Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.The activation of signal transduction pathways by hormonal and developmental stimuli ultimately leads to changes in the expression of specific genes. These changes often occur at the level of gene transcription and are mediated by rapid changes in the phosphorylation state of specific transcription factors. For example, the transcriptional transactivation function of the cyclic AMP (cAMP)-regulatory element binding protein (CREB) is stimulated by phosphorylation (15,28,45). CREB phosphorylation by the cAMP-dependent protein kinase A (PKA) is sufficient for the transcriptional transactivation in response to elevated intracellular cAMP levels (15,18,28,45).The stimulation of transcription of certain genes by cAMP is generally characterized by a rapid increase in the rate of transcription to a maximal level, followed by a slow decline in transcription to basal levels (29,38). The decrease in the rate of transcription from stimulated levels occurs even under conditions where PKA activity is elevated, and it is refractory to the addition of agents that ...

Protein phosphatase 1 catalytic subunit (PP1c) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)-labeled PP1revealed four major rat brain PP1~-binding proteins (PP1 bps) with molecular masses of~m216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PPlbpl75, PPlbpl34, PPlbp75, and PP1~were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size-exclusion chromatography of solubilized extracts separated two main PP1 activities (r~n600and nslOO kDa). PPlbps and PPlcy 1 were enriched in the rm600-kDa peak, but PP1~~3 was enriched in the snlOO-kDa peak. Furthermore, PPlbpl75 and PPlbpl34 exhibited lower binding of recombinant DIG-PP1c/J than recombinant DlG-PP1~y1 or DIGPPlca. Solubilized PPlbpl75 and PPlbpl34 interact with PP1~under native conditions, because they both (1) coeluted from size-exclusion and ion-exchange columns; (2) bound to microcystin-LR-Sepharose; and (3) coprecipitated using PP1~antibodies. Trypsinolysis of the ms600-kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1with these PP1 bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform-selective binding of PP1to these targeting/regulatory subunits, contributing to the specificity of its physiological roles. Key Words: Protein phosphatase-Localization -Postsynaptic density-Synaptic plasticity. J. Neurochem. 69, 920-929 (1997).Phosphorylation of key intracellular proteins is dependent~n appropriate coordination of the activities of cellular protein kinases and protein phosphatases. Although a great deal is known about protein kinase regulation, comparatively less is known about regulation of protein phosphatases, particularly in neuronal tissues. Four major classes of protein serine/threonine phosphatases (PPI, PP2A, PP2B, and PP2C) comprise most of the activity in extracts of brain and other tissues and many of their catalytic and regulatory subunits have been cloned. Several additional novel cDNAs encoding phosphatase catalytic subunits have been identified, although the contributions of these enzymes to phosphatase activities in extracts are generally unclear (for review, see Cohen, 1989;Bollen and Stalmans, 1992;Mumby and Walter, 1993;Brautigan, 1994; Depaoli-Roach et al., 1994; Wera and Hemmings, 1995). Although one phosphatase, PP2B or calcineurin, is directly regulated by a second messenger (Ca 2~),the emerging model is that most protein phosphatases are regulated by association of their catalytic subunits with specific proteins that target them to appropriate cellular locations. For example, the catalytic subunit of PP1 (PP1~)is targeted to substrates in skeletal muscle glycogen particles a...

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb [ PP2ACa [ PP4C [ PP6C), NRVM (PP2ACb [ PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa [ PP2ACb [ PP6C [ PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S protea-some inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomy-ocytes to H 2 O 2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium. Keywords Type 2A protein phosphatase Á Alpha4 Á Cardiac hypertrophy Á Hydrogen peroxide Á H2A.X

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