The mitogen-activated protein kinase cascade couples PKA and PKC to cAMP response element binding protein phosphorylation in area CA1 of hippocampus

Abstract

Activation of the mitogen-activated protein kinase (MAPK) cascade recently was discovered to play an important role in synaptic plasticity in area CA1 of rat hippocampus. However, the upstream mechanisms regulating MAPK activity and the downstream effectors of MAPK in the hippocampus are uncharacterized. In the present studies we observed that hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, we found that a wide variety of neuromodulatory neurotransmitter receptors (metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, and beta-adrenergic receptors) couple to MAPK activation via these two cascades. In additional studies we observed that PKC is a powerful regulator of CREB phosphorylation in area CA1. MAPK plays a critical role in transcriptional regulation by PKC, because MAPK activation is a necessary component for increased CREB phosphorylation in response to the activation of this kinase. Surprisingly, we also observed that MAPK activation is necessary for PKA coupling to CREB phosphorylation in area CA1. Overall, these studies indicate an unexpected richness of diversity in the regulation of MAPK in the hippocampus and suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of hippocampal synaptic plasticity.

Fig. 1.

MAP kinase signaling via Raf-1 and B-Raf. In the most well studied pathway for Raf-1 activation (left pathway), growth factor receptors, via the adapter protein Grb2, activate the guanine nucleotide exchange factor Sos and in turn the small GTP-binding protein, Ras; recruitment of Raf-1 to the plasma membrane by Ras leads to its activation. This pathway also is activated by Ca²⁺ influx in PC12 cells and cortical neurons (Rosen et al., 1994). PKC also activates this pathway, although it is unclear whether it activates Raf-1 directly or acts indirectly via Ras. Activation of this Raf-1-dependent pathway by either growth factor receptors or PKC is regulated negatively by PKA. One mechanism by which PKA exerts its effect is via phosphorylation of Ser⁴³ in Raf-1, which impairs its interaction with Ras, preventing its activation. In addition, PKA directly inhibits Raf-1 activity by the phosphorylation of its catalytic domain. The cAMP cascade also can activate p42 MAPK and p44 MAPK in PC12 cells and other cell types, but it has been demonstrated that, even in these cells, its effect on Raf-1 is inhibitory. The capacity for the cyclic AMP cascade to stimulate MAP kinase activity (right pathway) correlates with the expression of a tissue-specific Raf isoform, B-Raf. B-Raf does not contain the Raf-1 Ser⁴³ phosphorylation site, suggesting one reason why it may be resistant to inhibition by PKA. B-Raf expression, however, is not sufficient to confer the potential for PKA-stimulated p42 MAPK and p44 MAPK activation; the small GTP-binding protein, Rap-1, is required also. Rap-1 is a Ras homolog that, like Ras, can activate B-Raf. PKA phosphorylates Rap-1 at Ser¹⁷⁹ and leads to its activation. Thus, in cells expressing Rap-1 and B-Raf, PKA leads to the activation of MEK and its substrate MAP kinases via a pathway independent of Ras and Raf-1.

Fig. 2.

PKA coupling to p42 MAPK in area CA1.A, Representative ERK MAPK Western blots of area CA1 subregions from control slices (CTL) and slices treated with forskolin (FSK; 50 μm with 100 μm Ro20-1724 for 10 min). The α-ERK antiserum detects protein levels of p44 MAPK (ERK1) and p42 MAPK (ERK2), demonstrating equal protein loading. FSK treatment resulted in a selective increase in the tyrosine phosphorylation of a band that comigrates with p42 MAPK (anti-phosphotyrosine Western; APT) and a selective increase in p42 MAPK immunoreactivity to two different antisera that detect phosphorylated, activated MAPK (α-pY-ERK and α-dual-P-ERK). These Western blots thus provide three independent lines of evidence that FSK treatment leads to p42 MAPK activation in area CA1.B, Representative APT Western blots of p42 MAPK from control slices (CTL), slices treated with forskolin (FSK), the inactive forskolin analog dideoxyforskolin (ddFSK; 50 μm), or forskolin plus the MEK inhibitors PD 098059 (PD; 50 μm + FSK) or U0126 (20 μm + FSK). Note the diminished basal level of MAPK phosphorylation in the presence of the MEK inhibitors. C, Summary p42 MAPK APT immunoreactivity data: FSK, 236 ± 22% of control,n = 23 (p < 0.0001);ddFSK, 112 ± 6%, n = 7;FSK + PD, 162 ± 15%, n = 13;FSK + U0126, 112 ± 11%, n = 16. *Statistical significance. Error bars in this and all subsequent figures are ±SEM.

