The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 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 Belkowski, S. M.
Right arrow Articles by Prystowsky, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belkowski, S. M.
Right arrow Articles by Prystowsky, M. B.
The Journal of Immunology, 1998, 161: 659-665.
Copyright © 1998 by The American Association of Immunologists

Isolation and Analysis of a T Cell Clone Variant Exhibiting Constitutively Phosphorylated Ser133 cAMP Response Element-Binding Protein1

Stanley M. Belkowski*, Charles S. Rubin{dagger} and Michael B. Prystowsky2,*

Departments of * Pathology and {dagger} Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In driving T cell proliferation, IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen (PCNA), a requisite processivity factor for DNA polymerase {delta}. PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte, L2. After culturing L2 cells for greater than 91 days, we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation. L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity, PCNA expression and proliferation. In L2 cells, IL-2 stimulated and rapamycin inhibited the following: cAMP-independent CREB kinase activity, PCNA expression and proliferation. In L2 variant cells, CREB kinase activity was constitutively high; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation. These results indicate that IL-2 induces a rapamycin-sensitive, cAMP-independent CREB kinase activity in L2 cells. However, phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The macrolide rapamycin is a potent immunosuppressant that inhibits cytokine-dependent T cell proliferation (1). Rapamycin exerts its antiproliferative effect by binding to intracellular receptors generating complexes that inhibit progression from G1 to S phase of the cell cycle in IL-2-stimulated T-lymphocytes (2, 3). Inhibition of IL-2-induced proliferation by rapamycin is associated with a selective inhibition of protein synthesis, including proliferating cell nuclear Ag (PCNA)3 (4, 5).

PCNA is an auxiliary protein for DNA polymerase delta (6, 7, 8, 9, 10). The ability of PCNA-specific Abs to inhibit inducible DNA synthesis in isolated nuclei (11) and of PCNA antisense oligonucleotides to inhibit proliferation of BALB/c 3T3 cells (12) documents the role of this protein in DNA replication. PCNA transcription is increased in IL-2-stimulated T cells (13). The 5' regulatory region of the PCNA gene contains tandem cAMP response elements (CREs) and an E2F-like site that are critical for IL-2 induction and optimal transcriptional activity (14, 15). These CRE elements bind CREB and ATF1. Furthermore, IL-2 stimulates CREB and ATF1 phosphorylation in T cells during G1 activation; the ability of these transcription factors to bind to CRE elements in the promoter as measured using a gel-shift assay follows a similar time course as CREB/ATF1 phosphorylation (16).

CREs located in the control region of genes are recognized by several proteins that are members of the activating transcription factor subfamily, including CREB, ATF1, and others (17, 18, 19, 20). The best studied CRE-binding protein is a ~40-kDa protein termed CREB (17, 20). CREB transcriptional activity is regulated by phosphorylation at Ser133 by cAMP-dependent kinase A (PKA) (21, 22, 23, 24). Other kinases, including Ca2+/calmodulin dependent kinases (CAMK) (25), a Ras-dependent kinase (26), p90RSK (27), p70S6K (28), and RSK2 (29) have been shown to phosphorylate CREB at Ser133 in response to neurotrophic factors and Ca2+ fluxes (25, 26). Phosphorylation of CREB by PKA or cAMP-independent kinases is required for transcriptional activation (30, 31, 32, 33). Although cAMP has been implicated in the transcriptional regulation of many genes through CREs (34), increased levels of cAMP inhibit T cell proliferation (35), and IL-2 does not increase cellular levels of cAMP (16), suggesting that a kinase other than PKA is responsible for CREB activation in IL-2-stimulated T lymphocytes.

In this report we compare L2 cells with newly evolved L2 variant cells that exhibit constitutive Ser133 CREB phosphorylation. In parental L2 cells, IL-2 induces an increase in the levels of PKA, in the activity of a cAMP-independent CREB kinase and in an increase in CREB phosphorylation, all of which are inhibited by rapamycin. In L2 cells, we conclude that IL-2 regulates CREB phosphorylation through a cAMP-independent, rapamycin-sensitive kinase. In L2 variant cells, 1) basal levels of PKA, CREB kinase activity, and phosphorylated CREB are high and 2) PKA levels, CREB kinase activity, and CREB phosphorylation are not affected by IL-2 or rapamycin. In both L2 and L2 variant cells, rapamycin inhibits IL-2-stimulated PCNA expression and cellular proliferation. We conclude that Ser133 CREB phosphorylation and CREB kinase activity are not sufficient to drive PCNA expression and T cell proliferation and that rapamycin does not exert its inhibitory effect on PCNA expression primarily through inhibition of CREB activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The murine T helper lymphocyte clone, L2, was cultured with irradiated allogeneic spleen cells and 30 U/ml of rIL-2 (36). Cells were isolated by Ficoll-Hypaque density gradient centrifugation and incubated overnight in IL-2-free medium. Resting T cells (1 x 106/ml) were stimulated with 100 U/ml of IL-2 or 100 U/ml IL-2 plus rapamycin (20 ng/ml) (Research Biochemicals International, Natick, MA) for 24 h. Highly purified human recombinant IL-2 from Escherichia coli (37, 38) was a gift of Chiron (Emeryville, CA). During the last 2 years, L2 cells spontaneously converted to the variant phenotype (increased basal levels of PKA protein and CREB kinase activity) five times. Once the cells have undergone the transition to the variant phenotype, they have not reverted to the parental phenotype. Also, the variant state, having constitutively high phospho-CREB, is maintained with storage in liquid nitrogen. L2 variants were studied as a heterogenous population.

