrogen). Specific mutations within the ATF1 coding sequence were prepared by polymerase chain reaction mutagenesis(15) , and the complete coding sequence of the mutants was confirmed by nucleotide sequence analysis(13) . The construction of the GAL4-CREBDeltab-zip, CaM kinase II, CaM kinase IV, and PKA expression vectors and the 5xGAL4-TATA-luciferase reporter gene have been described previously(8, 16, 17) .

Cell Culture and Transfection

GH(3) cells were maintained in Dulbecco's modified Eagle's medium with 15% equine serum and 2.5% fetal bovine serum. GH(3) cells were transfected by electroporation using a single pulse at 220 V and 960 microfarads. In most experiments cells received 5 µg of the 5xGAL4-TATA-luciferase indicator DNA, 2 µg of the GAL4-CREBDeltab-zip or GAL4-ATF1Deltab-zip expression vector, and 5 µg of kinase expression vector. In some experiments, cells were treated with 80 mM KCl at 18 h after transfection. Cells were collected and lysates prepared 24 h post-transfection (6 h after KCl treatments). The protein concentration of the lysates was determined(18) , and luciferase activities were measured using a constant amount of protein(19) . Each experiment included three separate transfections for each experimental group, and the experiments have been repeated three to five times.

Expression and Purification of Recombinant ATF1

To facilitate isolation of recombinant ATF1, a poly(histidine)-tagged variant of ATF1 was prepared. DNA sequences encoding either full-length ATF1 or the transcriptional activation domain (amino acids 1-213) were cloned into the pET16b bacterial expression vector (Novagen), downstream and in-frame with the poly(histidine) segment and Factor Xa cleavage site. Recombinant poly(His)-ATF1 was expressed in Escherichia coli and a crude extract prepared as described previously(20) , except that 2 ml of 0.5 M isopropyl-D-thiogalactopyranoside was added to each liter of culture to induce the expression of ATF1 and the crude extract was not heat treated. The crude extract from each liter of culture was diluted with one volume of 8 M urea and mixed with 1 ml of Ni-NTA-agarose (Qiagen) in the presence of 0.8 mM imidazole. The mixture was rotated at 4 °C for 1 h before being packed into a column. The column was washed sequentially with 0.8, 8, and 40 mM imidazole in 20 mM Tris-HCl (pH 7.4) and 4 M urea. Poly(His)-ATF1 was eluted with 100 mM imidazole in the same buffer. The recombinant proteins containing ATF1 transcriptional activation domain were expressed and purified essentially in the same way, except that bacterial cells were lysed in 20 mM Tris (pH 7.4) by two passes through a French pressure cell at 10,000 p.s.i., and that urea was not used during the purification process. Purified proteins were dialyzed against 20 mM HEPES (pH 7.9), 50 mM NaCl, 1 mM EDTA, 10 mM beta-mercaptoethanol, and 10% glycerol and stored at -80 °C until use.

Expression and Purification of Recombinant CaM Kinase I

The full-length CaM kinase I coding sequence was inserted into the baculovirus transfer vector pBlueBac1 (Invitrogen) and used to prepare recombinant baculovirus, which directs the synthesis of CaM kinase I. Recombinant CaM kinase I was expressed in Sf9 insect cells and purified by affinity chromatography on calmodulin-Sepharose 4B (Pharmacia Biotech Inc.), as described previously(21) . The eluate from the calmodulin-Sepharose 4B column appeared to contain 37-kDa CaM kinase I as well as a 49-kDa contaminant protein. CaM kinase I was further purified to near homogeneity by size exclusion chromatography on Superdex-75 (Pharmacia). The purified CaM kinase I was dialyzed against 10 mM HEPES (pH 7.6), 1 mM CaCl(2), 50 mM NaCl, 5 mM beta-mercaptoethanol, and 10% glycerol and stored at -80 °C until use.