Fig. 3.

Dopamine or isoproterenol application to hippocampal slices elicits p42 MAPK activation in area CA1.A, Representative anti-phosphotyrosine Western blots of p42 MAPK in area CA1 subregions from control slices (CTL) and slices treated with dopamine (DA) or isoproterenol (ISO).B, Summary data of p42 MAPK phosphotyrosine immunoreactivity. DA, Dopamine (50 μm, 10 min, 262 ± 50%; n = 19;p < 0.0001); DA + SCH, SCH23390 (DA receptor antagonist) + DA (133 ± 12%; n = 17); DA + H89, H89 (PKA inhibitor) + DA (105 ± 20%; n = 5); DA + PD, DA + PD098059 (50 μm, 112 ± 28%; n = 5);DA + U0126, DA applied in the presence of U0126 (119 ± 20%; n = 4). ISO, Isoproterenol (10 μm, 10 min, 188 ± 33%;n = 9; p < 0.05); ISO + TIM, timolol (2 μm, βAR receptor antagonist) + ISO (97 ± 19%; n = 4); ISO + H89, H89 (PKA inhibitor) + ISO (125 ± 6%;n = 3); ISO + PD, ISO + PD098059 (50 μm, 113 ± 9%; n = 3).

Fig. 4.

Stimulation of PKC leads to p42 MAPK activation in area CA1. A, Representative ERK MAPK Western blots of area CA1 subregions from control slices (CTL) and slices treated with the PKC activator phorbol diacetate (PDA). The α-ERK antiserum detects protein levels of p44 MAPK (ERK1) and p42 MAPK (ERK2), demonstrating equal protein loading. As with FSK, PDA treatment resulted in a selective increase in the tyrosine phosphorylation of a band that comigrates with p42 MAPK (anti-phosphotyrosine Western; APT) and a selective increase in p42 MAPK immunoreactivity to two different antisera that detect phosphorylated, activated MAPK (α-pY-ERK and α-dual-P-ERK). These Western blots thus provide three independent lines of evidence that PDA treatment leads to p42 MAPK activation in area CA1.B, Representative p42 MAPK anti-phosphotyrosine Western blots in area CA1 subregions from control (CTL) slices and slices treated with either phorbol diacetate (PDA) or the inactive analog 4-α-phorbol (4αP). Representative blots demonstrating the effect of the MEK inhibitors PD098059 (PD) and U0126 are shown below.C, Summary data of p42 MAPK phosphotyrosine immunoreactivity. PDA application produced a significant increase in p42 MAPK phosphorylation (646 ± 101% of control;n = 12; p < 0.001), whereas the inactive analog 4-α-phorbol had no effect (80 ± 18% of control; n = 4). PD 098059 significantly attenuated PDA-stimulated phosphorylation (276 ± 58% of control;n = 9), whereas U0126 completely abolished this activation (113 ± 21% of control; n = 16).

Fig. 5.

