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The Journal of Immunology, 2001, 166: 284-292.
Copyright © 2001 by The American Association of Immunologists

Multiple Signals Required for Cyclic AMP-Responsive Element Binding Protein (CREB) Binding Protein Interaction Induced by CD3/CD28 Costimulation1

Cheng-Tai Yu*,{dagger}, Hsiu-ming Shih{ddagger} and Ming-Zong Lai2,*,{dagger},§,¶

* Graduate Institute of Life Science, National Defense Medical School; {dagger} Institute of Molecular Biology, Academia Sinica; {ddagger} Division of Molecular and Genomic Medicine, National Health Research Institute; § Graduate Institute of Immunology, National Taiwan University; and Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The optimal activation of cAMP-responsive element binding protein (CREB), similar to the full activation of T lymphocytes, requires the stimulation of both CD3 and CD28. Using a reporter system to detect interaction of CREB and CREB-binding protein (CBP), in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28. CD3/CD28-promoted CREB-CBP interaction was dependent on p38 mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase (CaMK) IV in addition to the previously identified extracellular signal-regulated kinase pathway. Extracellular signal-regulated kinase, CaMKIV, and p38 MAPK were also the kinases involved in CREB Ser133 phosphorylation induced by CD3/CD28. A reconstitution experiment illustrated that optimum CREB-CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells. Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB. Notably, CD28 ligation activated p38 MAPK and CaMKIV, the kinases stimulated by CD3 engagement, suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV. These results support the model of a minimum activation threshold for CREB-CBP interaction that can be reached only when both CD3 and CD28 are stimulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyclic AMP-responsive element binding protein (CREB)3 mediates cAMP-responsive gene expression in a well-characterized scheme (1, 2, 3). CREB binds to the cAMP-responsive element (CRE) located in the promoter of most cAMP-inducible genes (4, 5). The phosphorylation of CREB Ser133 by protein kinase A (6, 7) promotes the binding of the kinase-inducible domain of CREB to the KIX domain of CREB binding protein (CBP) through electrostatic interaction (8, 9, 10, 11). CBP then initiates the transcription of CRE-containing genes by the associated RNA polymerase II and histone acetyl transferase activities (9, 12). Ser133 phosphorylation hence plays a critical role in CREB activation. Mutation of serine to alanine at position 133 converts CREB into a dominant negative transcriptional regulator.

In addition to cAMP, CREB is activated by Ca2+, growth factors, and stress signals (1, 2, 3). A number of different kinases have been shown to stimulate CREB phosphorylation and several CREB kinase candidates have been identified. The calcium signaling that activates CREB can be traced back to calcium/calmodulin-dependent protein kinase I (CaMKI), CaMKII, and CaMKIV, which are activated by the increase in the calcium/calmodulin complex. Even though CaMKI and CaMKII phosphorylate CREB in vitro, CaMKI is not translocated into the nucleus, and CaMKII does not activate CREB transcriptionally (13, 14, 15). CaMKIV is thus the kinase that mediates calcium-induced CREB phosphorylation in vivo. In the other major pathway involved in CREB activation, the mitogen-activated protein kinase (MAPK) kinase (MAPKK)-extracellular signal-regulated kinase (ERK) cascade, the downstream p90rsk2 phosphorylates CREB (16). Other ribosomal S6 kinase (RSK) family members, RSK1 and RSK3, also phosphorylate CREB (17). Along the p38 MAPK cascade, which is the third major signaling pathway that contributes to CREB activation, MAPK-activated protein kinase 2 mediates stress- and fibroblast growth factor-induced phosphorylation of CREB (18). CREB is also directly phosphorylated by mitogen- and stress-activated kinase 1 (19), the kinase activated by stress stimuli in the p38 MAPK-dependent pathway or by growth factor in the ERK-dependent cascade. A third CREB kinase controlled by p38/SAPK2, RSK-B, has been identified (20). In addition to the calcium, ERK, and p38 signal pathways, CREB is phosphorylated by Akt (21). The CREB analogue CREM can also be phosphorylated by PKC and p70 S6 kinase (22, 23). The presence of a large number of CREB kinases indicates that phosphorylation of CREB may be mediated by two or more kinases under the same stimulus. For example, nerve growth factor-induced CREB phosphorylation involves both RSK and MAPK-activated protein kinase (17).

TCR engagement leads to CREB phosphorylation (24, 25). The transcriptional activation of CREB is essential for T cell activation and development. Blockage of CREB transcriptional activation by T cell-specific transgenic expression of the dominant negative CREB or dominant negative CaMKIV greatly impairs thymocyte proliferation and IL-2 production (26, 27). Moreover, genetic knockout of CREB abolishes {alpha}{beta} T cell development in mice (28). CREB is required for the expression of proliferating cell nuclear Ag (PCNA) which contributes to cell cycle progression (29). CREB may also participate in Th cell development through regulated expression of {gamma}-IFN (30).

We previously illustrated that full CREB activation, similar to T cell activation, requires dual signals from CD3 and CD28 (25). CD28 is a T cell-specific cell surface molecule that interacts with B7 on APCs. The costimulation signal from CD28 acts in synergy with the mitogenic signal from TCR to promote cell cycle progression and to increase IL-2 secretion (for reviews, see Refs. 31, 32). CD28 costimulation also prevents induction of T cell tolerance (33) and cell death (34). Even though the essential role of CD28 is well recognized, the CD28-mediated costimulatory signals are not well understood. CD28-mediated signals are known to involve phosphatidylinositol 3-kinase (PI 3-kinase) (35, 36, 37), but PI 3-kinase alone cannot account for all CD28-mediated costimulatory events (38, 39, 40). CD28 costimulation results in full activation of c-Jun N-terminal kinase (JNK) (41), yet signals other than JNK are required for the induction of IL-2 production (42). Additional candidates of CD28 costimulatory signals include I{kappa}B kinase (43) and p38 MAPK (44, 45, 46). Engagement of both CD3 and CD28 also induces PDE7, which decreases cAMP and increases T cell activation (47).

