cAMP-dependent protein kinase 1


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PKA targeting of CREB

A-kinase anchor protein 75 (AKAP75) binds regulatory subunits (RIIalpha and RIIbeta) of type II protein kinase A (PKAII) isoforms and targets the resulting complexes to sites in the cytoskeleton that abut the plasma membrane. Co-localization of AKAP75-PKAII with adenylate cyclase (Drosophila homolog: Ratabaga) and PKA substrate/effector proteins in the cytoskeleton and the plasma membrane effects a physical and functional integration of up-stream and downstream signaling proteins, thereby ensuring efficient propagation of signals carried by locally generated cyclic AMP (cAMP). An important (but previously untested) prediction is that efficient, cyclic nucleotide-dependent liberation of diffusible PKA catalytic subunits from cytoskeleton-bound AKAP75-PKAII complexes will also enhance signaling to distal organelles, such as the nucleus. This idea was tested by using HEK-A75 cells, in which PKAII isoforms are immobilized in cortical cytoskeleton by AKAP75. A comparison was made of the abilities of HEK-A75 and control cells (with cytoplasmically dispersed PKAII isoforms) to respond to increases in cAMP content. Cells with anchored PKAII exhibit a threefold higher level of nuclear catalytic subunit content and 4-10-fold greater increments in phosphorylation of a regulatory serine residue in cAMP response element binding protein (CREB) and in phosphoCREB-stimulated transcription of the c-fos gene. Each effect occurs more rapidly in cells containing targeted AKAP75-PKAII complexes. Thus, anchoring of PKAII in actin cortical cytoskeleton increases the rate, magnitude and sensitivity of cAMP signaling to the nucleus (Feliciello, 1997).

In chickens, PKA and PKC are involved in intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of developing dermis. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode epithelia (but not in interplacode epithelia). P-CREB is also expressed transiently in bud mesenchyme between stages 33 and 36, but not in the interbud mesenchyme. PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. It is likely that activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions (Noveen, 1995)

Cyclic AMP (cAMP) regulates a number of eukaryotic genes by mediating the protein kinase A (PKA)-dependent phosphorylation of the CREB transcription factor at Ser-133. The stoichiometry and kinetics of CREB phosphorylation are determined by the liberation and subsequent translocation of PKA catalytic subunit (C subunit) into the nucleus. PKA is activated in a stimulus-dependent fashion that leads to nuclear entry of C subunit over a 30-min period. The degree of CREB phosphorylation correlates with the amount of PKA liberated. The time course of phosphorylation closely paralleled the nuclear entry of the catalytic subunit. There is a linear relationship between the subsequent induction of the cAMP-responsive somatostatin gene and the degree of CREB phosphorylation, suggesting that each event--kinase activation, CREB phosphorylation, and transcriptional induction--was tightly coupled to the next. In contrast to other PKA-mediated cellular responses which are rapid and quantitative, the slow, incremental regulation of CREB activity by cAMP suggests that multifunctional kinases like PKA may coordinate cellular responses by dictating the kinetics and stoichiometry of phosphorylation for key substrates like CREB (Hagiwara, 1993).

Transcription factor CREB regulates cyclic AMP-dependent gene expression by binding to and activating transcription from cAMP response elements (CREs) in the promoters of target genes. The transcriptional transactivation functions of CREB are activated by means of phosphorylation carried out by cAMP-dependent protein kinase A (PKA). In studies of many different phenotypically distinct cells, the CRE of the somatostatin gene promoter is a prototype of a highly cAMP-responsive element regulated by CREB. Paradoxically, in a somatostatin-producing rat insulinoma cell line, transcription from the somatostatin gene promoter is repressed by CREB. CREB fails to transactivate a CRE-containing somatostatin promoter even when coexpressed with the catalytic subunit of PKA. CAAT box/enhancer-binding protein beta (C/EBP beta) and C/EBP-related activating transcription factor bind to the CRE in the promoter of the somatostatin gene and transactivate transcription. CREB binds competitively with C/EBP beta to the somatostatin CRE in vitro and represses C/EBP beta-induced transcription of the CRE-containing somatostatin promoter. The lack of CREB-mediated transcriptional stimulation is due to the presence of a heat-stable inhibitor of PKA that prevents activation of PKA and subsequent CREB phosphorylation in the nucleus. These findings indicate that dephosphorylated CREB is a negative regulator of C/EBP-activated transcription of the somatostatin gene promoter (Vallejo, 1995).

Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 (see Drosophila RSK) by Ca2+ leads to the phosphorylation and transactivation of CREB. The Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. Interestingly, Ca2+-mediated CREB phosphorylation in wild-type PC12 cells is decreased by a selective PKA inhibitor. With a high efficiency transfection protocol, expression of dominant negative PKA also attenuates Ca2+-stimulated CREB phosphorylation. In addition, treatment with the PKA inhibitors also inhibits depolarization-mediated CREB phosphorylation in primary hippocampal neurons. These results suggest that in PC12 cells and hippocampal neurons, PKA activity is required for Ca2+-induced CREB phosphorylation (Impey, 1998).