Carbachol (CCh) or DHPG application to hippocampal slices elicits p42 MAPK activation in area CA1.A, Representative anti-phosphotyrosine Western blots of p42 MAPK in area CA1 subregions from control (CTL) slices and slices treated with either CCh or DHPG. Representative blots showing the effect of receptor antagonists, PKC inhibitor, and the MEK inhibitors PD098059 (PD) and U0126 are shownbelow. B, Summary data of p42 MAPK phosphotyrosine immunoreactivity. CCh, Carbachol (50 μm, 10 min, 223 ± 46%; n = 7;p < 0.001); CCh + Atr, 50 μm atropine (muscarinic receptor antagonist) + CCh (92 ± 22%; n = 7); CCh + Chel, chelerythrine (PKC inhibitor) + CCh (106 ± 30%;n = 6); CCh + PD, CCh + PD098059 (50 μm, 107 ± 24%; n = 4);CCh + U0126, CCh applied in the presence of U0126 (109 ± 31%; n = 2). DHPG (10 μm, 10 min, 173 ± 23%; n = 12;p < 0.05). MCPG (DHPG + MCPG; 2 μm, 85 ± 8%; n = 3) blocked this response, as did chelerythrine (DHPG + chel; 87 ± 8%; n = 4) and U0126 (DHPG + U0126; 71 ± 20%; n = 4).

Fig. 6.

Activated p42 MAPK stimulates CREB phosphorylation. A, Selectivity of the anti-phospho-CREB antibody. Hippocampal nuclear extract, either untreated or phosphorylated in vitro with PKA, was Western-blotted with the anti-phospho-CREB antibody (left), as described in Materials and Methods. Then the blot was stripped and reprobed with the nonphospho-selective anti-CREB antibody (right), demonstrating that although CREB was present in both samples the anti-phospho-CREB antibody recognized only CREB in the PKA-phosphorylated extract; no signal was detected in the unphosphorylated sample even after prolonged exposures.B, Top, Representative Western blots from experiments in which activated p42 MAPK (2 ng/ml; Stratagene, La Jolla, CA) was added to either native or boiled hippocampal homogenate with Mg-ATP and phosphatase inhibitors and incubated at 30°C for 30 min. Then the samples were Western-blotted with anti-phospho-CREB antiserum. The blots demonstrate an increase in CREB phosphorylation with p42 MAPK that is not blocked by the inhibitors of the calcium/calmodulin-dependent protein kinases known to phosphorylate CREB (KN-62) or a PKA inhibitor (IP₂₀ fragment of the Walsh inhibitor). p42 MAPK does not trigger CREB phosphorylation in boiled homogenate, indicating that CREB is not a substrate for p42 MAPK, consistent with the hypothesis that CREB activation by p42 MAPK is mediated by RSK2. PKA did phosphorylate CREB in the boiled homogenate (data not shown). B, Bottom, Summary data (n = 6).

Fig. 7.

PKA activation increases CREB phosphorylation in area CA1. A dual approach was used to test the ability of PKA to phosphorylate CREB in area CA1 of hippocampal slices in situ. First, forskolin was applied to slices (50 μm with 100 μm Ro20-1724 for 10 min), the slices were frozen, and area CA1 was dissected and homogenized. Then phosphorylation of CREB was assayed by Western blotting with the anti-phospho-CREB antibody. A, Representative anti-phospho-CREB Western blots in control (CTL) slices and slices treated with forskolin (FSK). Thefar left lane (⊕) represents the phospho-CREB positive control sample, consisting of hippocampal nuclear extract phosphorylated in vitro with PKA. B, p42 MAPK immunoreactivity from representative anti-phospho-CREB Western blots of control (CTL) slices and slices treated with forskolin (FSK) in either the absence (No Inh) or the presence of the MEK inhibitor U0126.Below, an inactive forskolin analog, dideoxyforskolin (ddFSK), has no effect. C, Group data. Forskolin elicited a significant increase in CREB phosphorylation (173 ± 17% of control; n = 17;p < 0.0012), indicating that PKA is coupled to CREB in area CA1. This effect was not mimicked by the inactive analog dideoxyforskolin (ddFSK; CREB phosphorylation, 82 ± 12% of control; n = 3) and was attenuated by U0126 (FSK + U0126; CREB phosphorylation, 117 ± 9% of control; n = 13; p < 0.012). To gather more specific information about the cell types in which the effect occurred, we used an immunohistochemical approach. Hippocampal slices were exposed to forskolin (50 μm for 30 min); then they were fixed in paraformaldehyde, frozen, and sectioned with a cryostat. The 20-μm sections were stained with the anti-phospho-CREB antibody. In area CA1 the increase in CREB phosphorylation was prominent in the nuclei of the pyramidal cells as well as in CA3 pyramidal cells and dentate granule cells (data not shown). Together, these data demonstrate that, within the pyramidal neurons in area CA1, PKA is coupled to phosphorylation of CREB at the critical Ser¹³³ site.