In the present study we found that in addition to ERK, p38 MAPK and CaMKIV are activated by CD3/CD28. ERK, p38 MAPK, and CaMKIV are required for both CREB-CBP interaction and CREB trans-activation, which were significantly compromised when any single kinase cascade was inhibited. Our findings suggest that multiple signals are required to coordinate a stable CREB-CBP interaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

A23187, tetradecanoyl phorbol 13-acetate, Con A, and prestained m.w. markers (SDS-7B) were purchased from Sigma (St. Louis, MO). H89 was obtained from Seikagaku (Tokyo, Japan). DEAE-dextran (m.w., 5 x 105) was purchased from Pharmacia (Uppsala, Sweden). Rapamycin was obtained from Biomol (Plymouth Meeting, PA). PD 98059, KN-62, KN-93, Ly 294002, wortmannin, and SB 203580 were purchased from Calbiochem (La Jolla, CA). Anti-CREB Ab recognizing peptide 136–150 of CREB was previously described (48). Anti-phosphorylated CREB Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-CD28 Ab 37.51, generated by Dr. James Allison (University of California, Berkeley, CA) (49), was obtained through Dr. Nan-Shih Liao (Academia Sinica, Taipei, Republic of China). Anti-phospho-Akt (Ser473) Ab was purchased from New England Biolabs (Beverly, MA).

T cell lines and cell culture

EL4 T lymphoma cells (ATCC TIB39; American Type Culture Collection, Manassas, VA) were a gift from Dr. Nan-Shih Liao. Jurkat cells (ATCC TIB 152) were obtained from Dr. Chung-Yi Wang (National Yang-Ming University, Taipei, Republic of China). All cultures were performed in RPMI with 10% FCS (both from Life Technologies, Grand Island, NY), 10 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 x 10-5 M 2-ME.

Plasmids

CRE-chloramphenicol acetyltransferase (CRE-CAT) was previously described (25). pVP16-CREB and pVP16-CREB[S133A] were previously described (50). pGAL-CREB-binding domain of CBP (pGAL-CBD) encodes fusion protein of Gal1–147 and CBD of CBP (aa 461–682). RSV-CBP was a gift from Dr. Richard H. Goodman (Oregon Health Sciences University, Portland, OR). pG5B-CAT (51) was obtained from Dr. Mark Ptashne (Harvard University, Boston, MA). PCNA-EH-CAT (abbreviated as PCNA-CAT here) containing a 213-bp 5' flanking sequence plus intron sequences (52) and ÆCRE-PCNA-EH-CAT were gifts from Dr. Sun-Yu Ng (Institute of Molecular Medical Sciences, Palo Alto, CA). The constitutively active form of MAPK kinase 1 (MKK1), pMCL-MKK1-N3/S218E/S222D (53), was a gift from Dr. Natalie G. Ahn (University of Colorado, Boulder, CO). The active mutant of MKK3b (MKK3b(Glu189, Glu193) (54) and MKK7 were gifts from Dr. Jiahuai Han (Scripps Research Institute, La Jolla, CA). Active PI 3-kinase p110* (55) was a gift from Dr. Ruey-Hwa Chen (National Taiwan University, Taipei, Republic of China).

Nuclear extract and total cell extract

T cells were lysed in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40. The lysates were incubated on ice for 5 min and centrifuged at 1500 rpm for 5 min at 4°C. The pellet (nuclei) was suspended in hypertonic buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.3 M KCl, 0.5 mM DTT, 1 mM PMSF, 20% glycerol, and 0.4 mM EDTA and were rocked at 4°C for >30 min. The mixture was centrifuged at 13,000 rpm for 10 min. The supernatant was then mixed with 2 vol of the above buffer (without KCl) and immediately frozen. Total cell extracts were prepared by resuspending cells in hypotonic buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 100 µg/ml aprotinin, and 1.25 µg/ml leupeptin and were lysed by three cycles of freezing and thawing. One-tenth volume of 3 M KCl was then added, and the lysates were rocked at 4°C for 30 min. The same isolation procedures as those for the nuclear extract were then followed. Protein concentration was determined by Bradford assay (Bio-Rad, Richmond, CA).

Immunoblots

Cell extracts (10–30 µg) were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) for 4 h at 20 V. Membranes were washed in rinse buffer (PBS with 2% Tween 20) at room temperature for 15 min and incubated in blocking buffer (5% nonfat milk in rinse buffer) for 1.5 h. The membrane was then incubated with 0.1 µg/ml anti-phosphorylated CREB or 2 µg/ml anti-CREB. After washing, the membrane was incubated with HRP-conjugated anti-rabbit Ig Ab (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer for 1.5 h. After washing three times, membranes were developed by ECL Western blot detection reagents (Amersham, Aylesbury, U.K.). The developed membrane was detected using x-ray film and was quantitated using a densitometer (Molecular Dynamics, Sunnyvale, CA).

Transfection

EL4 cells (1.6 x 107) were washed once with STBS (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, and 0.5 mM MgCl2) and incubated with a total of 10 µg of DNA in 1.2 ml of STBS containing 0.5 mg/ml DEAE-dextran for 20 min at room temperature. One microgram of pGreen Lantern-1 (Life Technologies) was included in all transfections. EL4 cells were then treated with 15% DMSO for 3 min and washed once with STBS. The transfection efficiency was determined 24 h after transfection by counting the fraction of green fluorescence cells. The cells were activated and harvested 6–24 h after activation. The CAT activities were measured as previously described (56) and were normalized by the transfection efficiency.

Protein kinase assays

T cells were treated with immobilized anti-CD3 and anti-CD28 Ab, and cell lysates were prepared 20 min after activation. One hundred to 200 µg of lysate was precipitated with 1 µg of anti-ERK2 Ab (Santa Cruz Biotechnology; C-14) for ERK assay (57), 1 µl of anti-JNK1 Ab101 (58) for JNK assay, 1 µg of anti-p38{alpha} polyclonal Ab (59) for p38 MAPK assay, or 1 µg of anti-CaMKIV Ab (Santa Cruz Biotechnology; C-20) for CaMKIV assay followed by 20 µl of protein A-Sepharose. The kinase activity of the immune complexes was determined using myelin basic protein as substrate for the ERK assay, GST-c-Jun1–79 for the JNK assay, GST-ATF-21–109 for the p38 MAPK assay, and GST-CREB for the CaMKIV assay. The reaction mixtures were resolved on SDS-PAGE followed by autoradiography and were quantitated by PhosphorImager (Molecular Dynamics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Requirement of CD3/CD28 costimulation for CREB-CBP interaction

We used reporters pGAL-CBD and pVP16-CREB (50) to measure the interaction of CREB and CBP (Fig. 1GoA). pGAL-CBD encodes the fusion protein of Gal1–147 and the CBD of CBP (aa 461–682). The activation of pG5B-CAT upon binding of CBP to CREB was used as a readout for the assay. Stimulation through CD3 alone or CD28 alone failed to promote the binding of CREB to CBP (Fig. 1GoB). CREB-CBP interaction was detectable only upon costimulation of CD3 and CD28. The interaction was dependent on the phosphorylation of CREB Ser133. Substitution of pVP16-CREB with pVP16-CREB[S133A] abrogated the CREB-CBP binding induced by CD3/CD28 (Fig. 1GoC).