Because the nuclear translocation of ERK may be necessary for ERK-activated transcription, and PKA is required for Ca2+ stimulation of CREB phosphorylation, nuclear translocation of ERK was monitored when PKA was inhibited. To efficiently induce the nuclear translocation of ERK by Ca2+, PC12 cells were treated with KCl and a direct activator of L-type Ca2+ channels. Depolarization induces the phosphorylation of ERK and its translocation to the nucleus in both PC12 cells and hippocampal neurons. The specific PKA inhibitors inhibited the nuclear translocation of Erk in PC12 cells and hippocampal neurons. Western blotting of cytosolic fractions shows that the inhibition of Erk translocation by treatment with PKA inhibitors is not the result of an effect on Erk activation. To verify that PKA is required for the nuclear translocation of ERK, the cytosolic-to-nuclear ratio of phospho-ERK in KCl-stimulated hippocampal neurons was also quantitated. A specific PKA inhibitor significantly inhibited the translocation of ERK to the nucleus. The importance of PKA activity for ERK nuclear translocation was confirmed by transiently transfecting PC12 cells with a dominant negative PKA fused to green fluorescent protein. Only cells that express dominant negative PKA-GFP show impaired nuclear translocation of phospho-ERK. These results suggest that PKA is required for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK. However, since Rsk2 [a member of the pp90(RSK) family] is a major Ca2+-activated CREB kinase in PC12 cells, inhibition of Erk translocation should also block the activation of nuclear but not cytosolic Rsk2. Accordingly, inhibition of PKA blocks the activation of Rsk2 in the nuclear fraction but not in the cytosolic fraction. Treatment with PKA inhibitors attenuates the nuclear translocation of Rsk2. This is not surprising, because it is known that both ERK and Rsk2 are tightly associated in vivo and that they cotranslocate to the nucleus. Collectively, these data indicate that PKA may be necessary for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK and subsequent nuclear activation of the CREB kinase Rsk2. Inhibition of PKA also significantly impairs the translocation of ERK to the nucleus in response to NGF. Interestingly, coexpression of dominant negative PKA attenuates NGF-stimulated Elk1 transcriptional activation. Evidently, the modulation of ERK translocation by PKA activity plays a general role in the activation of transcription by mitogens and neurotrophic factors. NGF does not detectably elevate intracellular cAMP, suggesting that basal PKA activity is sufficient for neurotrophic factors and other strong ERK activators to induce nuclear translocation of ERK. Nevertheless, in the case of depolarization, which activates ERK to a lesser degree, the concomitant depolarization-mediated increase in cAMP levels enhances ERK translocation. These results may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity (Impey, 1998).

Activation of the mitogen-activated protein kinase (MAPK) cascade plays 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. Hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, 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. 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, 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 (Roberson, 1999).

Numerous in vivo studies have demonstrated that psychostimulant drugs such as amphetamine and cocaine can induce the expression of the immediate early gene c-fos in striatal neurons via the activation of D1 dopamine receptors. NMDA receptor activation is also known to induce c-fos in the striatum. A primary striatal neuronal culture preparation was used to examine the mechanisms whereby these stimuli lead to changes in gene expression. Direct application of NMDA to striatal cells in culture causes a rapid increase in the expression of c-fos, as well as an increase in the phosphorylation of the transcription factor CRE binding protein (CREB). This is prevented by NMDA receptor antagonists, and requires extracellular calcium, but does not involve L-type calcium channels. The induction of c-fos and CREB phosphorylation following NMDA are unaffected by inhibition of protein kinase C, tyrosine kinases or nitric oxide synthase. However, the response to NMDA is blocked by KN62, a selective inhibitor of calcium/calmodulin-dependent protein kinase. The application of the D1 agonist SKF 38393, or the direct stimulation of adenylyl cyclase with forskolin, also results in the phosphorylation of CREB and the induction of c-fos in striatal neurons. These effects are blocked by the protein kinase A inhibitor H89. These observations are consistent with the hypothesis that calcium/calmodulin-dependent phosphorylation of CREB induced by NMDA, or cAMP-dependent phosphorylation of CREB induced by D1 agonists, underlie the induction of c-fos seen following activation of these receptors in striatal neurons (Das, 1997).

Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response element-binding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. The morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. Antisense oligonucleotides directed against CREB were used to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduces levels of CREB immunoreactivity. This effect is sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely block the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Gialpha, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies reveal that intra-LC antisense oligonucleotide infusions completely prevent the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade is completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduces the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence (Lane-Ladd, 1997).

The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength-dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras-MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription (Hu, 1999).

Table of contents

cAMP-dependent protein kinase 1: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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