Fig. 8.

PKC activation in situ triggers CREB phosphorylation. PKC stimulation leads to p42 MAPK activation and CREB phosphorylation in area CA1. A, Representative anti-phospho-CREB Western blots in control (CTL) slices and slices treated with the PKC activator phorbol diacetate (PDA; 10 μm for 10 min). The far left lane (⊕) represents the phospho-CREB positive control sample, consisting of hippocampal nuclear extract phosphorylatedin vitro with PKA. B, p42 MAPK immunoreactivity from representative anti-phospho-CREB Western blots of control (CTL) slices and slices treated with PDA in either the absence (No Inh) or the presence of the MEK inhibitor U0126. Below, an inactive forskolin analog, 4-α-phorbol (4αP), has no effect. C, Group data. PDA elicited a significant increase in CREB phosphorylation (264 ± 55% of control; n = 7;p < 0.05), indicating that PKC is coupled to CREB in area CA1. This effect was not mimicked by the inactive analog 4-α-phorbol (4αP; 10 μm; CREB phosphorylation, 97 ± 27% of control; n = 4) and was attenuated significantly by U0126 (PDA + U0126; CREB phosphorylation, 170 ± 14% of control;n = 16; p < 0.05).

Fig. 9.

Signal transduction pathways operating in hippocampal synaptic plasticity. This schematic diagram, which places components postsynaptically for the convenience of presentation, diagrams several of the signal transduction pathways documented as operating in hippocampal area CA1. Although a static diagram is presented, it should be kept in mind that the effects that are described can be divided into two broad temporal categories: (1) short-term effects caused by second messenger-dependent protein kinase activation and (2) longer-term effects caused by the generation of persistently activated second messenger-independent forms of PKC and CaMKII, such as occurs in LTP. Although all of the ultimate effectors of these various pathways are not known, nor have the genes downstream of CREB been identified, several candidate categories are documented, e.g., glutamate receptors, K⁺ channels, and cell surface molecules. As indicated, multiple receptor subtypes are coupled to a variety of downstream kinases, and the kinase cascades may interact extensively with each other. In many cases the interactions may serve as a signal amplification mechanism, as multiple upstream regulators converge on final common effectors such as MAPK and CREB. The convergence on final common effectors has an interesting implication: the capacity of multiple cascades to elicit the activation of the same downstream effectors may provide strong stimuli with a fail-safe mechanism wherein the failure of one pathway may be compensated for by the functionally redundant pathway. Finally, signal amplification may not be limited to the simultaneous activation of two pathways. Because termination of kinase activation is not instantaneous, temporally spaced stimuli may serve to amplify each other, allowing for temporal integration. A/K, AMPA/KA receptors; CaM, calmodulin; DAR, dopamine receptor D1/D5; βAR, β-adrenergic receptor;mGLUR, metabotropic glutamate receptor;mAChR, muscarinic acetylcholine receptor;PLC, phospholipase C; AC, adenylyl cyclase; DAG, diacylglycerol; PKC, persistently or transiently activated PKC; RSK2, ribosomal S6 kinase, also known as CREB kinase (Xing et al., 1996);CaMKII/IV, CaMKII or CaMKIV, which also have been implicated in the CREB-mediated regulation of gene expression in the hippocampus (Deisseroth et al., 1996).