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  		FIGURE 1. Costimulation of CD3 and CD28 is required for binding of CREB to CBP. A, Interaction of CBP and CREB as determined by pGAL-CBD, pVP16-CREB, and pG5B-CAT. B, EL4 T cells were transfected with pGAL-CBD, pVP16-CREB, pG5B-CAT, and 1 µg of pGreen Lantern-1 by DEAE-dextran methods. Twenty-four hours later T cells were stimulated with immobilized anti-CD3 (2C11; 5 µg/ml) and/or anti-CD28 (38.51; 2.5 µg/ml). T cells extract were prepared after another 24 h. CAT activities were determined and normalized by the transfection efficiency assessed through green fluorescence protein expression. C, EL4 cells were treated as described in B, except that pVP16-CREB was replaced with pVP16-CREB[S133A]. The value is the mean of three measurements. SD is expressed as an error bar.

 
CD3/CD28 costimulation-promoted CREB-CBP interaction involves ERK, p38 MAPK, and CaMKIV pathways

Inhibitors for different kinases were used to assess the contribution of each kinase to CREB-CBP interaction. As illustrated in Fig. 2GoA, PD 98059 (10 µM) reduced the activation of pGAL-CBD by 50%, supporting the previous note that the ERK cascade is an essential pathway for CREB trans-activation (25). KN-62 (10 µM), the antagonist of both CaMKII and CaMKIV, was as potent as PD 98059 in reducing CREB-CBP interaction. In contrast, CaMKII-specific inhibitor KN-93 did not interfere with the binding of CREB to CBP (Fig. 2GoA). The inhibition by KN-62, but not KN-93, thus suggests the involvement of CaMKIV in CREB trans-activation. The inhibitor SB 203590 (10 µM) was found to suppress >60% of the CREB-CBP interaction. A combination of PD 98059, KN 62, and SB 203580 completely blocked CD3/CD28-promoted CREB-CBP binding (Fig. 2GoA). The specificity of PD 98059, SB 203590, and KN-62 was confirmed using kinase assays (Fig. 2GoC). PD 98059 selectively inhibited the activation ERK, but not p38 MAPK and CaMKIV, induced by CD3/CD28. Similarly, SB 203580 antagonized only 38 MAPK and KN-62 specifically suppressed CaMKIV.



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  		FIGURE 2. Involvement of ERK, p38 MAPK, and CaMKIV in CREB-CBP interaction induced by CD3/CD28. A, EL4 T cells were transfected with pGAL-CBD, CREB-VP16, pG5B-CAT, and 1 µg of pGreen Lantern-1 by DEAE-dextran method and then activated with anti-CD3 plus anti-CD28 after 24 h. Kinase inhibitors were added immediately after T cell activation. CAT activities were determined 24 h after T cell activation. The inhibitors used were PD 098059 (PD; 10 µM), KN-62 (10 µM), KN-93 (10 µM), and SB 203580 (10 µM). B, CREB-CBP interaction was not mediated by PKA, p70 S6 kinase, or PI 3-kinase. EL4 T cells were treated as described in A with the following inhibitors: LY 294002 (LY; 10 µM), H89 (1 µM), calphostin C (CC; 2 µM), or rapamycin (R; 50 ng/ml). C, EL4 T cells were pretreated with PD 098059, KN-62, SB 203580, or 0.1% DMSO (control) for 20 min. T cells were then stimulated with anti-CD3 plus anti-CD28, and the cell extracts were prepared 20 min after activation. ERK, p38 MAPK, and CaMKIV activities were determined by immunoprecipitation of the cell lysates with specific Abs. One hundred to 200 µg of lysate was precipitated with 1 µg of anti-ERK2 C-14 Ab (Santa Cruz Biotechnology), anti-p38{alpha} Ab (59 ), or anti-CaMKIV C-20 Ab (Santa Cruz Biotechnology) followed by protein A-Sepharose. The kinase activity of the immune complexes was determined by the phosphorylation of myelin basic protein (MBP) for ERK, GST-ATF-21–109 for p38 MAPK, and GST-CREB for CaMKIV, as resolved on SDS-PAGE.

 
In contrast, inhibition of PI 3-kinase, protein kinase A, protein kinase C, and p70 S6 kinase only weakly interfered with CREB-CBP interaction (Fig. 2GoB). Because PI 3-kinase is known to be activated by CD28 (35, 36, 37), and PI 3-kinase activation promotes CREB phosphorylation (21), we also examined binding with another PI 3-kinase inhibitor, wortmannin, and did not detect any inhibitory effect on CREB-CBP binding (Not shown). Therefore, CD28 costimulation-induced CREB-CBP binding is mainly dependent on the ERK, CaMKIV, and p38 MAPK pathways.

CD3/CD28-costimulated CREB trans-activation requires ERK, p38 MAPK, and CaMKIV pathways

Because CREB-CBP binding may not necessarily represent the activation of CREB (24, 60, 61), we further used CRE-CAT to assess the transcription activity of CREB (Fig. 3Go). We previously illustrated that CRE-CAT is activated by CD3 plus CD28, but not by CD3 or CD28 alone (25). The signaling requirement of the CD3/CD28 costimulation-induced CRE-CAT expression was similarly determined. The presence of PD 98059 or KN-62 reduced the activation of CRE-CAT by 35%, while SB 203580 suppressed >45% of CRE-CAT expression. The combination of PD 98059, KN-62, and SB 203580 abrogated the activation of CRE-CAT stimulated by CD3/CD28 (Fig. 3Go). ERK, p38 MAPK, and CaMKIV were similarly involved in the trans-activation of CREB induced by CD3/CD28.