Phosphorylation of ATF1 and Phosphopeptide Mapping

Recombinant ATF1 protein (2 µg) was phosphorylated in vitro using recombinant CaM kinase I, CaM kinase IIalpha (21) , CaM kinase IV or the catalytic subunit of PKA in the presence of [-P]ATP (1000-5000 cpm/pmol). The preparation of recombinant PKA, CaM kinase IIalpha, and CaM kinase IV has been described previously(21, 22) . For phosphorylation by CaM kinase I, II, or IV, the reaction mixtures contained 400 µM [-P]ATP (5000 cpm/pmol), 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 50 nM kinase, 0.5 mM CaCl(2), and 1 µM calmodulin. Reactions containing PKA were performed in 400 mM [-P]ATP, 50 nM PKA, 25 mM Tris (pH 7.4), 5 mM magnesium acetate, and 0.5 mM dithiothreitol. Reactions were initiated by addition of protein kinase and incubated for 45 min at 30 °C. For two-dimensional phosphopeptide mapping, ATF1 was fractionated by electrophoresis using a 12% polyacrylamide denaturing gel and phospho-ATF1 was identified by autoradiography of the dried gel. The phospho-ATF1 bands were cut from the gel, and two-dimensional tryptic phosphopeptide maps were prepared on the extracted proteins as described(23) . Briefly, the gel pieces were rehydrated in 50 mM ammonium bicarbonate. Phosphorylated ATF1 was eluted from the gel, precipitated in 16% trichloroacetic acid, and oxidized with performic acid. Samples were lyophilized, resuspended in 50 mM ammonium bicarbonate, and digested with 30 mg of L-1-tosylamido-2-phenylethylchloromethyl ketone-treated trypsin (U. S. Biochemical Corp.) for three 8-h periods at 37 °C. The peptides were lyophilized and resuspended in the pH 1.9 buffer (2.5% of formic acid and 7.8% of acetic acid in water). Equal amounts of radioactivity for each sample was loaded on a cellulose plate and fractionated by high voltage electrophoresis in the pH 1.9 buffer for 25 min at 1 kV using the Hunter thin layer electrophoresis system (HTLE-7000, CBS Scientific Co., Del Mar, California). Samples were separated in the second dimension by thin layer chromatography using n-butanol:pyridine:acetic acid:water in volume ratios of 0.375:0.25:0.075:0.30. The phosphopeptides were visualized by autoradiography.


RESULTS

Expression Vectors for Constitutively Active Forms of CaM Kinase I and CaM Kinase IV Can Activate Both ATF1 and CREB

It has been reported that in vitro, CaM kinase I can phosphorylate both ATF1 and CREB(2) . However, the ability of CaM kinase I to activate ATF1 and CREB has not been tested. To examine this issue, we prepared an expression vector for a constitutively active form of CaM kinase I. Previous studies have shown that truncation of CaM kinase II at Leu(24) or CaM kinase IV at Leu(5) removes an autoinhibitory-regulatory region of the enzyme and results in a constitutively active protein kinase, which no longer requires Ca and calmodulin for activity. We aligned the amino acid sequences of CaM kinase I with those of CaM kinase II and CaM kinase IV and found that Lys of CaM kinase I corresponded to Leu of CaM kinase II or Leu of CaM kinase IV. Therefore the coding sequence of CaM kinase I was truncated at Lys and used to prepare a mammalian expression vector. To specifically determine if CaM kinase I could alter the activity of ATF1 and CREB, expression vectors for GAL4-ATF1 or GAL4-CREB fusion proteins (2, 11, 25, 26) were transfected with a luciferase reporter gene containing five copies of a GAL4 binding site (5xGAL4-TATA-luciferase). To avoid possible formation of heterodimers with endogenous members of the CREB/ATF family, the GAL4-ATF1 and GAL4-CREB coding sequences were truncated to remove the carboxyl-terminal basic+leucine zipper DNA binding region (GAL4-ATF1Deltab-zip, GAL4-CREBDeltab-zip). Transfection of expression vectors for constitutively active forms of CaM kinase I or CaM kinase IV increased the ability of GAL4-ATF1Deltab-zip or GAL4-CREBDeltab-zip to stimulate expression of the reporter gene (Fig. 1, A and B). The kinase expression vectors did not substantially alter the expression of the luciferase reporter gene driven by the thymidine kinase promoter (Fig. 1C), demonstrating promoter specificity and suggesting that transcriptional activation of GAL4-ATF1 and GAL4-CREB are not due to general effects on the transcription apparatus. These findings provide evidence that both CaM kinase I and CaM kinase IV can increase the transcriptional activity of ATF1 and CREB. Surprisingly, an expression vector for the catalytic subunit of PKA was not able to activate GAL4-ATF1Deltab-zip (Fig. 1A), although PKA strongly activated GAL4-CREBDeltab-zip (Fig. 1B).


Figure 1: Regulation of ATF1 and CREB activity by PKA, CaM kinase I, and CaM kinase IV. GH(3) cells were transfected with 2 µg of an expression vector for the DNA binding domain of GAL4 (GAL4), GAL4-ATF1Deltab-zip (GAL4-ATF1), or GAL4-CREBDeltab-zip (GAL4-CREB) as indicated. Cells also received 5 µg of an expression vector for PKA, CaM kinase I, or CaM kinase IV and 5 µg of the 5xGAL4-TATA-luciferase reporter gene (A and B) or a reporter gene containing the thymidine kinase promoter linked to luciferase (TK-lucif) (C). Luciferase activity was determined 24 h after transfection. Values are means ± S.E. for three separate transfections.