Kinase analysis

Cell lysates were processed by the method of Schwartz et al. (39). Briefly, cells were collected by centrifugation and washed twice in PBS. The pellets were resuspended in lysis buffer (10 mM potassium phosphate, pH 7.5, containing 1 mM EDTA, 1 mM 2-ME, 0.1 PMSF, and 200 nM methyl isobutyl xanthene) at 0°C. After 2 min, magnesium acetate was added to a final concentration of 0.5 mM. The cells were disrupted by passage through a 26-gauge needle. The lysate was centrifuged at 100,000 x g for 15 min at 4°C. The supernatant was collected and stored at -80°C until further analysis. Lysates were analyzed by the method of Roskowski (40). Lysates were added to the protein kinase reaction mixture: 50 mM MOPS, pH 7.0, 10 mM magnesium chloride, 0.25 mg/ml BSA, 100 µM acceptor peptide, kemptide (LRRASLG) (Bachem, King of Prussia, PA) (41) or crebtide (SRRPSYRK), 100 µM [{gamma}-32P]ATP, and, when indicated, 10 µM cAMP, 5 µM PKI, a PKA specific inhibitor (42), and/or 20 ng/ml rapamycin. To terminate the reaction, 25 µl of sample was spotted onto Whatman P81 phosphocellulose discs (Maidstone, U.K.) and immersed in 75 mM phosphoric acid for 2 min. The discs were washed twice more in phosphoric acid. To determine the total amount of phosphate available for transfer, 25 µl from a sample without kinase was spotted onto a phosphocellulose disc and left unwashed. The discs were placed in scintillation mixture, and the amount of radioactivity was assessed using a Wallac 1410 scintillation counter. The dose dependence of protein lysate on kinase activity was measured for kemptide and crebtide at 1 to 13 µg of protein lysate at a constant reaction time of 10 min at room temperature. These reactions yielded a sigmoid curve for each substrate with 2 to 10 µg of protein lysate defining the linear portion of the curve. The assays were also performed with a constant amount of protein (2.5 µg) for reaction times of 2 to 15 min. These reactions yielded a linear curve for each substrate. Based on this information, the experiments shown in this paper were performed using 2.5 to 5.0 µg of protein for 10 min.

Immunostaining

Cells were collected by centrifugation and washed twice with PBS. Cells for immunostaining were prepared by cytocentrifugation of 50,000 cells per slide and by fixation with 4% paraformaldehyde. The slides were incubated with 0.2% Triton X-100 for 15 min to permeabilize the cells, were blocked with normal goat serum for 20 min, and incubated with rabbit anti-P-CREB (Upstate Biotechnology, Lake Placid, NY) (1:500) or rabbit anti-CREB-1 Ab (Santa Cruz, Santa Cruz, CA) (1:500) overnight at 4°C. After washing, the slides were incubated sequentially with a biotinylated goat anti-rabbit Ab (Pierce, Rockford, IL)(1:400), with ABC peroxidase reagent (Pierce) for 30 min, and with 3,3'-diaminobenzine tetrahydrochloride (Sigma, St. Louis, MO) as a peroxidase substrate that forms an insoluble reaction product. Positive staining was visualized using differential interference contrast optics (43).

Western blot analysis

Cells were collected by centrifugation and washed with PBS. Lysis buffer (0.05 M HEPES, 1% Triton X-100, 0.01 M sodium fluoride, 10 mM sodium vanadate, 25 mM benzamidine, 10 µg/ml aprotonin, 30 mM sodium pyrophosphate, 20 µg/ml PMSF, and 1 µg/ml leupeptin) was added to cells at a final concentration of 1 x 107 cells/ml, and the lysate was stored at -80°C. Cell lysates (10 µg) were suspended in Laemmli gel sample buffer, boiled for 5 min, analyzed by SDS-PAGE, and electrotransferred onto Immobilon P membranes (Millipore, Bedford, MA). The membrane was blocked with PBS-5% milk for 1 h and then immunoblotted with anti-mouse PCNA Ab (Sigma) (1:2000) or anti-PCREB (UBI, Lake Placid, NY) (1:2000) overnight at 4°C. The blots were washed in PBS-tween, blocked with PBS-5% milk, and incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated Ab (Sigma) (1:10,000). The immunoreactive band was visualized using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).

Cellular proliferation

After treatment with IL-2 and rapamycin, as described above, L2 cells were plated on 96-well plates using 150 µl of each culture (1.5 x 105 cells/well) and were pulsed for 2 h with 1 µCi of [3H]thymidine/well. Cells were harvested with a PHD cell harvester (Cambridge Technology, Cambridge, MA). The filters were placed in scintillation mixture, and [3H]thymidine incorporation was assessed using a Wallac 1410 scintillation counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-2-stimulated CREB phosphorylation

Resting murine-cloned T cells, L2 cells, were stimulated with IL-2 or treated with both IL-2 and rapamycin for 24 h. When total immunoreactive CREB was examined (Figs. 1Go and 2), both the parental and the variant L2 cells contained similar amounts of CREB localized both to the perinuclear space and the nucleus for all conditions consistent with our previous findings (16). We examined Ser133 CREB phosphorylation in L2 cells by immunohistochemistry and Western blot analysis (Figs. 1Go and 3Go). In an experiment representative of cells passed for less than 61 days, phosphorylated CREB was present in nuclei, and levels of phosphorylation increased following IL-2 stimulation. IL-2-induced CREB phosphorylation was inhibited by rapamycin. After greater than 91 days in passage, variant L2 cells emerged that had high basal levels of nuclear Ser133-phosphorylated CREB, with no increase following treatment with IL-2 and no decrease following treatment with rapamycin (Figs. 2Go and 3Go). The size of the variant cells appears to be reduced when compared with the parent cells; the significance of this observation is not known.