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  		FIGURE 3. Involvement of ERK, p38 MAPK, and CaMKIV in CREB trans-activation. EL4 T cells were transfected with CRE-CAT and pGreen Lantern-1, and then activated with CD3/CD28 24 h later. Different inhibitors were added immediately after T cell activation. CAT activity was determined after another 24 h.

 
p38 MAPK and CaMKIV are activated by CD3 or CD28 alone and are further stimulated by CD3/CD28 costimulation

We next determined the activation of MAPKK, p38 MAPK, and CaMKIV when T cells were stimulated by CD3, CD28, or both (Fig. 4Go). ERK is activated by CD3 and is not affected by CD28 costimulation (41). Fig. 4GoA confirms that CD28 engagement did not stimulate ERK. ERK activation was identical when T cells were stimulated by CD3 or by CD3/CD28 (Fig. 4GoB). We and others have shown that p38 MAPK activity is stimulated by CD3 and is further enhanced by CD3/CD28 costimulation (43, 44). Here we observed that p38 MAPK was also activated by CD28 alone (Fig. 4GoA). CD28-stimulated p38 activation was slightly lower than that stimulated by CD3 (Fig. 4GoB). Both CD3 and CD28 ligation alone led to activation of CaMKIV to a similar extent. CaMKIV activation was further augmented by CD3/CD28 costimulation (Fig. 4Go). Of the three kinases required for CREB-CBP binding, p38 MAPK and CaMKIV were activated by both CD3 and CD28 alone.



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  		FIGURE 4. Activation of p38 MAPK and CaMKIV by CD3 or CD28 alone and synergistic activation by CD3/CD28. A, EL4 T cells were stimulated with anti-CD3, anti-CD28, or the combination of both, and the cell extracts were prepared 20 min after activation. ERK, p38 MAPK, and CaMKIV activities were determined as described in Fig. 2GoC. B, Quantitation of the results obtained from A by densitometer. Numbers are the average of at least two independent kinase assays.

 
ERK, p38 MAPK, and CaMKIV are also essential for CD3/CD28-induced CREB Ser133 phosphorylation

Ser133 phosphorylation of CREB is the primary step that leads to CBP binding (6, 7, 8, 9, 10, 11). Because the ERK, p38 MAPK, and CaMKIV pathways mediate CREB phosphorylation induced by other stimuli (13, 14, 15, 16, 17, 18, 19, 20), we further examined whether all three of these signaling pathways were involved in CREB phosphorylation induced by CD3/CD28. Stimulation of T cells by CD3 promoted the Ser133 phosphorylation of CREB (Fig. 5GoA). CD3-induced phosphorylation was partially inhibited by PD 98059 and SB 203580, and was almost abolished by KN-62 (Fig. 5GoA), suggesting that all three kinase cascades are involved in CD3-induced CREB phosphorylation. CD28 engagement also led to extensive CREB phosphorylation at Ser133 (Fig. 5GoB). Consistent with the inability of CD28 to stimulate ERK (Fig. 4Go), CD28-stimulated CREB phosphorylation was not affected by PD 98059. In contrast, CD28-induced CREB phosphorylation was suppressed by SB 203580 and KN-62 (Fig. 5GoB). We repeatedly observed that SB 203580 (10 µM) was a more effective inhibitor of CD28-initiated CREB phosphorylation than was KN-62 (10 µM). CREB Ser133 phosphorylation was increased by costimulation of CD3 and CD28 (Fig. 5GoC). CD3/CD28-induced CREB phosphorylation was equally sensitive to PD 98059, KN-62, and SB 203580, suggesting that ERK, p38 MAPK, and CaMKIV are also essential for CD3/CD28-induced CREB Ser133 phosphorylation. The synergistic effect of CD3 and CD28 in CREB phosphorylation is supported by the fact that none of the three inhibitors was as effective as in the inhibition of CREB phosphorylation triggered by sole CD3 or CD28 (Fig. 5GoC).



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  		FIGURE 5. Signal requirements for CD3/CD28-induced CREB phosphorylation were similar to those for CREB-CBP binding. Nuclear extracts were prepared 1 h after anti-CD3/anti-CD28 treatment of EL4 T cells in the presence of the inhibitors indicated. The concentrations of the inhibitors were the same as those in Fig. 2Go. Thirty micrograms of each extract was resolved by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was detected with anti-phosphorylated CREB (Upstate Biotechnology) or anti-CREB (25 ) using ECL reagents (Amersham). The doublet of CREB corresponding to CREB341 and CREB327 was previously reported (70 ). pCREB, CREB phosphorylated at Ser133.

 
Synergistic effect of ERK, p38 MAPK, and Ca2+ on CREB-CBP interaction

The observed signaling requirement for CREB-CBP binding was further examined by reconstitution experiments. ERK was activated through expression of active MKK1 (53), p38 MAPK was stimulated through expression of active MKK3b (54), and CaMKIV was activated by calcium ionophore A23187 in EL4 T cells. Kinase assay confirmed the activation of the respective kinase (Fig. 6GoA). Transfection of either active MKK1 or MKK3 alone weakly stimulated pGAL-CBD plus pVP16-CREB (Fig. 6GoB). MKK1 stimulated a 2-fold activation of pG5B-CAT, while MKK3 led to a 3.6-fold activation. CREB-CBP interaction was increased to 13.8-fold when stimulated by a combination of MKK1 and A23187. Combination of MKK3 plus A23187 further increased the binding of VP16-CREB to Gal4-CBD by 23.9-fold (Fig. 6GoB). Up to 37-fold activation of pG5B-CAT was observed when it was stimulated by a combination of MKK1, MKK3, and A23187, indicating a very strong CREB-CBP interaction. The CREB-CBP binding stimulated by reconstitution of the individual signals was similarly dependent on Ser133 phosphorylation. pG5B-CAT was no longer activated when VP16-CREB was replaced with VP16-CREB[S133A] (Fig. 6GoC). We also tested the ability of JNK and PI 3-kinase, two kinases that are activated by CD3/CD28, to promote CREB-CBP binding. JNK was activated by expression of active MKK7, as indicated by the phosphorylation of GST-c-Jun, and PI 3-kinase was activated by expression of p110* (55), as indicated by the phosphorylation of Akt (data not shown). MKK7 or p110* alone did not stimulate the interaction of CREB and CBP (Fig. 7Go). Moreover, in contrast to the effective activation of pG5B-CAT by the combination of MKK1 and A23187, the inclusion of PI 3-kinase or MKK7 in the stimulation cocktail did not add to the interaction of CREB and CBP.