Previous studies have shown that phosphorylation of Ser of CREB is crucial for transcriptional activation by PKA and CaM kinase IV(3, 8) . A large portion of the transcriptional activation domain of ATF1 is similar to the transcriptional activation domain of CREB, including a conserved PKA site in which Ser of ATF1 corresponds to Ser of CREB. Mutation of Ser of ATF1 to alanine (ATF1-S63A) essentially blocked the ability of CaM kinase I and CaM kinase IV to activate GAL4-ATF1. Similarly, mutation of Ser of CREB to alanine (CREB-S133A) blocked activation in response to PKA, CaM kinase I, and CaM kinase IV. These findings demonstrate that CaM kinase I and CaM kinase IV can enhance the ability of ATF1 and CREB to stimulate transcription, likely involving a crucial phosphorylation event at Ser of ATF1 or Ser of CREB.

As indicated above, it was surprising that expression vectors for constitutively active forms of CaM kinase I and CaM kinase IV were able to activate GAL4-ATF1Deltab-zip, but that a PKA expression vector was not able to substantially increase the activity of this fusion protein. To examine this issue further, we performed a titration experiment using increasing concentrations of expression vectors for each of three protein kinases (Fig. 2). We found that at all of the tested concentrations of DNA, expression vectors for CaM kinase I and CaM kinase IV enhanced the ability of GAL4-ATF1Deltab-zip to stimulate reporter gene expression. At lower concentrations of the expression vectors, CaM kinase I consistently resulted in greater activation of ATF1 than CaM kinase IV. In contrast, PKA had little effect on activation of GAL4-ATF1Deltab-zip at any of the tested concentrations of expression vector. The failure of PKA to substantially activate GAL4-ATF1 might be due to inhibitory effects of the kinase. To address this issue, a PKA expression vector was co-transfected with a vector for a constitutively active form of CaMKI (Fig. 3). As observed above, the CaMKI expression vector enhanced the ability of GAL4-ATF1Deltab-zip to stimulate reporter gene activity while the PKA vector by itself had little effect. Co-transfection of the two kinase expression vectors demonstrated that PKA does not block activation by CaMKI. Overall these studies provide evidence that the transcription stimulating activity of ATF1 appears to be responsive to CaM kinases I and IV and relatively unresponsive to PKA.


Figure 2: Dose-response analysis of the ability of PKA, CaM kinase I, and CaM kinase IV to activate ATF1 in GH(3) cells. GH(3) cells were transfected with 2 µg of an expression vector for GAL4-ATF1Deltab-zip fusion protein, 5 µg of the 5xGAL4-TATA-luciferase reporter gene and increasing amounts (0, 3, 5, 10, or 20 µg) of the expression plasmid for PKA (circles), CaM kinase I (squares), or CaM kinase IV (triangles). The total amount of DNA for each transfection was equalized using pBSSK(-). Luciferase activity was determined 24 h after transfection. Values are the mean for two separate transfections.




Figure 3: Transfection of an expression vector for PKA does not block the ability of CaM kinase I to activate ATF1. GH(3) cells were transfected with 2 µg of an expression vector for GAL4-ATF1Deltab-zip (GAL4-ATF1) or GAL4-CREBDeltab-zip (GAL4-CREB) and 5 µg of expression vectors for PKA and CaM kinase I as indicated, as well as 5 µg of the 5xGAL4-TATA-luciferase reporter gene. Luciferase activity was determined 24 h after transfection. Values are means ± S.E. for three separate transfections.



We also examined the ability of endogenous kinases to activate GAL4-ATF1Deltab-zip (Fig. 4). To stimulate endogenous CaM kinases, transfected GH(3) cells were depolarized by KCl treatment, which results in Ca influx through voltage-dependent Ca channels. We previously found that in GH(3) cells, KCl-induced activation of GAL4-CREB is dependent on the presence of extracellular Ca(8) . KCl treatment enhanced the ability of GAL4-ATF1 to stimulate reporter gene expression. These findings provide evidence that endogenous Ca-responsive enzymes can activate GAL4-ATF1. Treatment of transfected cells with chlorophenylthio-cAMP had a modest effect on the ability of GAL4-ATF1Deltab-zip to stimulate reporter gene expression.


Figure 4: Activation of ATF1 by KCl-induced Ca influx. GH(3) cells were transfected with 6 µg of an expression vector for GAL4-ATF1Deltab-zip as indicated and 15 µg of the 5xGAL4-TATA-luciferase reporter gene. After transfection the cells were divided into three dishes. Cells were treated by the addition of 80 mM KCl or 0.5 mM chlorophenylthio-cAMP (cAMP) at 18 h after transfection. The cells were collected and luciferase activity determined 6 h after treatment. Values are means ± S.E. for three separate transfections.



