The cAMP signalling pathway activates CREB through PKA, p38 and MSK1 in NIH 3T3 cells

doi:10.1016/j.cellsig.2005.02.003

Cellular Signalling

Volume 17, Issue 11, November 2005, Pages 1343-1351

https://doi.org/10.1016/j.cellsig.2005.02.003 Get rights and content

Abstract

Cyclic adenosine 3′,5′-monophosphate (cAMP) was originally shown to induce gene transcription through activation of cAMP-dependent protein kinase (PKA), and subsequent phosphorylation of the transcription factor cAMP response element-binding protein, CREB, at serine-133. However, elevated cAMP levels may activate multiple signalling pathways with protein kinases that can phosphorylate CREB at serine-133. We analysed the pathways involved in CREB phosphorylation and activation in NIH 3T3 cells exposed to the cAMP elevating agent forskolin. PKA represented the predominant pathway during the burst phase, while the mitogen-activated protein kinase p38 pathway became activated in a PKA-dependent fashion in forskolin treated cells. The phosphorylation kinetics of p38 was delayed compared to PKA activation. Activated p38 stimulated CREB-mediated transcription and potentiated the transcriptional strength of CREB provoked by forskolin. The p38-mediated activation of CREB was inhibited by dominant negative mutants of MSK-1 and by the PKA/MSK-1 inhibitor H89, but not by dominant negative mutants of MSK-2/RSK-B and MAPKAPK2. Our results suggest that forskolin-induced CREB phosphorylation and activation in NIH 3T3 cells is mediated directly by PKA and by a time-delayed PKA-dependent p38/MSK-1 pathway. This bifurcation and time-dependent regulation of the cAMP-responsive signalling pathways may enable the cell to endure and/or enforce a cellular response provoked by a cAMP-elevating stimulus.

Introduction

It is generally accepted that cyclic adenosine 3′,5′-monophosphate (cAMP)-induced gene transcription is mediated through activation of cAMP-dependent protein kinase or protein kinase A (PKA), and subsequent phosphorylation of the transcription factor cAMP response element-binding protein, CREB. CREB binds the conserved cAMP response element TGACGTCA typically found in the promoter of many cAMP-responsive genes. The bipartite transactivator domain of CREB is involved in basal and signal-inducible transcription activation by CREB. The glutamine-rich, constitutive activation domain (Q2 or CAD) interacts with the general transcription factor TAF4 (formerly known as TAF_II130/135) and allows recruitment of the RNA polymerase II initiation complex. The activity of the kinase-inducible domain (KID) of CREB's transactivation domain was found to be regulated by PKA, thus forming the missing link in a linear signalling cAMP–PKA–CREB pathway. It was shown that serine residue 133 in KID was a unique phosphoacceptor site for PKA in vitro and in vivo, and that phosphorylation of CREB at this residue promotes recruitment of the general co-activator CBP and its paralogue p300. CBP and p300 stimulate CREB-mediated transcription through their interaction with the RNA polymerase II complex and through their intrinsic histon acetyltransferase activity [1], [2], [3], [4].

Although correct, cAMP/PKA-dependent phosphorylation of CREB at Ser-133 is not the only mechanism that activates CREB-mediated transcription. Ramification of the cAMP pathway contributes to the diversity of signalling pathways that can modulate CREB activity. Indeed, it was originally believed that PKA was the only target for cAMP. However, cAMP was also found to bind and activate the guanine nucleotide-exchange factors Epac1 and Epac2. These proteins can activate the GTPase Rap1, which in turn binds B-Raf. cAMP-induced activation of Rap1 through Epac is clearly independent of PKA [5], [6]. The B-Raf–Rap1 complex has been shown to promote activation of the mitogen-activated protein kinases (MAPK) ERK1/2 in different cells and to regulate CREB-dependent gene expression [7], [8], [9], [10]. ERK1/2 can activate mitogen-activated protein kinase activated protein kinases. Some of these, including mitogen and stress-activated kinase 1 (MSK1), RSK2 (also called MAPKAPK1b), and MSK2 (= RSK-B) have been shown to phosphorylate CREB at serine 133 [11], [12], [13]. Elevated cAMP levels can also activate the p38 and the MEK5/ERK5 MAPK pathways and CREB has been shown to be a substrate for the p38-regulated MAPKAPK2 and MAPKAPK3 [14], [15], [16]. Although ERK5 has been shown to be involved in neurotrophin-induced CREB phosphorylation [17], a role for MEK5/ERK5 in cAMP-induced CREB phosphorylation is missing. Serine 133 turned out to be a target for protein kinase B (PKB), but the exact signalling pathway that leads to PKB-induced CREB phosphorylation remains unclear [18]. Interestingly, cAMP activates PKB through a PI3-K dependent mechanism in a cell-type-specific manner [19], [20], [21], opening for an alternative pathway for cAMP-induced CREB phosphorylation. Another target for cAMP are the cyclic-nucleotide-gated ion channels [22]. They conduct the release of Ca²⁺, which may result in stimulation of calmodulin-dependent protein kinases (CaMK) and conventional protein kinases C (PKC). Both CaMK and PKC can modulate CREB phosphorylation at ser-133 and enhance CREB-mediated transcription, although the exact role of these kinases in CREB phosphorylation remains elusive [1].