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  		FIGURE 6. Synergistic effect of ERK, p38 MAPK, and CaMKIV on CREB-CBP interaction. A, Activation of ERK, p38 MAPK, or JNK by transfection with active kinase. EL4 T cells were transfected with active MKK1, active MKK3b, or active MKK7, and the activation of the respective kinase was determined 16 h later. B, EL4 T cells were transfected with pGAL-CBD, pVP16-CREB, pG5B-CAT, and pGreen Lantern-1 in the absence or the presence of the indicated kinase/reagent. CAT activities were determined 24 h after transfection. C, Same as A, except that pVP16-CREB was replaced with pVP16-CREB[S133A]. A, A23187.

 


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  		FIGURE 7. Inability of JNK and PI 3-kinase to increase CREB-CBP binding. EL4 T cells were transfected with pGAL-CBD, pVP16-CREB, pG5B-CAT, and pGreen Lantern-1 in the absence or the presence of MKK1, p110* (PI3K), MKK7, A23187, or their combinations. CAT activities were determined 24 h after transfection

 
Synergistic effect of ERK, p38 MAPK, and Ca2+ on CREB trans-activation

The synergistic effect of ERK, p38 MAPK, and CaMKIV on CRE-CAT was further examined to evaluate the trans-activation of CREB. Similar to pVP16-CREB/pGAL-CBD, the expression of CRE-CAT was weakly stimulated by transfection with MKK1 and MKK3b (Fig. 8GoA). Expression of either MKK1 or MKK3b in the presence of A23187 further increased the activation of CRE-CAT. CRE-CAT was optimally activated in the presence of MKK1, MKK3, and A123817. We also used a promoter of PCNA, in which the activation is dependent on the binding of CREB to its CRE site (62). The same synergistic activation by ERK, p38 MAPK, and CaMKIV was observed with PCNA-CAT (Fig. 8GoB). The observed activation was CREB dependent, as deletion of the CRE element in the PCNA promoter eliminated the coordinated inducibility mediated by ERK, p38 MAPK, and CaMKIV (Fig. 8GoC).



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  		FIGURE 8. Induction of CREB-dependent expression of CRE-CAT and PCNA-CAT. A, EL4 cells were transfected with CRE-CAT together with the indicated kinase/reagent as described in Fig. 6Go. Cell lysates were prepared after another 24 h, and CAT activity was determined. B, EL4 cells were transfected with PCNA-CAT (5 µg) together with the indicated kinase/reagents, and the CAT activities were determined. C, EL4 cells were transfected with ÆCRE-PCNA-CAT (5 µg) and the indicated kinase, and the CAT activities were determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we found that either TCR (CD3) alone or CD28 alone induced phosphorylation of CREB (Fig. 5Go), yet costimulation of both receptors was essential for the binding of CREB to CBP (Fig. 1Go). In addition, CD3/CD28 costimulation-induced CREB-CBP interaction involves ERK, p38 MAPK, and CaMKIV. The inhibition of any single kinase significantly prevented CREB-CBP binding (Fig. 2Go). Therefore, signaling from ERK, p38 MAPK, and CaMKIV is required for a stable CREB-CBP interaction when costimulated by CD3 and CD28. This finding was supported by a reconstitution experiment in which ERK, p38, and calcium signal acted together to promote strong CREB-CBP binding in T cells (Fig. 6GoB). Coordination of these three signals appears to be required for optimum binding of CREB. We also found that the signal requirements for CREB-CBP binding and for CRE-CAT expression were nearly identical (Fig. 3Go). This is consistent with the suggestion that CRE-mediated gene expression are activated if a stable CREB-CBP complex can be formed (63, 64).

We have further illustrated that multiple signals are required both in the CREB Ser133 phosphorylation step (Fig. 5Go) and in the CREB-CBP binding stage (Fig. 2Go) stimulated by CD3/CD28. ERK, CaMKIV, and p38 were involved in CD3-induced CREB phosphorylation, with a higher contribution from CaMKIV (Fig. 5GoA). CaMKIV and p38 were participated in CD28-triggered phosphorylation, with a slightly increased contribution from p38 (Fig. 5GoB). Costimulation of CD3 and CD28 led to a synergistically elevated CREB phosphorylation that was almost equally dependent on ERK, CaMKIV, and p38 (Fig. 5GoC). Inhibition of ERK, CaMKIV, and p38 each led to a >50% decrease in CREB Ser133 phosphorylation, which is well correlated with the extent of suppression observed on CREB-CBP binding by PD 98059, KN-62, or SB 203580 (Fig. 2GoA). A full CREB Ser133 phosphorylation and an optimum CREB-CBP interaction in T cells display a similar dependence on simultaneous activation of ERK, p38, and CaMKIV.

However, CREB Ser133 phosphorylation was induced by either anti-CD3 or anti-CD28 alone (Fig. 5Go), yet no CREB-CBP interaction was detectable in EL4 cells (Fig. 1Go). Recent studies have suggested that the trans-activation of CREB may involve multiple stages. Our results are identical with the report that anti-CD3 stimulates CREB phosphorylation, but not CRE-CAT in Jurkat cells (24). CREB also remains inactive even though Ser133 phosphorylation is induced by Ca2+ (60). The activation of CREB needs an additional signal (24, 60). Therefore, despite the early illustration that the phosphorylation event is directly coupled to CREB transcriptional activation (65), phosphorylation of CREB at Ser133 is not sufficient to activate CRE-dependent gene expression (24, 60, 66). It has been suggested that CREB-CBP interaction is uncoupled from CBP activation. The second signal is to activate CBP or other cofactors for assembly of complete transcriptional complexes or to dissociate a hypothetical CBP inhibitor (24, 60). The multiple signal requirement for CREB activation may be partly explained by the fact that different signals are required to phosphorylate CREB and to activate CBP. Alternatively, the possible involvement of Ser133-independent mechanisms (61) in CD3/CD28-mediated CREB activation needs to be elucidated. Efforts are currently devoted to dissect these events.