CaM Kinase I Phosphorylates CREB on Ser and ATF1 on Ser in Vitro

To explore the biochemical events that are likely important in mediating the ability of CaM kinase I to activate ATF1, we characterized the in vitro phosphorylation of ATF1 and CREB using two-dimensional tryptic phosphopeptide mapping (Fig. 5). As has been observed previously(3) , phosphorylation of CREB by PKA results in a major phosphopeptide (Fig. 5A). We frequently observed a minor phosphopeptide after phosphorylation by PKA. Mutation of Ser of CREB to alanine eliminated the major phosphopeptide as well as the minor phosphopeptide (data not shown). This observation supports previous conclusions that PKA phosphorylates a single site, Ser, on CREB. Presumably, the minor phosphopeptide that we frequently detect is the result of partial digestion with trypsin. A very similar phosphopeptide map was obtained when CREB was phosphorylated with CaM kinase I, consistent with studies by Sheng et al.(2) showing that CaM kinase I can phosphorylate CREB on Ser. Phosphorylation of ATF1 by PKA or CaM kinase I (Fig. 5, C and D) yielded phosphopeptide maps that were very similar to those obtained after phosphorylation of CREB with PKA. The region surrounding Ser of ATF1 is identical to the region surrounding Ser of CREB, and phosphorylation of these sites would result in identical tryptic phosphopeptides (Fig. 6). To further examine the phosphopeptides obtained from ATF1, a mixing experiment was performed. Phosphopeptides obtained from ATF1 which had been phosphorylated by PKA were mixed with peptides derived from CREB that had been phosphorylated with PKA (Fig. 5E). The mixing experiment demonstrates that after phosphorylation by PKA, the resulting ATF1 and CREB phosphopeptides migrate indistinguishably in the two-dimensional map. A similar mixing experiment demonstrated that the phosphopeptides obtained after phosphorylation of ATF1 with CaM kinase I co-migrated with CREB PKA-derived peptides (Fig. 5F). As phosphorylation of Ser of ATF1 would produce these identically migrating peptides, these findings support the view that both PKA and CaM kinase I phosphorylate a single major site in ATF1 at Ser. Similar findings were obtained for ATF1 after phosphorylation by CaM kinase IV (data not shown). Thus it is likely that CaM kinase I, CaM kinase IV, and PKA all phosphorylate ATF1 at Ser.


Figure 5: Two-dimensional phosphopeptide mapping of the phosphorylation of CREB and ATF1 by PKA and CaM kinase I. Recombinant poly(His)-CREB or poly(His)-ATF1 were phosphorylated in vitro by PKA or CaM kinase I in the presence of [-P]ATP as indicated. The phosphorylation products were purified by denaturing polyacrylamide gel electrophoresis, oxidized, and then digested with trypsin. Phosphopeptides were separated by high voltage electrophoresis in the first dimension and by thin layer chromatography in the second dimension. The phosphopeptides were visualized by autoradiography.




Figure 6: Comparison of the amino acid sequence surrounding phosphorylation sites in CREB and ATF1. The amino acid sequence of residues 127-150 of CREB are aligned with the sequence of residues 57-80 of ATF1. Positions where amino acids are identical for CREB and ATF1 are indicated by a dot between the two sequences. PKA and CaM kinase II phosphorylation sites at Ser and Ser of CREB (3, 8) are indicated with an asterisk, and the predicted tryptic phosphopeptides are enclosed by boxes. For ATF1, the PKA phosphorylation site at Ser(11) is indicated as well as a putative CaM kinase II site at Ser and the predicted phosphopeptides are enclosed by boxes.



Full-length CaM Kinase I as Well as CaM Kinase IV Can Augment the Activation of CREB by Ca Influx in Vivo

The preceding experiments demonstrated that a truncated, constitutively active form of CaM kinase I was able to activate both ATF1 and CREB. Of course, it is possible that although the truncated form of the enzyme can activate these transcription factors, the full-length enzyme might not be able to function in this manner. We previously found that an expression vector for full-length CaM kinase IV was able to augment the ability of Ca influx to stimulate the activity of GAL4-CREB(8) . We transfected GH(3) cells with expression vectors for full-length CaM kinase I or CaM kinase IV and examined the ability of KCl to enhance GAL4-CREBDeltab-zip mediated activation of the GAL4-dependent luciferase reporter gene (Fig. 7). We found that both CaM kinase I and CaM kinase IV had effects to augment activation of CREB by KCl-induced Ca influx in a concentration-dependent manner. Interestingly, CaM kinase I was found to stimulate reporter gene activity in the absence of KCl treatment. This effect was not observed with the CaM kinase IV expression vector. At similar concentrations of expression vector, CaM kinase I and CaM kinase IV resulted in similar levels of KCl-stimulated reporter gene activity. Because CaM kinase I increased both basal and KCl stimulated reporter gene activity, the calculated -fold inductions did not increase. In contrast, the CaM kinase IV vector resulted in a substantial increase in KCl-induced -fold activation. The reason for CaM kinase I-induced CREB activation in non KCl-treated cultures is not clear. None the less, the finding that full-length CaM kinase I can alter maximal KCl-induced activity of GAL4-CREB suggests that this enzyme as well as CaM kinase IV is a candidate for mediating transcriptional activity in response to Ca influx. Neither kinase expression vector had effects on transcriptional activation mediated by the GAL4 DNA binding domain alone (Fig. 7, C and D), demonstrating that the observed effects are specific for CREB.