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 1. IL-2-stimulated CREB phosphorylation in L2 cells. L2 cells were rested in IL-2-free medium for 24 h and then stimulated for 24 h with 100 units of IL-2 or 100 units of IL-2 with rapamycin (20 ng/ml). L2 cells were labeled for phosphorylated CREB (PCREB) and total CREB using specific polyclonal Abs and a second Ab (HRP-conjugated donkey anti-rabbit) as described in Materials and Methods. As a negative control, cells were labeled with only the donkey anti-rabbit Ab to detect nonspecific secondary Ab binding.

 


View larger version (89K):
[in this window]
[in a new window]
  		FIGURE 3. Ser133 CREB phosphorylation and PCNA expression in L2 and L2 variant cells. L2 and L2 variant cells were rested for 24 h in IL-2-free medium and left unstimulated (0), stimulated with IL-2 (100 U/ml) for 24 h (IL-2), or stimulated with IL-2 in the presence of rapamycin (20 ng/ml) for 24 h (R). Cells were lysed as described in Materials and Methods. Ten micrograms of total protein were separated by acrylamide gel electrophoresis and transferred to a membrane. Blots were incubated with HRP-conjugated anti-P-CREB or anti-PCNA Ab and developed by enhanced chemiluminescence (ECL).

 


View larger version (64K):
[in this window]
[in a new window]
  		FIGURE 2. IL-2-stimulated CREB phosphorylation in L2 variant cells. L2 variant cells were rested for 24 h in IL-2-free medium and then were treated for 24 h with 100 U of IL-2 or 100 U of IL-2 with rapamycin (20 ng/ml). L2 variant cells were labeled for phosphorylated CREB and total CREB using specific polyclonal Abs and a second Ab (HRP-conjugated donkey anti-rabbit) as described in Materials and Methods. As a negative control, cells were labeled with only the donkey anti-rabbit Ab to detect nonspecific secondary Ab binding.

 
Relationship of phosphorylated CREB to PCNA expression

Biochemical and molecular consequences of IL-2 stimulation and rapamycin inhibition are assessed in the context of lymphocyte proliferation. Previous studies suggested that CREB activation may be a key regulatory step in IL-2-stimulated PCNA expression and T cell proliferation (14, 16). Since the late passage L2 variant cells contain constitutively high levels of phosphorylated Ser133 CREB, resting late passage variant cells were examined for potential increases in basal PCNA expression and the effect of IL-2 and rapamycin on PCNA expression and cellular proliferation (Fig. 3Go and Table IGo). Resting late passage variant cells had low levels of PCNA similar to those found in parental L2 cells. Furthermore, IL-2 induced, and rapamycin inhibited, both PCNA expression and cellular proliferation. These findings indicate that CREB phosphorylation is not sufficient to drive these processes and that rapamycin does not inhibit PCNA expression primarily through inhibition of CREB activation.


View this table:
[in this window]
[in a new window]
  		Table I. The effect of IL-2 and rapamycin treatment on proliferation of L2 and L2 variant cellsa

 
IL-2-inducible kinase activity

To determine the mechanism responsible for IL-2-inducible and constitutive phosphorylation of CREB, we assessed kinase activities in L2 and L2 variant cells. PKA phosphorylation of CREB at Ser133 is well known (21, 22, 23, 24); therefore, lysates were assayed for kinase activity using kemptide, a protein kinase A specific substrate. PKA exists as an inactive holoenzyme that is dissociated into an active form in the presence of cAMP. Assays done in the presence of cAMP reflect total kinase levels in the cell, and assays done in the absence of cAMP reflect the amount of kinase activity at the time of cell lysis. The amount of kinase activity is expressed as a percent of the amount of activity in unstimulated L2 or L2 variant cells plus cAMP. In early passage L2 cells, total PKA increases following stimulation with IL-2 for 24 h (Fig. 4Go). However PKA activity in the absence of cAMP reveals that only 27% of the total kinase is active in resting cells. Despite the substantial increase in PKA levels in IL-2-stimulated cells, the amount of active PKA remains unchanged. Rapamycin treatment inhibited the IL-2-induced increase in PKA but had no effect on levels of active kinase. Inhibition by PKI, a PKA-specific kinase inhibitor, indicates that the majority of kinase activity measured using kemptide can be attributed to PKA.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 4. Protein kinase A activity in L2 and L2 variant. L2 cells from short time of passage (21–63 days) and L2 variant cells (91–140 days) were rested for 24 h in IL-2-free medium and left unstimulated (0), stimulated with IL-2 (100 U/ml) alone for 24 h (IL-2), or stimulated with IL-2 in the presence of rapamycin (20 ng/ml) for 24 h (IL-2+R). Total protein was extracted from the cells, and 5.0 µg of protein was assayed for the ability to phosphorylate kemptide (LRRASLG), a PKA-selective substrate. Enzyme assays were done in the presence (+cAMP) or absence (-cAMP) of cAMP (10 µM). Some enzyme assays were performed in the presence of 5 µM PKI, a PKA-specific inhibitor. Samples were assayed in duplicate, and an average of three experiments are shown. Values are expressed as percent of 0 h + cAMP kinase activity, which for the three experiments was 84, 94, and 562 pmol phosphate transferred/min/mg protein for L2 cells and 301, 711, and 826 pmol phosphate transferred/min/mg protein for L2 variant cells.