CaMKIV is supposed to be an exception to the above multisignal scenario for CREB activation. CaMKIV alone not only phosphorylates CREB, but also activates CBP (61, 66), suggesting that CaMKIV alone should be sufficient to phosphorylate CREB and to promote CREB-CBP binding. This is in contrast to our observations that CD3/CD28 activated CaMKIV (Fig. 4GoA), yet the CD3/CD28-induced CREB-CBP interaction still required signaling from both ERK and p38 (Fig. 2Go). We speculate that there are two possible causes for this seeming discrepancy. First, the CaMKIV activated by CD3/CD28 may not reach a threshold for full phosphorylation of CREB and activation of CBP. It has been shown that a low concentration of glutamate induces CREB phosphorylation in neurons, but CREB trans-activation occurs only when CBP is also activated by a high concentration of glutamate (65). Under such circumstances, activation signals from the ERK and p38 MAPK pathways are required for sufficient phosphorylation of CREB and formation of an active transcription complex. Second, kinases such as CaMKIV are regulated by tightly associated phosphatases, such that inactivation of CaMKIV occurs in the presence of sustained increases in Ca2+ (67). Signals from ERK or p38 MAPK may help maintain the activation level of CaMKIV through antagonism of the specific phosphatases. Similar reasons may explain why ERK or p38 cascade alone is insufficient to activate CREB in T cells stimulated through CD3/CD28, even though a weak CREB-CBP binding (2- to 3-fold) was detected when MKK1 or MKK3 was overexpressed (Figs. 6Go and 7Go).

Both CBP binding and activation of CREB by CD3/CD28 costimulation were insensitive to LY 294002 and wortmannin, inhibitors of PI 3-kinase. In addition, PI 3-kinase, by itself or in combination with MKK1/A23187, did not contribute to CREB-CBP binding (Fig. 7Go). Even though PI 3-kinase is activated by CD28 ligation (33, 34), and PI 3-kinase activates Akt, which has been shown to phosphorylate CREB (21), our results suggest that CD3/CD28 costimulation of CREB is mediated by a PI 3-kinase-independent pathway. Similarly, JNK, another kinase activated by CD3/CD28 costimulation, was not able to stimulate CREB-CBP interaction (Fig. 7Go). These results clearly illustrate that not all CD28-activated signals are involved in CREB trans-activation.

We have also found, probably for the first time, that CD28 engagement alone stimulates CaMKIV and p38 MAPK in T cells (Fig. 4Go). CD28 costimulatory signal has been defined mostly for its convergence with TCR signals, for example, on the activation of JNK and I{kappa}B kinase (41, 43). Few activation events stimulated by CD28 ligation in the absence of CD3 engagement have been reported (68, 69). Together with the activation of PI 3-K (35), the present study supports the idea that CD28 engagement independently activates selective kinase pathways in T cells. Our results thus suggest that CD28 contributes to CREB activation in a quantitative manner rather than in a qualitative manner. CD3 alone stimulates ERK, p38 MAPK, and CaMKIV, but CD3 alone does not promote CREB-CBP binding (Fig. 4Go). The signals activated by CD28 costimulation that lead to CREB-CBP interaction are p38 MAPK and CaMKIV, kinases that are also activated by CD3. Therefore, CD28 costimulation acts by synergistic enhancement of activation signals from CD3. In such a model, signals from CD3 engagement alone do not reach a threshold sufficient to activate transcription factor such as CREB. Only through the augmentation from CD28 costimulation are T cell activation signals sufficient to trigger CREB-CBP binding. We are in the progress of determining whether other T cell activation events induced by CD3/CD28 costimulation may be mediated by a mechanism similar to the one that activates CREB.


   Acknowledgments
 
We thank Drs. Daniel Olive and Jean Imbert for helpful discussion, Dr. Nataline Ahn for active MKK1, Dr. Jiahuai Han for active MKK3b and MKK7, Dr. Sun-Yu Ng for PCNA-CAT, Dr. Michael Karin for HA-JNK1, Dr. Melanine Cobb for ERK2, Dr. Ruey-Hwa Chen for p110*, Dr. Tse-Hua Tam for anti-JNK1 Ab, and Dr. Mark Ptashne for pG5B-CAT. We also thank Douglas Platt for the editorial correction.


   Footnotes
 
1 This work was supported by Grant 89-2311-B001-109 from the National Science Council and a grant from Academia Sinica, Taiwan, Republic of China. Back

2 Address correspondence and reprint requests to Dr. Ming-Zong Lai, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China. Back

3 Abbreviations used in this paper: CREB, cAMP-responsive element binding protein; CaMK, calcium/calmodulin-dependent protein kinase; CAT, chloramphenicol acetyltransferase; CRE, cAMP-responsive element; CBP, CREB binding protein; CBD, CREB-binding domain of CBP; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; PCNA, proliferating cell nuclear Ag; PI 3-kinase, phosphatidylinositol 3-kinase; RSK, ribosomal S6 kinase; STBS, 25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, and 0.5 mM MgCl2. Back