Figure 7: Expression vectors encoding full-length CaM kinase I or CaM kinase IV augment activation of CREB in response to depolarization-induced Ca influx. GH(3) cells were transfected with 2 µg of an expression vector for either the DNA binding domain of GAL4 (GAL4) or the GAL4-CREBDeltab-zip fusion protein (GAL4-CREB) and 5 µg of the 5xGAL4-TATA-luciferase reporter gene. The cells also received increasing amounts (0, 1, 5, or 10 µg) of an RSV expression vector for full-length CaM kinase I or CaM kinase IV. The total amount of DNA for each transfection was equalized using an RSV-globin expression vector. After transfection the cells were divided into two 60-mm dishes, one of which was treated by addition of 80 mM KCl to the medium 18 h after transfection. Luciferase activity was determined 6 h after KCl treatment. Values are means ± S.E. for three separate transfections. The numbers at the top of each histogram indicate the -fold activation by KCl treatment.



An Expression Vector for a Constitutively Active Form of CaM Kinase II Is Unable to Activate ATF1

We previously found that although CaM kinase II can phosphorylate CREB, it is not able to activate CREB (8) . These studies provided evidence that CaM kinase II is not able to activate CREB because it phosphorylates Ser as well as Ser. Phosphorylation of Ser was found to block the activation of CREB, which would otherwise occur following phosphorylation of Ser. As a serine residue is found in ATF1 at a location corresponding to Ser of CREB (Fig. 6), it seemed possible that CaM kinase II might also fail to activate ATF1. GH(3) cells were transfected with expression vectors for constitutively active CaM kinases, GAL4-ATF1Deltab-zip or GAL4-CREBDeltab-zip and the GAL4-dependent luciferase reporter gene (Fig. 8). These studies revealed that the CaM kinase II expression vector was unable to activate GAL4-ATF1 or GAL4-CREB. In the same experiment, expression vectors for constitutively active CaM kinase I or CaM kinase IV substantially activated GAL4-ATF1 or GAL4-CREB. These findings extend our previous observation that CaM kinase II cannot active CREB and demonstrate that CaM kinase II also cannot activate ATF1.


Figure 8: An expression vector for a constitutively active form of CaM kinase II does not activate ATF1 or CREB. GH(3) cells were transfected with 2 µg of an expression vector for GAL4-ATF1Deltab-zip (GAL4-ATF1) fusion protein (A) or GAL4-CREBDeltab-zip (GAL4-CREB) fusion protein (B), 5 µg of the 5xGAL4-TATA-luciferase reporter gene, and either 5 µg of pBSSK(-) as control (box) or 5 µg of an expression vector for PKA (&cjs2112;), CaM kinase I (&cjs2098;), CaM kinase II (&cjs2110;), or CaM kinase IV (). Luciferase activity was determined 24 h after transfection. Values are means ± S.E. for three separate transfections.



CaM Kinase II Phosphorylates ATF1 at Two Sites in Vitro

Two-dimensional phosphopeptide mapping experiments were performed to examine sites on ATF1 that are phosphorylated by CaM kinase II in vitro (Fig. 9). After phosphorylation of ATF1 by CaM kinase II, two major phosphopeptides and several minor phosphopeptides were observed (Fig. 9A). One of the major phosphopeptides migrated similarly to the major peptide that is obtained after phosphorylation of ATF1 or CREB with PKA (data not shown), suggesting that this peptide likely represents phosphorylation of Ser. The identity of this site as Ser was further supported by mutation of Ser to alanine, which eliminated this major phosphopeptide (Fig. 9B). We suspected that the other major phosphopeptide might result from phosphorylation of Ser of ATF1 which appears to correspond to Ser of CREB (Fig. 6). To further examine this possibility, we mutated Ser as well as the adjacent Ser to alanine and used the mutant coding sequence to produce recombinant ATF1 in E. coli. Unfortunately, despite several attempts, we were unable to obtain sufficient quantities of highly purified ATF1-S72A to permit phosphopeptide mapping experiments. The reason for this difficulty is not clear, although we have found it much more difficult to express and purify ATF1 than CREB. We were able to express and purify ATF1-S73A and ATF1-S72A-S73A and use these proteins for phosphopeptide mapping experiments. While mutation of Ser to alanine did not substantially alter the phosphopeptide maps (Fig. 9C), mutation of both Ser and Ser eliminated one of the major ATF1 phosphopeptides (Fig. 9D). These findings suggest that CaM kinase II probably phosphorylates Ser of ATF1. However, we cannot exclude the possibility that either Ser or Ser of ATF1 can be phosphorylated by CaM kinase II.