 
In the L2 variant, passed for greater than 91 days, a different pattern of kinase activity was seen after IL-2 stimulation. Instead of an increase in PKA levels after IL-2 treatment, a decrease was observed. The amount of active kinase remained low at 33% and decreased after IL-2 treatment. Rapamycin treatment had no effect on total or active PKA levels. The reaction done in the presence of PKI again revealed that most of the kinase activity measured was PKA.

To determine the presence of other kinases that phosphorylate CREB, similar experiments were performed using crebtide as a substrate (Fig. 5Go). Crebtide contains the Ser133 phosphorylation site of the CREB protein, which is required for transcriptional activation (30, 31, 32, 33). In early passage L2 cells, IL-2 stimulation resulted in an increase in CREB kinase activity that was inhibited by rapamycin. In contrast to PKA (kemptide substrate), most of this kinase activity was cAMP independent and inhibited to a much lesser extent by PKI, indicating that CREB kinase activity is not PKA.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 5. CREB kinase activity in L2 and L2 variant cells. L2 cells from short time of passage (21–63 days) and L2 variant cells (91–140 days) were rested for 24 h in IL-2-free medium and left unstimulated (0), stimulated with IL-2 (100 U/ml) for 24 h (IL-2), or stimulated with IL-2 in the presence of rapamycin (20 ng/ml) for 24 h (IL-2+R). Total protein was extracted from the cells, and 5.0 µg of protein was assayed for the ability to phosphorylate crebtide (SRRPSYRK), a substrate that contains the Ser133 phosphorylation site of CREB. Enzyme assays were done in the presence (+cAMP) or absence (-cAMP) of cAMP (10 µM). Some enzyme assays were performed in the presence of 5 µM PKI, a PKA-specific inhibitor. Samples were assayed in duplicate, and an average of three experiments are shown. Values are expressed as percent of 0 h + cAMP kinase activity, which for the three experiments was 93, 99, and 391 pmol phosphate transferred/min/mg protein for L2 cells and 274, 870, and 1206 pmol phosphate transferred/min/mg protein for L2 variant cells.

 
In L2 variant cells passed for greater than 91 days, IL-2 did not cause an increase in CREB kinase activity. The kinase activity was cAMP independent, and there was a greater resistance to inhibition with PKI than that seen with kemptide as a substrate. In contrast to L2 cells, rapamycin had no inhibitory effect on the kinase activity.

Effect of cell aging on kinase activity

The inability of IL-2 to induce kinase activity in older cultures appears to result from a change in basal kinase activity as the cultures age (Fig. 6Go). PKA activity in cells passed for less than 63 days was at an average of 250 pmol phosphate transferred/min/mg protein and was inducible to an average of 640 pmol phosphate transferred/min/mg protein. Cells that were passed for greater than 91 days had an average resting PKA level of 610 pmol phosphate transferred/min/mg protein and could not be induced. When crebtide was used as the substrate, resting kinase activity in cells passed for fewer than 63 days was at an average of 90 pmol phosphate transferred/min/mg protein and was inducible to an average value of 500 pmol phosphate transferred/min/mg protein. The average resting kinase activity of cells that were passed for greater than 91 days was 780 pmol phosphate transferred/min/mg protein and also could not be induced. Thus, in aged cultures, basal kinase activity for each substrate was equal to or greater than the induced levels in younger cultures.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 6. Basal kinase levels are elevated in L2 variant cells. L2 cells from short time of passage (21–63 days) and L2 variant cells (91–140 days) were rested for 24 h in IL-2-free medium and left unstimulated (0) or stimulated with IL-2 (100 U/ml) for 24 h (24). Total protein was extracted from the cells, and 5.0 µg of protein was assayed for the ability to phosphorylate kemptide (LRRASLG), a PKA-selective substrate, or crebtide (SRRPSYRK), a substrate that contains the Ser133 phosphorylation site of CREB. Samples were assayed in duplicate, and an average of three experiments is shown. Results are expressed as picomole of phosphate transferred in a 10-min reaction. Protein (2.5–5.0 µg) was used for each reaction.

 
Rapamycin effect on kinase activity in vitro

To determine whether rapamycin has a direct effect on the kinases studied, kinase activity was measured in the presence and absence of rapamycin (Fig. 7Go). PKA and CREB kinase activities were not affected by rapamycin. This implies that rapamycin exerts its inhibitory effect on the increase in kinase levels or kinase activation but not directly on kinase activity.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 7. Rapamycin does not inhibit kinase activity in vitro. L2 cells were rested for 24 h in IL-2-free medium and left unstimulated (0) or stimulated with IL-2 (100 U/ml) for 24 h (24). Total protein was extracted from the cells, and 5.0 µg of protein was assayed for the ability to phosphorylate kemptide (LRRASLG), a PKA-selective substrate, or crebtide (SRRPSYRK), a substrate that contains the Ser133 phosphorylation site of CREB. Untreated and 24-h IL-2-stimulated L2 cells were assayed for kinase activity in the presence or absence of rapamycin (20 ng/ml). Results are expressed as picomole of phosphate transferred in a 10-min reaction. Protein (2.5–5.0 µg) was used for each reaction.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Stimulation of the murine-cloned T lymphocyte L2 with IL-2 drives entry and progression through the cell cycle (36, 37). Increased PCNA expression has been shown in IL-2-stimulated T cells (13); there appears to be a close temporal link with the phosphorylation of CREB protein and the ability of proteins to bind to the PCNA CRE sequences (14, 16). CRE sequences are required for optimal promoter activity, and rapamycin inhibits IL-2-stimulated CREB binding to these elements (5). The discovery of an L2 cell variant that has constitutively phosphorylated CREB allowed us to test the hypothesis that CREB activation is a key rapamycin-sensitive step in IL-2-stimulated PCNA expression and proliferation.