Received for publication July 21, 2000.
   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Montiminy, M.. 1997. Transcriptional regulation by cyclic AMP. Annu. Rev. Biochem. 66:807.[Medline]
  2. De Cesare, D., G. M. Fimia, P. Sassone-Corsi. 1999. Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem. Sci. 24:281.[Medline]
  3. Shaywitz, A. J., M. E. Greenberg. 1999. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68:821.[Medline]
  4. Hoeffler, J. P., T. E. Meyer, Y. Yun, 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]
  5. Yamamoto, K. K., G. A. Gonzalez, III W. H. Biggs, M. R. Montminy. 1988. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334:494.[Medline]
  6. Hagiwara, M., P. Brindle, A. Harootunian, R. Armstrong, J. Rivier, W. Vale, R. Tsien, M. R. Montiminy. 1993. Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol. Cell. Biol. 13:4852.[Abstract/Free Full Text]
  7. Gonzalez, G. A., M. R. Montminy. 1989. Cyclic AMP-regulated stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675.[Medline]
  8. Chrivia, J. C., R. P. S. Kwok, N. Lamb, M. Hagiwara, M. R. Montminy, R. H. Goodman. 1993. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855.[Medline]
  9. Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G. Brennan, S. G. E. Roberts, M. R. Green, R. H. Goodman. 1994. Nuclear factor CBP is a coactivator for the transcription factor CREB. Nature 370:223.[Medline]
  10. Mestas, S. P., K. J. Lumb. 1999. Electrostatic contribution of phosphorylation to the stability of the CREB-CBP activator-coactivator complex. Nat. Struct. Biol. 6:613.[Medline]
  11. Radhakrishnan, I., G. C. Perez-Alvarado, D. Parker, H. J. Dyson, M. R. Montminy, P. E. Wright. 1999. Structural analyses of CREB-CBP transcriptional activator-coactivator complexes by NMR spectroscopy: implications for mapping the boundaries of structural domains. J. Mol. Biol. 287:859.[Medline]
  12. Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smeal, M. Karin, J. Feramisco, M. Montminy. 1994. Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370:226.[Medline]
  13. Sun, P., H. Enslen, P. S. Myung, R. A. Maurer. 1994. Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. Genes Dev. 8:2527.[Abstract/Free Full Text]
  14. Matthews, R. P., C. R. Guthrie, L. M. Wailes, X. Zhao, A. R. Means, G. S. Knight. 1994. Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression. Mol. Cell. Biol. 14:6107.[Abstract/Free Full Text]
  15. Bito, H., K. Deisseroth, R. W. Tsien. 1996. CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87:1203.[Medline]
  16. 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]
  17. Xing, J., J. M. Kornhauser, Z. Xia, E. A. Thiele, M. E. Greenberg. 1998. Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation. Mol. Cell. Biol. 18:1946.[Abstract/Free Full Text]
  18. Tan, Y., J. Rouse, A. Zhang, S. Cariati, P. Cohen, M. J. Comb. 1996. FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. EMBO J. 15:4629.[Medline]
  19. Deak, M., A. D. Clifton, L. M. Lucocq, D. R. Alessi. 1998. Mitogen- and stress-activated protein kinase-1 (MSK-1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 17:4426.[Medline]
  20. Pierrat, B., J. S. Correia, J. L. Mary, M. Tomas-Zuber, W. Lesslauer. 1998. RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38{alpha} mitogen-activated protein kinase (p38{alpha} MAPK). J. Biol. Chem. 273:29661.[Abstract/Free Full Text]
  21. Du, K., M. Montminy. 1998. CREB is a regulatory target for the protein kinase Akt/PKB. J. Biol. Chem. 273:32377.[Abstract/Free Full Text]
  22. de Groot, R. P., J. den Hertog, J. R. Vandenheede, J. Goris, P. Sassone-Corsi. 1993. Multiple and cooperative phosphorylation events regulate the CREM activator function. EMBO J. 12:3903.[Medline]
  23. de Groot, R. P., L. M. Ballow, 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]
  24. 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]
  25. Hsueh, Y.-P., H.-E. Liang, S.-Y. Ng, M.-Z. Lai. 1997. CD28-costimulation activates cyclic AMP-responsive element binding protein in T lymphocytes. J. Immunol. 158:85.[Abstract]
  26. Barton, K., N. Muthusamy, M. Chanyanggam, C. Fischer, C. Cledenin, J. M. Leiden. 1996. Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB. Nature 379:81.[Medline]
  27. Anderson, K. A., T. J. Ribar, M. Illario, A. R. Means. 1997. Defective survival and activation of thymocytes in transgenic mice expressing a catalytic inactive form of Ca2+/calmodulin-dependent protein kinase IV. Mol. Endocrinol. 11:725.[Abstract/Free Full Text]
  28. Rudolph, D., A. Tafuri, P. Gass, G. J. Hammerling, B. Arnold, G. Schutz. 1998. Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein. Proc. Natl. Acad. Sci. USA 95:4481.[Abstract/Free Full Text]
  29. Belkowski, S. M., C. S. Rubin, M. B. Prystowsky. 1998. Isolation and analysis of a T cell clone variant exhibiting constitutively phosphorylated Ser133 cAMP responsive element-binding protein. J. Immunol. 161:659.[Abstract/Free Full Text]
  30. Zhang, F., D.-Z. Wang, M. Boothby, L. Penix, R. A. Flavell, T. M. Aune. 1998. Regulation of the activity of IFN-{gamma} promoter elements during Th cell differentiation. J. Immunol. 161:6105.[Abstract/Free Full Text]
  31. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14:233.[Medline]
  32. Chambers, C. A., J. P. Allison. 1999. Costimulatory regulation of T cell function. Curr. Opin. Cell Biol. 11:203.[Medline]
  33. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison. 1992. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature 356:607.[Medline]
  34. Boise, L. H., A. J. Minn, P. J. Noel, C. H. June, M. A. Accavitti, T. Lindsten, C. B. Thompson. 1995. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3:87.[Medline]
  35. Pages, F., M. Ragueneau, R. Rottapel, A. Truneh, J. Nunes, J. Imbert, D. Olive. 1994. Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling. Nature 369:327.[Medline]
  36. Truitt, K. E., C. M. Hicks, J. B. Imboden. 1994. Stimulation of CD28 triggers an association between CD28 and phosphatidylinositol 3-kinase in Jurkat T cells. J. Exp. Med. 179:1071.[Abstract/Free Full Text]
  37. Prasad, K. V. S., Y.-C. Cai, M. Raab, B. Duckworth, L. Cantely, S. E. Shoelson, C. E. Rudd. 1994. T cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proc. Natl. Acad. Sci. USA 91:2834.[Abstract/Free Full Text]
  38. Hutchcroft, J. E., D. P. Franklin, B. Tsai, D. Harrison-Findik, L. Varticovski, B. E. Bieber. 1995. Phorbol ester treatment inhibits phosphatidylinositol 3-kinase activation by, and association with, CD28, a T-lymphocyte surface receptor. Proc. Natl. Acad. Sci. USA 92:8808.[Abstract/Free Full Text]