Figure 9: Two-dimensional phosphopeptide mapping of the phosphorylation of ATF1 by CaM kinase II. Recombinant poly(His)-ATF1 (wild-type or mutants as indicated) were phosphorylated in vitro with CaM kinase II in the presence of [-P]ATP. The phosphorylation products were purified by denaturing polyacrylamide gel electrophoresis, oxidized and digested with trypsin. Peptides were separated by high voltage electrophoresis in the first dimension and by thin layer chromatography in the second dimension. The phosphopeptides were visualized by autoradiography. The open arrows indicate the phosphopeptide that appears to contains Ser, and the closed arrows indicate the phosphopeptide that probably contains Ser.



Mutation of Ser of ATF1 Permits CaM Kinase II to Activate ATF1

The phosphopeptide mapping experiments identified two sites within ATF1 that are phosphorylated by CaM kinase II. This finding raised the possibility that the inability of CaMKII to activate ATF1 might involve phosphorylation of a site that inhibits transcriptional activation, similar to our previous findings concerning CaMKII effects on activation of CREB. As Ser of ATF1 is crucial for phosphorylation mediating transcriptional activation, it seemed likely that phosphorylation of the second site, presumably Ser, mediates the inhibitory effects of CaM kinase II on ATF1 activation. To test this possibility, a mutant ATF1 coding sequence was prepared in which Ser was replaced with an alanine. Mutation of Ser to alanine greatly enhanced activation of GAL4-ATF1Deltab-zip as compared to the wild type construct (Fig. 10). This finding strongly supports a model in which phosphorylation of Ser serves to inhibit activation of ATF1.


Figure 10: Mutation of Ser of ATF1 permits activation by CaM kinase II. GH(3) cells were transfected with 2 µg of an expression vector for GAL4-ATF1Deltab-zip (GAL4-ATF1) or a mutant in which the codon for Ser of ATF1 was replaced with a codon for alanine (GAL4-ATF1-S72A), 5 µg of a 5xGAL4-TATA-luciferase reporter gene, and increasing concentrations of an expression vector for a constitutively active form of CaM kinase II (CaMKII). The total amount of DNA for each transfection was equalized using pBSSK(-). Luciferase activity was determined 24 h after transfection. Values are means ± S.E. for three separate transfections.




DISCUSSION

We have compared the ability of CaM kinases I, II, and IV to alter the activity of ATF1 and CREB. We find that CaM kinase I or IV can activate either ATF1 or CREB. In contrast, CaM kinase II cannot activate either ATF1 or CREB. These findings provide further insight into the signal transduction pathways that are important for mediating the ability of Ca to activate CREB and ATF1. The results provide strong evidence that CaM kinase I as well as CaM kinase IV can activate CREB and ATF1. In titration experiments we found that low concentrations of the CaM kinase I vector were considerably more effective than the CaM kinase IV vector in activating CREB. While we have not determined that equal concentrations of expression vector produce equivalent amounts of CaM kinase I and IV, these studies at least raise the possibility that CaM kinase I may be more effective than CaM kinase IV in activating CREB. The finding that full-length CaM kinase I or IV can augment the activation of ATF1 or CREB in response to Ca influx supports the view that both of these enzymes are good candidates as physiological regulators of CREB and ATF1 activity.

In contrast to previous studies of ATF1(10, 11) , we found that PKA was unable to enhance the ability of GAL4-ATF1Deltab-zip to stimulate reporter gene activity. The earlier studies utilized either full-length ATF1 or full-length ATF1 fused to the DNA binding domain of GAL4. As ATF1 can form heterodimers with CREB(11) , the activation observed with full-length ATF1 may actually reflect phosphorylation and activation of CREB as part of an ATF1bulletCREB heterodimer. Studies utilizing leucine zipper variants of CREB which are engineered to form specific heterodimers have shown that a hemiphosphorylated CREB dimer or a CREBbulletCREMalpha heterodimer can mediate PKA-induced transcriptional activation(27, 28) . Our use of a GAL4-ATF1Deltab-zip construct would eliminate formation of CREBbulletATF1 heterodimers, and this might account for the failure to respond to PKA. The finding that ATF1 has only a limited ability to mediate transcriptional responses to PKA would be consistent with recent studies, which concluded that ATF1 is a specific antagonist of CREB-mediated transcriptional activation(29) . On the other hand, we found that both CaM kinase I and CaM kinase IV were able to activate GAL4-ATF1Deltab-zip. This finding is quite surprising as phosphopeptide mapping experiments demonstrated that PKA, CaM kinase I, and CaM kinase IV all appear to phosphorylate ATF1 at the same site in vitro, Ser. How is it that all three kinases appear to phosphorylate ATF1 on Serin vitro, yet only CaM kinase I or IV can activate GAL4-ATF1Deltab-zip in a transfection experiment? We have used co-transfection experiments to demonstrate that PKA cannot block the ability of CaMKI to activate ATF1. Thus, it seems unlikely that PKA phosphorylates a factor that inhibits responses to ATF1. It may be that CaM kinase I and IV phosphorylate other transcription factors or coactivators, which are required to mediate responses to phospho-ATF1. Microinjection experiments have shown that phosphorylation of CREB on Ser is sufficient for transcriptional activation and thus other PKA-dependent phosphorylations are not required for a response to phospho-CREB. Similar microinjection experiments might yield insight into the differential activation of ATF1 by PKA and CaM kinase I or IV.