Phosphorylation of the Ser133 residue on CREB has been shown to have great importance in gene expression and proliferation as shown by the inability of T cells expressing the dominate-negative form of CREB to produce IL-2 and undergo cell cycle progression (44). Phosphorylation of CREB has been shown to regulate binding of the protein to CRE sites (21, 23, 26, 45, 46). Maximal transcriptional activation of the PCNA promoter requires CREB/ATF1 transcription factors following IL-2 stimulation (14). Previous data generated using the L2 cell line have shown an increase in PCNA expression after IL-2 stimulation and a decrease in the presence of rapamycin (5). Despite the high constitutive kinase activity and CREB phosphorylation in variant cells, PCNA expression was low in untreated and rapamycin-treated cells (Fig. 3Go). Thus, CREB activation alone is insufficient for PCNA gene expression.

CREB transcriptional activation has been shown to require other signals in T cells. For example, CD28 signaling was required for full transcriptional activity of CREB in EL4 cells, whereas Ser133 phospho-CREB alone was insufficient for CRE-CAT (chloramphenicol acetyltransferase) gene expression (47). Similarly in Jurkat cells CREB phosphorylation alone was insufficient for transcriptional regulation; PKA-mediated phosphorylation of the CREB binding protein (CBP) was required. (48). CBP phosphorylation allows a complex to be formed with phospho-CREB, which then binds to the CRE. Therefore, a similar form of control may be active in both L2 cells and L2 variant cells where a secondary IL-2-mediated phosphorylation event is required for PCNA transcription.

While the variant phenotype showed that CREB phosphorylation alone was insufficient for PCNA expression, there is strong evidence for an IL-2-inducible, rapamycin-sensitive kinase responsible for CREB activation that is required for optimal PCNA promoter activity (14). cAMP-dependent protein kinase A (PKA) was the first enzyme identified to phosphorylate CREB at Ser133 (21, 22, 23, 24). Increased intracellular levels of cAMP occur following mitogenic stimulation of splenic B lymphocytes and T lymphocytes (49, 50). Elevation of cAMP induces the translocation of PKA to the nucleus (51). While this information suggests that IL-2-stimulation may act in part through PKA activation, other data argue against the role of PKA in CREB activation in T cells. For example, IL-2 does not induce an increase in intracellular levels of cAMP during cell cycle progression in L2 cells (16), and T cell proliferation is inhibited by high cAMP levels (35, 52), potentially through cAMP-blocking Raf-1 kinase (53). These conflicting data create a paradox; two possible explanations include 1) a transient activation of PKA that is supported by the data, which show that there must be a rise followed by a decrease in cAMP levels for progression of T cells into S phase (50), or 2) a kinase other than PKA activates CREB in IL-2-stimulated T cells.

This paradox led us to examine PKA levels in L2 and L2 variant cells. The kinase activity responsible for kemptide phosphorylation is PKA, as demonstrated by the dependence on cAMP (Fig. 4Go) and by the sensitivity of kinase activity to the inhibitor peptide, PKI (54). Since PKA activity was low and uninducible by IL-2 (Fig. 4Go), it seems likely that another kinase may be phosphorylating CREB in IL-2-stimulated cells. To determine other possible kinase activities, the substrate crebtide was used in the kinase assays. Crebtide is a 9-amino acid peptide (SRRPSYRK) containing the Ser133 phosphorylation site shown to be important in the regulation of CREB interactions with DNA and other regulatory proteins (21, 22, 23, 24, 55). Phosphorylation of crebtide reflects phosphorylation of endogenous CREB protein (56). When using crebtide as the substrate, the presence of a cAMP-independent, IL-2-inducible, rapamycin-sensitive kinase was shown in the parental cell line (Fig. 5Go). This was demonstrated by the observation that almost 100% of the CREB kinase was active (-cAMP) on the crebtide substrate (Fig. 5Go), while only about 25% of the PKA kinase was active (-cAMP) on the kemptide substrate (Fig. 4Go). This kinase was relatively resistant to the PKA inhibitor, PKI, further supporting the notion that it is not PKA. Kinases other than PKA can mediate phosphorylation of CREB at Ser133 or the corresponding Ser residue on CRE modulator (CREM), such as p90RSK (27), RSK2 (29), Ca2+/calmodulin kinase II(26, 57), a Ras-dependent protein kinase (25), PKC (58, 59), and p70S6 kinase (59). Of these kinases, the activities of p70S6 kinase(60, 61, 62) and PKC (63) are affected by rapamycin. While PKC can mediate p70S6 kinase phosphorylation, rapamycin appears to act downstream of PKC-mediated p70S6 kinase activation (64). A rapamycin-sensitive kinase in T cells in the pathway to activation of p70S6 kinase is FKBP12-rapamycin-associated protein (FRAP) (65). p70S6 kinase has been shown to be stimulated in murine T cells by IL-2 (66) and inhibited by rapamycin (60, 61). Interestingly, p70S6 kinase activity can be inhibited by cAMP acting through PKA (64). In our cells, active PKA levels are only slightly above baseline in resting and IL-2-stimulated cells (Fig. 4Go), which would potentially allow for normal activity of p70S6 kinase. Our results indicate that the increase in CREB kinase activity is sensitive to rapamycin (Fig. 5Go), but the kinase is not affected directly by rapamycin (Fig. 7Go). Previous studies have shown that p70S6 kinase activation is rapamycin sensitive, but enzyme activity is not inhibited directly by rapamycin (67). Thus one possibility would be that rapamycin inhibits FRAP blocking p70S6 kinase activation and CREB phosphorylation.