  39. Crooks, M. E. C., D. R. Littman, R. H. Carter, D. T. Fearson, A. Weiss, P. H. Stein. 1995. CD28-mediated costimulation in the absence of phosphatidylinositol 3-kinase association and activation. Mol. Cell. Biol. 15:6820.[Abstract]
  40. Truitt, K. E., J. Shi, S. Gibson, L. G. Segal, G. B. Mills, J. B. Imboden. 1995. CD28 delivers costimulatory signals independently of its association with phosphatidylinositol 3-kinase. J. Immunol. 155:4702.[Abstract]
  41. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, Y. Ben-Nerian. 1994. JNK is involved in the signal integration during costimulation of T lymphocytes. Cell 77:727.[Medline]
  42. Barz, C., T. Nagel, K. E. Truitt, J. B. Imboden. 1998. Mutational analysis of CD28-mediated costimulation of Jun-N-terminal kinase and IL-2 production. J. Immunol. 161:5366.[Abstract/Free Full Text]
  43. Harhaj, E. W., S.-C. Sun. 1998. I{kappa}B kinases serve as a target of CD28 signaling. J. Biol. Chem. 273:25185.[Abstract/Free Full Text]
  44. Matsuda, S., T. Moriguchi, S. Koyasu, E. Nishida. 1998. T lymphocyte activation signal for interleukin-2 production involve activation of MKK6–p38 and MKK7-SAPK-JNK signaling pathways sensitive to cyclosporin A. J. Biol. Chem. 273:12378.[Abstract/Free Full Text]
  45. Hsu, S.-C., M. Gavrilin, M.-H. Tsai, J. Han, M.-Z. Lai. 1999. p38 mitogen activated protein kinase is involved in Fas ligand expression. J. Biol. Chem. 274:25769.[Abstract/Free Full Text]
  46. Zhang, J., K. V. Salojin, J.-X. Gao, M. J. Cameron, I. Bergerot, T. Delovitch. 1999. p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. J. Immunol. 162:3819.[Abstract/Free Full Text]
  47. Li, L., C. Yee, J. A. Beavo. 1999. CD3- and CD28-dependent induction of PDE7 required for T cell activation. Science 283:848.[Abstract/Free Full Text]
  48. Hsueh, Y.-P., M.-Z. Lai. 1995. Overexpression of activation transcriptional factor 1 in lymphomas and in activated lymphocytes. J. Immunol. 154:5675.[Abstract]
  49. Gross, J. A., E. Callas, J. P. Allison. 1992. Identification and distribution of the costimulatory receptor CD28 in the mouse. J. Immunol. 149:380.[Abstract]
  50. Shih, H., P. Goldman, A. J. DeMaggio, S. M. Hollenberg, R. H. Goodman, M. F. Hoesktra. 1996. A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with coactivator CBP. Proc. Natl. Acad. Sci. USA 93:13896.[Abstract/Free Full Text]
  51. Sadowski, I., J. Ma, S. Triezenberg, M. Ptashne. 1988. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335:563.[Medline]
  52. Lee, H.-H., W.-H. Chiang, S.-H. Chaing, Y.-C. Liu, J. Huang, S.-Y. Ng. 1995. Regulation of cyclin D1, DNA topoisomerase I, and proliferating cell nuclear antigen promoters during the cell cycle. Gene Expression 4:95.[Medline]
  53. Mansour, S. J., W. T. Matten, A. S. Hermann, J. M. Candia, S. Rong, K. Fukasawa, G. F. Vande Woude, N. G. Ahn. 1994. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966.[Abstract/Free Full Text]
  54. Jiang, Y., C. Chen, Z. Li, W. Guo, J. A. Gegner, S. Lin, J. Han. 1996. Characterization of the structure and function of a new mitogen-activated protein kinase (p38{beta}). J. Biol. Chem. 271:17920.[Abstract/Free Full Text]
  55. Klippel, A., C. Reinhard, W. M. Kavanaugh, G. Apell, M.-A. Escobedo, L. T. Williams. 1996. Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transduction kinase pathways. Mol. Cell. Biol. 16:4117.[Abstract]
  56. Ho, H.-Y., H.-H. Lee, M.-Z. Lai. 1997. Overexpression of mitogen-activated protein kinase kinase kinase reversed cAMP inhibition of NF-{kappa}B in T cells. Eur. J. Immunol. 27:222.[Medline]
  57. Hsueh, Y.-P., M.-Z. Lai. 1995. JNK but not MAP kinase is sensitive to cAMP in T lymphocytes. J. Biol. Chem. 270:18094.[Abstract/Free Full Text]
  58. Meyer, C. F., X. Wang, C. Chang, D. Templeton, T.-H. Tan. 1996. Interaction between c-Rel and the mitogen-activated protein kinase kinase kinase 1 signaling cascade in mediating B enhancer activation. J. Biol. Chem. 271:8971.[Abstract/Free Full Text]
  59. Jiang, Y., H. Gram, M. Zhao, L. New, J. Gu, L. Feng, F. D. Padova, R. J. Ulevitch, J. Han. 1997. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38{delta}. J. Biol. Chem. 272:30122.[Abstract/Free Full Text]
  60. Thompson, M. A., D. D. Ginty, A. Bonni, M. E. Greenberg. 1995. L-type voltage-sensitive Ca channel activation regulates c-fos transcription at multiple level. J. Biol. Chem. 270:4224.[Abstract/Free Full Text]
  61. Chawla, S., G. E. Hardingham, D. R. Quinn, H. Bading. 1998. CBP: a signal-regulated transcriptional coactivator controlled by nuclear calcium and CaM kinase IV. Science 281:1505.[Abstract/Free Full Text]
  62. Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrich, 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]
  63. Cardinaux, J.-R., J. C. Notis, O. Zhang, N. Vo, J. C. Craig, D. M. Fass, R. G. Brennan, R. H. Goodman. 2000. Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation. Mol. Cell. Biol. 20:1546.[Abstract/Free Full Text]
  64. Goodman, R. H., S. Smolik. 2000. CBP/p300 in cell growth, transformation, and development. Genes Dev. 14:1553.[Free Full Text]
  65. Alberts, A. S., J. Arias, M. Hagiwara, M. R. Montminy, J. R. Feramisco. 1994. Recombinant cyclic AMP response element binding protein (CREB) phosphorylated on Ser-133 is transcriptionally active upon its introduction into fibroblast nuclei. J. Biol. Chem. 269:7623.[Abstract/Free Full Text]
  66. Hu, S.-C., J. Chrivia, A. Ghosh. 1999. Regulation of CBP-mediated transcription by neuronal calcium signaling. Neuron 22:799.[Medline]
  67. Westphal, R. S., K. A. Anderson, A. R. Means, B. E. Wadzinski. 1998. A signal complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A. Science 280:1258.[Abstract/Free Full Text]
  68. Ledbetter, J. A., J. B. Imboden, G. L. Schieven, L. S. Grosmaire, P. S. Rabinovitch, T. Lindsten, C. B. Thompson, C. H. June. 1990. CD28 ligation in T-cell activation: evidence for two signal transduction pathways. Blood 75:1531.[Abstract/Free Full Text]
  69. Siefken, R., S. Klein-He{beta}ling, E. Serfling, R. Kurrle, R. Schwinzer. 1998. A CD28-associated signaling pathway leading to cytokine gene transcription and T cell proliferation without TCR engagement. J. Immunol. 161:1645.[Abstract/Free Full Text]
  70. Berkowtiz, L. A., M. Z. Gilman. 1990. Two distinct forms of active transcriptional factor CREB (cAMP responsive element binding protein). Proc. Natl. Acad. Sci. USA 87:5258.[Abstract/Free Full Text]



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