The use of expression vectors for full-length CaM kinase I and IV demonstrated that both of these kinases can participate in Ca-regulated activation of CREB. Most of our studies relied upon the use of truncated, constitutively active forms of these enzymes. It is possible that the truncated forms of the kinase may have enhanced access to the nucleus and therefore might alter nuclear events which are not normally regulated by the holoenzyme. The finding that full-length CaM kinase I and IV can activate CREB supports a role for these enzymes in the physiological regulation of transcription. It is interesting that transfection of an expression vector for full-length CaM kinase I stimulated basal, CREB-mediated transcriptional activation in the absence of depolarization-induced Ca influx. On the other hand, full-length CaM kinase IV appeared to be much more Ca-dependent, suggesting that there may be differences in Ca-mediated activation of the two enzymes. There is evidence that activation of both CaM kinase I and IV requires activator protein, which appears to be another protein kinase(30, 31, 32, 33, 34) . Our findings suggest that some step in the activation of CaM kinase I may be more sensitive to Ca than occurs for activation of CaM kinase IV. It seems likely that this altered Ca sensitivity occurs at the level of the CaM kinase activator kinase. Thus our findings suggest possible differences in the enzymes which mediate activation of CaM kinase I and IV.

Our studies have shown that CaM kinase II is unable to activate ATF1. This finding extends our previous observation that CaM kinase II cannot activate CREB (8) and demonstrates that the failure to respond to CaM kinase is conserved for both proteins. Unlike CaM kinase I and IV, which phosphorylate ATF1 or CREB at a single major site, CaM kinase II phosphorylates both ATF1 and CREB at two sites. One of the sites, Ser of CREB or Ser of ATF1, is the same site that is recognized by PKA, CaM kinase I, and CaM kinase IV. Phosphorylation of Ser of CREB or Ser of ATF1 is crucial for transcriptional activation. The second site in ATF1 that is phosphorylated by CaM kinase II appears to be Ser, which corresponds to Ser, the inhibitory CaM kinase II site in CREB. Neither Ser of CREB nor Ser of ATF1 match a consensus CaM kinase II phosphorylation site, which is Arg-X-X-Ser/Thr (35) . Phosphorylation at non-consensus sites by CaM kinase II has been reported for several proteins(36, 37) . It will likely require experiments exploring the phosphorylation of an extensive set of peptides based on these proteins to further define the alternative consensus sites recognized by CaM kinase II. In any case, the present findings and our previous studies (8, 17) suggest that the ability of CaM kinase II to phosphorylate a second, non-consensus site in ATF1, and CREB appears to inhibit activation of these transcription factors.

In conclusion, we have demonstrated differential activation of ATF1 and CREB by specific CaM kinases. Both CaM kinase I and IV can activate ATF1 and CREB. CaM kinase II is unable to activate either transcription factor. Based on these studies, it seems likely that tissue-specific or developmental changes in the concentrations of individual CaM kinases permits variations in the ability of the Ca signaling pathway to regulate the activity of specific genes.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant DK 49995 and American Heart Association Grant-in-aid 93006350. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, L215, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201.

(^1)
The abbreviations used are: CREB, cAMP response element-binding protein; PKA, cAMP-dependent protein kinase; CaM kinase, Ca/calmodulin-dependent protein kinase; ATF1, activating transcription factor-1; GAL4, DNA binding domain of the yeast GAL4 transcription factor; pBSSK(-), pBlueScript, SK(-); RSV, Rous sarcoma virus.


ACKNOWLEDGEMENTS

We thank Drs. Richard Goodman, Thomas Soderling, and Anthony Means for helpful discussions. We also thank Bobbi Maurer for aid in preparing this manuscript.