We have demonstrated the existence of an L2 variant expressing constitutively phosphorylated Ser133 CREB. In this variant, constitutive CREB phosphorylation alone is not sufficient to drive PCNA expression and cellular proliferation. L2 cells express a kinase other than PKA that is capable of phosphorylating CREB at Ser133; based on the IL-2 inducibility and sensitivity to rapamycin, p70S6 kinase is a candidate kinase. Thus, while CREB phosphorylation plays a role in the control of gene expression and proliferation of T cells, there are other rapamycin-sensitive events that are the rate limiting steps in IL-2-driven T cell proliferation.


   Footnotes
 
1 This work was supported by Grant RO1 MH51327-02 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Michael B. Prystowsky, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: Back

3 Abbreviations used in this paper: PCNA, proliferating cell nuclear Ag; CRE, cAMP response element; CREB, CRE-binding protein; CBP, CREB-binding protein; ATF1, activating transcription factor-1; PKA, cAMP-dependent protein kinase A; RSK2, ribosomal S6 kinase-2; PKI, PKA-specific kinase inhibitor; HRP, horseradish peroxidase; FRAP, FKBP12-rapamycin-associated protein. Back

Received for publication December 8, 1997. Accepted for publication March 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sigal, N. H., F. J. Dumont. 1992. Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction. Annu. Rev. Immunol. 10:519.[Medline]
  2. Schreiber, S. L.. 1991. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251:283.[Abstract/Free Full Text]
  3. Dumont, F. J., M. J. Staruch, S. L. Koprak, M. R. Melino, N. H. Sigal. 1990. Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin. J. Immunol. 144:251.[Abstract]
  4. Jefferies, H. B. J., C. Reinhard, S. C. Kozma, G. Thomas. 1994. Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family. Proc. Natl. Acad. Sci. USA 91:4441.[Abstract/Free Full Text]
  5. Feuerstein, N., D. Huang, M. B. Prystowsky. 1995. Rapamycin selectively blocks IL-2 induced PCNA gene expression in T lymphocytes: evidence for inhibition of CREB/ATF binding activities. J. Biol. Chem. 270:9454.[Abstract/Free Full Text]
  6. Prelich, G., C. K. Tan, M. Kostura, M. B. Mathews, A. G. So, K. M. Downey, B. Stillman. 1987. Functional identity of proliferating cell nuclear antigen and a DNA polymerase delta auxiliary protein. Nature 326:517.[Medline]
  7. Bravo, R., R. Frank, P. A. Blundell, H. Macdonald-Bravo. 1987. Cyclin/PCNA is the auxiliary protein of DNA polymerase delta. Nature 326:515.[Medline]
  8. Prelich, G., B. Stillman. 1988. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell 53:117.[Medline]
  9. Matthews, M. B.. 1989. The proliferating cell nuclear antigen, PCNA, a cell growth-regulated DNA replication factor. E. Wang, and H. R. Warner, eds. Growth Control During Cell Aging 89. CRC Press, Boca Raton, Fla.
  10. Krishna, T. S. R., X. P. Kong, S. Gary, P. M. Burgers, J. Kuriyan. 1994. : crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79:1233.[Medline]
  11. Wong, R. L., M. E. Katz, K. Ogata, E. M. Tan, S. Cohen. 1987. Inhibition of nuclear DNA synthesis by an autoantibody to proliferating cell nuclear antigen/cyclin. Cell. Immunol. 110:443.[Medline]
  12. Jakulski, D., J. K. DeRiel, W. E. Mercer, B. Calabretta, R. Baserga. 1988. Inhibition of cellular proliferation by antisense to oligodeoxinucleotides to PCNA/cyclin. Science 240:1544.[Abstract/Free Full Text]
  13. Shipman-Appasamy, P., K. Cohen, M. B. Prystowsky. 1990. Interleukin 2-induced expression of proliferating cell nuclear antigen is regulated by transcriptional and post-transcriptional mechanisms. J. Biol. Chem. 265:19180.[Abstract/Free Full Text]
  14. Huang, D., P. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, M. B. Prystowsky. 1994. Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes. Mol. Cell. Biol. 14:4233.[Abstract/Free Full Text]
  15. Huang, D. Y., M. B. Prystowsky. 1996. Identification of an essential cis-element near the transcription start site for transcriptional activation of the proliferating cell nuclear antigen gene. J. Biol. Chem. 271:1218.[Abstract/Free Full Text]
  16. Feuerstein, N., D. Huang, S. H. Hinrichs, D. J. Orten, N. Aiyar, M. B. Prystowsky. 1995. Regulation of cAMP-responsive enhancer binding proteins during cell cycle progression in T lymphocytes stimulated by IL-2. J. Immunol. 154:68.[Abstract]
  17. Meyer, T. E., J. F. Habener. 1993. Cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) and related transcription activating deoxyribonucleic acid binding proteins. Endocr. Rev. 14:269.[Medline]
  18. Hoeffler, J. P.. 1992. Structure/function relationships of CREB/ATF proteins. J. Invest. Dermatol. 98:21S.[Medline]