The adenylyl cyclase activator forskolin is an extremely valuable tool to study cellular processes in response to increased intracellular cAMP levels [23]. Hence, forskolin has often been employed in studies with CREB. Because of the ramification of the cAMP signalling pathway, we wanted to identity which of the cAMP transduction pathways that could mediate forskolin-induced CREB phosphorylation in NIH 3T3 cells. Our results show that cAMP/PKA is the major pathway explored in forskolin-treated NIH 3T3 cells, but that PKA may also activate p38, which in turn can phosphorylate and activate CREB through the MAPKAPK MSK1. The PKA-induced activation of the p38-MSK1 is delayed compared to the cAMP–PKA activation, suggesting that the former pathway may be used when the cAMP–PKA pathway is attenuated. The cross talk between PKA and p38 may allow the cells to prolong the effect of an incoming signal.

Section snippets

Reagents

Forskolin was purchased from Sigma (MO, USA), and newborn Calf Serum (NCS) was from Bio Whittaker (Verviers, Belgium). Cell culture medium was obtained from Gibco/BRL (MD, USA). CDP-Star and MagicMark Western Standard were from Applied Biosystems (CA, USA) and Invitrogen (Oslo, Norway), respectively. Antibodies against CREB, phospho-CREB, p38 MAPK, phospho-p38 MAPK, and the monoclonal antibody phospho-p44/42 MAPK were all obtained from Cell Signaling Technology (CA, USA). Luciferase assay kit

Specific inhibition of cAMP-regulated signalling pathways indicates that PKA is the major pathway that mediates forskolin-induced CREB phosphorylation in NIH 3T3 cells

The adenylate cyclase activator forskolin (FSK) is generally applied as a cAMP-elevating agent to induce cAMP-dependent protein kinase or PKA [23]. However, cAMP has been demonstrated to activate also the MAP kinase p38, MEK1/2-ERK1/2 and MEK5-ERK5 modules, and the PI3K-PKB/Akt1 pathway [32], [33], [34], [35]. Moreover, cAMP-induced release of Ca²⁺ from cyclic-nucleotide-gated ion channels may stimulate CaMK and conventional PKC [36], [37]. We first set out to determine which signalling

Discussion

The adenylate cyclase activator forskolin is commonly used to increase intracellular cAMP levels and is generally applied as a specific activator of the PKA pathway [23]. However, other pathways such as the PI3K→PKB, the Epac→B-Raf→MEK1/2→ERK1/2, the MEK5→ERK5, the c-Raf→MEK1/2→ERK1/2, and the p38 pathways can also be induced by cAMP in a cell-specific fashion [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [32], [33], [34], [35]. Moreover,

Conclusions

The second messenger cAMP can activate multiple signalling pathways that can converge to CREB. The aim of this study was to identify the cAMP signalling pathways involved in forskolin-induced CREB activation in NIH3T3 cells. Our findings, which are summarized in Fig. 7, demonstrate that PKA is the major CREB kinase exploited by NIH 3T3 cells after forskolin treatment. Forskolin also induces phosphorylation of p38 in a PKA-dependent way, and the forskolin-induced p38 phosphorylation kinetics is

Acknowledgements

The authors thank Dr. R.A. Maurer, Dr. R.J. Davis, Dr. M. Deak, Dr. T. Jahnsen, Dr. K. Taskén, Dr. A. Brunet, Dr. D. Zechner, and Dr. E. Sontag for kindly providing plasmids used in this study.

This work was supported by grants from the Norwegian Cancer Society (Kreftforeningen, project A01037/004), the Norwegian Research Council (NFR projects S5168 and S5228), and the Olav and Aakre foundation (A5048).

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The cAMP signalling pathway activates CREB through PKA, p38 and MSK1 in NIH 3T3 cells

Abstract

Introduction

Section snippets

Reagents

Specific inhibition of cAMP-regulated signalling pathways indicates that PKA is the major pathway that mediates forskolin-induced CREB phosphorylation in NIH 3T3 cells

Discussion

Conclusions

Acknowledgements

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