REFERENCES

  1. Sheng, M., McFadden, G., and Greenberg, M. E. (1990) Neuron 4, 571-582 [Medline] [Order article via Infotrieve]
  2. Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991) Science 252, 1427-1430 [Medline] [Order article via Infotrieve]
  3. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59, 675-680 [Medline] [Order article via Infotrieve]
  4. Dash, P. K., Karl, K. A., Colicos, M. A., Prywes, R., and Kandel, E. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5061-5065 [Abstract]
  5. Cruzalegui, F. H., and Means, A. R. (1993) J. Biol. Chem. 268, 26171-26178 [Abstract/Free Full Text]
  6. Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269, 15520-15527 [Abstract/Free Full Text]
  7. Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R., and McKnight, G. S. (1994) Mol. Cell. Biol. 14, 6107-6116 [Abstract]
  8. Sun, P., Enslen, H., Myung, P. S., and Maurer, R. A. (1994) Genes & Dev. 8, 2527-2539
  9. Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262, 7273-7281 [Abstract/Free Full Text]
  10. Rehfuss, R. P., Walton, K. M., Loriaux, M. M., and Goodman, R. H. (1991) J. Biol. Chem. 266, 18431-18434 [Abstract/Free Full Text]
  11. Liu, F., Thompson, M. A., Wagner, S., Greenberg, M. E., and Green, M. R. (1993) J. Biol. Chem. 268, 6714-6720 [Abstract/Free Full Text]
  12. Picciotto, M. R., Czernik, A. J., and Nairn, A. C. (1993) J. Biol. Chem. 268, 26512-26521 [Abstract/Free Full Text]
  13. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  14. Gorman, C., Padmanabhan, R., and Howard, B. H. (1983) Science 221, 551-553 [Medline] [Order article via Infotrieve]
  15. Steinberg, R. A., and Gorman, K. B. (1994) Anal. Biochem. 219, 155-157 [CrossRef][Medline] [Order article via Infotrieve]
  16. Maurer, R. A. (1989) J. Biol. Chem. 264, 6870-6873 [Abstract/Free Full Text]
  17. Sun, P., and Maurer, R. A. (1995) J. Biol. Chem. 270, 7041-7044 [Abstract/Free Full Text]
  18. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  19. de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S. (1987) Mol. Cell. Biol. 7, 725-737 [Medline] [Order article via Infotrieve]
  20. Pei, L., Dodson, R., Schoderbek, W. E., Maurer, R. A., and Mayo, K. E. (1991) Mol. Endocrinol. 5, 521-534 [Abstract]
  21. Brickey, D. A., Colbran, R. J., Fong, Y. L., and Soderling, T. R. (1990) Biochem. Biophys. Res. Commun. 173, 578-584 [Medline] [Order article via Infotrieve]
  22. Howard, P. W., and Maurer, R. A. (1994) J. Biol. Chem. 269, 28662-28669 [Abstract/Free Full Text]
  23. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110-149 [Medline] [Order article via Infotrieve]
  24. Kapiloff, M. S., Mathis, J. M., Nelson, C. A., Lin, C. R., and Rosenfeld, M. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3710-3714 [Abstract]
  25. Berkowitz, L. A., and Gilman, M. Z. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5258-5262 [Abstract]
  26. Lee, C. Q., Yun, Y., Hoeffler, J. P., and Habener, J. F. (1990) EMBO J. 9, 4455-4465 [Abstract]
  27. Loriaux, M. M., Rehfuss, R. P., Brennan, R. G., and Goodman, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9046-9050 [Abstract]
  28. Loriaux, M. M., Brennan, R. G., and Goodman, R. H. (1994) J. Biol. Chem. 269, 28839-28843 [Abstract/Free Full Text]
  29. Ellis, M. J. C., Lindon, A. C., Flint, K. J., Jones, N. C., and Goodbourn, S. (1995) Mol. Endocrinol. 9, 255-265 [Abstract]
  30. Tokumitsu, H., Brickey, D. A., Glod, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269, 28640-28647 [Abstract/Free Full Text]
  31. Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164 [Abstract/Free Full Text]
  32. Sugita, R., Mochizuki, H., Ito, T., Yokokura, H., Kobayashi, R., and Hidaka, H. (1994) Biochem. Biophys. Res. Commun. 203, 694-701 [CrossRef][Medline] [Order article via Infotrieve]
  33. Enslen, H., Tokumitsu, H., and Soderling, T. R. (1995) Biochem. Biophys. Res. Commun. 207, 1038-1043 [CrossRef][Medline] [Order article via Infotrieve]
  34. Lee, J. C., and Edelman, A. M. (1995) Biochem. Biophys. Res. Commun. 210, 631-637 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kennelly, P. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 15555-15558 [Free Full Text]
  36. Ikebe, M., and Reardon, S. (1990) J. Biol. Chem. 265, 17607-17612 [Abstract/Free Full Text]
  37. Ando, S., Tokui, T., Yamauchi, T., Sugiura, H., Tanabe, K., and Inagaki, M. (1991) Biochem. Biophys. Res. Commun. 175, 955-962 [Medline] [Order article via Infotrieve]

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