  19. Lee, K. A. W., N. Masson. 1993. Transcriptional regulation by CREB and its relatives. Biochim. Biophys. Acta 1174:221.[Medline]
  20. Brindle, P. K., M. R. Montminy. 1992. The CREB family of transcription activators. Curr. Opin. Gene. Dev. 2:199.[Medline]
  21. Gonzalez, G. A., M. R. Montminy. 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675.[Medline]
  22. Montiminy, M. R., L. M. Bilezikjian. 1987. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328:175.[Medline]
  23. Yamamoto, K. K., G. A. Gonzalez, W. H. Biggs, M. R. Montminy. 1988. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334:494.[Medline]
  24. Hoeffler, J. P., T. E. Meyer, Y. Yub, J. L. Jameson, J. F. Habener. 1988. Cyclic AMP-responsive DNA binding protein: structure based on a cloned placental cDNA. Science 242:1430.[Abstract/Free Full Text]
  25. Ginty, D. D., A. Bonni, M. E. Greenberg. 1994. Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77:713.[Medline]
  26. Sheng, M., M. A. Thompson, M. E. Greenberg. 1991. CREB: A Ca2+-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252:1427.[Abstract/Free Full Text]
  27. Bohm, M., G. Moellmann, E. Cheng, M. Alverez-Fronco, S. Wagner, P. Sassone-Corsi, R. Halaban. 1995. Identification of p90rsk as the probable CREB-ser133 kinase in human melanocytes. Cell Growth Differ. 6:291.[Abstract]
  28. deGroot, R. P., L. M. Ballou, P. Sassone-Corsi. 1994. Positive regulation of the cAMP-responsive activator CREM by the p70 S6 kinase: an alternative route to mitogen-induced gene expression. Cell 79:81.[Medline]
  29. Xing, J., D. D. Ginty, M. E. Greenberg. 1996. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273:959.[Abstract]
  30. Nichols, M., F. Weih, W. Schmid, C. DeVack, E. Kowenz-Leutz, B. Luckow, M. Boshart, G. Schutz. 1992. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription. EMBO J. 11:3337.[Medline]
  31. Kramer, I. M., I. Koornneef, S. W. deLaat, A. van den Eijnden-van Raaij. 1991. TGF-ß1 induces phosphorylation of the cyclic AMP responsive element binding protein in ML-CC164 cells. EMBO J. 10:1083.[Medline]
  32. Dean, D. C., J. J. McQuillan, S. Weintraub. 1990. Serum stimulation of fibronectin gene expression appears to result from rapid serum induced binding of nuclear proteins to a cAMP response element. J. Biol. Chem. 265:3522.[Abstract/Free Full Text]
  33. Englander, E. W., S. G. Widen, S. H. Wilson. 1991. Mammalian B-polymerase promoter: phosphorylation of ATF/CRE binding protein and regulation of DNA binding. Nucleic Acids Res. 19:3369.[Abstract/Free Full Text]
  34. Roesler, W. J., G. R. Vandenbark, R. W. Hanson. 1988. Cyclic AMP and the induction of eukaryotic gene transcription. J. Biol. Chem. 263:9063.[Free Full Text]
  35. Tamir, A., Y. Granot, N. Isakov. 1996. Inhibition of T lymphocyte activation by cAMP as associated with down-regulation of two parallel mitogen-activated protein kinase pathways, the extracellular signal-related kinase and c-Jun N-terminal kinase. J. Immunol. 157:1514.[Abstract]
  36. Glasebrook, A. L., F. W. Fitch. 1980. Alloreactive cloned T cell lines. I. Interactions between cloned amplifier and cytolytic T cell lines. J. Exp. Med. 151:876.[Abstract/Free Full Text]
  37. Sabath, D. E., D. S. Monos, S. C. Lee, C. Deutsch, M. B. Prystowsky. 1986. Cloned T-cell proliferation and synthesis of specific proteins are inhibited by quinine. Proc. Natl. Acad. Sci. USA 83:4739.[Abstract/Free Full Text]
  38. Lee, S. C., D. E. Sabath, C. Deutsch, M. B. Prystowsky. 1986. Increased voltage-gated potassium conductance during interleukin 2-stimulated proliferation of a mouse helper T lymphocyte clone. J. Cell Biol. 102:1200.[Abstract/Free Full Text]
  39. Schwartz, D. A., C. S. Rubin. 1985. Identification and differential expression of two forms of regulatory subunits (RII) of cAMP-dependent protein kinase II in friend erythroleukemic cells. J. Biol. Chem. 260:6296.[Abstract/Free Full Text]
  40. Roskowski, R.. 1983. Assays of protein kinase. Methods Enzymol. 99:3.[Medline]
  41. Kemp, B. E., D. J. Graves, E. Benjamini, E. G. Krebs. 1977. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J. Biol. Chem. 252:4888.[Free Full Text]
  42. Glass, D. B., L. J. Lundquist, B. M. Katz, D. A. Walsh. 1989. Protein kinase inhibitor-(6–22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. J. Biol. Chem. 264:14579.[Abstract/Free Full Text]
  43. Prystowsky, M. B., K. M. Khan, W. F. Marovitz. 1978. Detection of H2-b and Thy-1.2 surface antigens on the differentiating murine otocyst using Nomarski optics. Differentiation 12:53.[Medline]
  44. Barton, K., N. Muthusamy, M. Chanyangam, C. Fischer, C. Clendenin, J. M. Leiden. 1996. Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominate-negative form of CREB. Nature 379:81.[Medline]
  45. Dash, P. K., K. A. Karl, M. A. Colicos, R. Prywes, E. R. Kandel. 1991. cAMP response element binding protein is activated by Ca2+/calmodulin as well as cAMP dependent protein kinase. Proc. Natl. Acad. Sci. USA 88:5061.[Abstract/Free Full Text]
  46. Sheng, M., G. McFadden, M. E. Greenberg. 1990. Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB. Neuron 4:571.[Medline]
  47. Hseuh, Y., H. Liang, S. Ng, M. Lai. 1997. CD28-costimulation activates cyclic AMP-responsive element-binding protein in T lymphocytes. J. Immunol. 158:85.[Abstract]
  48. Brindle, P., T. Nakajima, M. Montminy. 1995. Multiple protein kinase A-regulated events are required for transcriptional induction by cAMP. Proc. Natl. Acad. Sci. USA 92:10521.[Abstract/Free Full Text]
  49. Feuerstein, N., M. L. Lindsberg, L. Tung, M. L. Francis, J. J. Mond. 1991. Identification of a prominent 85-kDa cAMP-dependent phosphoprotein associated with late G1 phase in mitogen-stimulated B lymphocytes. J. Biol. Chem. 266:4746.[Abstract/Free Full Text]
  50. Wang, T., J. R. Sheppard, J. E. Foker. 1991. Rise and fall of cyclic AMP required for onset of lymphocyte DNA synthesis. Science 201:155.
  51. Nigg, E. A., H. Hilz, H. M. Eppenberger, F. Dutly. 1991. Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the golgi complex to the nucleus. EMBO J. 4:2801.[Medline]
  52. Kammer, G. M.. 1988. The adenylate cyclase-cAMP-protein kinase A pathway and regulation of the immune response. Immunol. Today 9:222.[Medline]
  53. Hafner, S., H. S. Adler, H. Mischak, P. Janosch, G. Heidecker, A. Wolfman, S. Pippig, M. Lohse, M. Ueffing, W. Kolch. 1994. Mechanism of inhibition of Raf-1 by protein kinase A. Mol. Cell. Biol. 14:6696.[Abstract/Free Full Text]
  54. Cheng, H.-C., B. E. Kemp, R. B. Pearson, A. J. Smith, L. Misconi, S. M. Van Patten, D. A. Walsh. 1986. A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. J. Biol. Chem. 261:989.[Abstract/Free Full Text]
  55. Montminy, M., P. Brindle, J. Arias, K. Ferreri, R. Armstrong. 1996. Regulation of somatostatin gene transcription by cyclic andenosine monophosphate. Metab. Clin. Exp. 45:4.
  56. Colbran, J. L., P. J. Roach, C. J. Fiol, J. E. Dixon, O. M. Andrisiani, J. D. Corbin. 1992. cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide. Biochem. Cell Biol. 70:1277.[Medline]
  57. Sheng, M., M. A. Thompson, M. E. Greenberg. 1992. CREB: a Ca++-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252:1427.
  58. Xie, H., and T. L. Rothstein. 1995. Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig. J. Immunol. 1717.
  59. de Groot, R. P., J. den Hertog, J. Vandenheede, J. Goris, P. Sassone-Corsi. 1993. Multiple and cooperative phosphorylation events regulate the CREM activator function. EMBO J. 12:3903.[Medline]
  60. Calvo, V., C. M. Crews, T. A. Vik, B. E. Bierer. 1992. Interleukin 2 stimulation of p70 S6 kinase activity is inhibited by the immunosuppressant rapamycin. Proc. Natl. Acad. Sci. USA 89:7571.[Abstract/Free Full Text]
  61. Kuo, C. J., J. Chung, D. F. Fiorentino, W. M. Flanagan, J. Blenis, G. R. Crabtree. 1992. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 358:70.[Medline]
  62. de Groot, R. P., L. M. BNallou, P. Sassone-Corsi. 1994. Positive regulation of the cAMP responsive activator CREM by the p70 S6 kinase: an alternative route to mitogen induced gene expression. Cell 79:81.
  63. Kimbal, P. M., R. K. Kerman, C. T. VanBuren, R. M. Lewis, S. Katz, B. D. Kahan. 1993. Cyclosporine and rapamycin affect protein kinase C induction of the intracellular activation signal, activator of DNA replication. Transplantation 55:1128.[Medline]
  64. Monfar, M., K. P. Lemon, T. C. Grammer, L. Cheatham, J. Chung, C. J. Vlahos, J. Blenis. 1995. Activation of pp70/85 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol. Cell. Biol. 15:326.[Abstract]
  65. Brown, E. J., P. A. Beal, C. T. Keith, J. Chen, T. B. Shin, S. L. Schreiber. 1995. Control of p70 S6 kinase by kinase activity of FRAP in vivo. Nature 377:441.[Medline]
  66. Evans, S. W., W. L. Farrar. 1987. Interleukin 2 and diacylglycerol stimulate phosphorylation of 40 S ribosomal S6 kinase activation. J. Biol. Chem. 262:4624.[Abstract/Free Full Text]
  67. Chung, J., C. J. Kuo, G. R. Crabtree, J. Blenis. 1992. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69:1227.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C.-T. Yu, H.-m. Shih, and M.-Z. Lai
Multiple Signals Required for Cyclic AMP-Responsive Element Binding Protein (CREB) Binding Protein Interaction Induced by CD3/CD28 Costimulation
J. Immunol., January 1, 2001; 166(1): 284 - 292.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 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 Belkowski, S. M.
Right arrow Articles by Prystowsky, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belkowski, S. M.
Right arrow Articles by Prystowsky, M. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS