Agents that elevate cAMP levels in platelets decrease thrombin binding

The effect of high intracellular levels of cAMP on the ability of rabbit and human platelets to bind and respond to thrombin was examined. Control rabbit platelets differed from human platelets in two interesting respects: they showed thrombin-dependent up-regulation of thrombin binding, but also a 3- to 5-fold lower thrombin-binding capacity. Nevertheless, treatment with prostaglandin E1 + theophylline or with forskolin decreased thrombin binding to both rabbit and human platelets by 60 to 70%. This effect was associated with a marked increase in the level of cAMP and seemed to depend on a decrease in number rather than affinity of thrombin-binding sites. Changes in thrombin binding correlated closely with changes in thrombin-stimulated incorporation of 32Pi into phosphatidic acid and a 40-kDa protein. However, regardless of the amount of thrombin that bound to treated platelets, thrombin-stimulated phosphorylation of a 20-kDa protein and serotonin secretion were severely inhibited. Thus, increased levels of platelet cAMP are associated with a reduced ability to bind and respond to thrombin. However, thrombin binding to platelets correlates more closely with some responses than others, presumably because cAMP inhibits additional platelet reactions.     

Growth and protein phosphorylation in the Nb2 lymphoma: effect of prolactin, cAMP, and agents that activate adenylate cyclase

The Nb2 T lymphoma is unique in that these lymphocytes proliferate in response to prolactin as well as in response to interleukin-2. In this study, we have examined the responsiveness of the adenylate cyclase system in Nb2 cells and the role of this signaling system in regulating proliferation and protein phosphorylation. An analog of cAMP inhibited prolactin-stimulated proliferation and blocked a prolactin-induced decrease in protein phosphorylation. Forskolin, a potent activator of adenylate cyclase in T lymphocytes, did not elevate cAMP levels in Nb2 cells and was not an effective inhibitor of prolactin-induced proliferation. In fact, one preparation of forskolin stimulated proliferation of quiescent Nb2 cells. Like forskolin, prostaglandin E2 did not stimulate cAMP production in Nb2 cells even though it increased cAMP in a preparation of rat peripheral blood lymphocytes. Cholera toxin appeared to ADP-ribosylate a stimulatory guanine nucleotide-binding protein in Nb2 cells, but the toxin did not increase intracellular levels of cAMP nor was it a potent anti-mitogenic agent. Pertussis toxin, an agent that can increase cAMP production through suppression of the inhibitory guanine nucleotide-binding protein, exerted only minor anti-proliferative actions on prolactin-stimulated Nb2 cells. These data suggest that cAMP inhibits Nb2 cell proliferation and prolactin-induced changes in protein phosphorylation but that the adenylate cyclase system in our clone of Nb2 cells responds poorly to agents that normally increase cAMP.     

Control of platelet protein kinase C activation by cyclic AMP

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.     

The effect of retinoic acid on protein phosphorylation in mouse melanoma cells

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.     

Adrenergic receptor agonists prevent bile duct injury induced by adrenergic denervation by increased cAMP levels and activation of Akt

Loss of parasympathetic innervation after vagotomy impairs cholangiocyte proliferation, which is associated with depressed cAMP levels, impaired ductal secretion, and enhanced apoptosis. Agonists that elevate cAMP levels prevent cholangiocyte apoptosis and restore cholangiocyte proliferation and ductal secretion. No information exists regarding the role of adrenergic innervation in the regulation of cholangiocyte function. In the present studies, we investigated the role of adrenergic innervation on cholangiocyte proliferative and secretory responses to bile duct ligation (BDL). Adrenergic denervation by treatment with 6-hydroxydopamine (6-OHDA) during BDL decreased cholangiocyte proliferation and secretin-stimulated ductal secretion with concomitant increased apoptosis, which was associated with depressed cholangiocyte cAMP levels. Chronic administration of forskolin (an adenylyl cyclase activator) or beta(1)- and beta(2)-adrenergic receptor agonists (clenbuterol or dobutamine) prevented the decrease in cholangiocyte cAMP levels, maintained cholangiocyte secretory and proliferative activities, and decreased cholangiocyte apoptosis resulting from adrenergic denervation. This was associated with enhanced phosphorylation of Akt. The protective effects of clenbuterol, dobutamine, and forskolin on 6-OHDA-induced changes in cholangiocyte apoptosis and proliferation were partially blocked by chronic in vivo administration of wortmannin. In conclusion, we propose that adrenergic innervation plays a role in the regulation of biliary mass and cholangiocyte functions during BDL by modulating intracellular cAMP levels.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

Calcium Requirement for α-MSH Action on Melanophores: Studies with Forskolin

Abstract

α-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca²⁺. To localize Ca²⁺ sites involved in the mechanism of action of α-MSH we studied the effects of Ca²⁺ deprivation on α-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system α-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein.

Forskolin induces a dose-dependent pigment dispersion (EC₅₀ 7 × 10^?7 M). In contrast to the dispersion induced by α-MSH forskolin-induced dispersion does not require extracellular Ca²⁺. Moreover, in a Ca²⁺-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dosedependent manner. Maximal stimulation with forskolin (10^?5 M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with α-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca²⁺ deprivation, whereas the forskolin-induced increase is unaffected. Our results suggest that α-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in α-MSH action in this system. We conclude that the Ca²⁺ sites in the mechanism of α-MSH action on melanophores precede adenylate cyclase activation.     

Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase

The activity of the serine/threonine kinase c-Raf (Raf) is inhibited by increased intracellular cAMP. This is believed to require phosphorylation with the cAMP-dependent protein kinase (PKA), although the mechanism by which PKA inhibits Raf is controversial. We investigated the requirement for PKA phosphorylation using Raf mutants expressed in HEK293 or NIH 3T3 cells. Phosphopeptide mapping of (32)P-labeled Raf (WT) or a mutant lacking a putative PKA phosphorylation site (serine to alanine, S43A) confirmed that serine 43 (Ser(43)) was the major cAMP (forskolin)-stimulated phosphorylation site in vivo. Interestingly, the EGF-stimulated Raf kinase activity of the S43A mutant was inhibited by forskolin equivalently to that of the WT Raf. Forskolin also inhibited the activation of an N-terminal deletion mutant Delta5-50 Raf completely lacking this phosphorylation site. Although WT Raf was phosphorylated by PKA, phosphorylation did not inhibit Raf catalytic activity in vitro, nor did forskolin treatment inhibit the activity of an N-terminally truncated Raf protein (Raf 22W) or a full-length Raf protein (Raf-CAAX) expressed in NIH 3T3 cells. In contrast, forskolin inhibited the EGF-dependent activation of a Raf isoform (B-Raf), lacking an analogous phosphorylation site to Ser(43). Thus, these results demonstrate that PKA exerts its inhibitory effects independently of direct Raf phosphorylation and suggests instead that PKA prevents an event required for the EGF-dependent activation of Raf.     

Elevated cyclic-AMP represses expression of exchange protein activated by cAMP (EPAC1) by inhibiting YAP-TEAD activity and HDAC-mediated histone deacetylation

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts.Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP.Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

Leptin potentiates antiproliferative action of cAMP elevation via protein kinase A down‐regulation in breast cancer cells

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT‐5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA‐MB‐231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT‐5720 blocked the forskolin‐induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin‐induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down‐regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin‐dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA‐MB‐231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed. J. Cell. Physiol. 225: 801–809, 2010. © 2010 Wiley‐Liss, Inc.     

The effect of Sildenafil on human platelet secretory function is controlled by a complex interplay between phosphodiesterases 2, 3 and 5

Human platelets contain the cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) 2, 3 and 5. The cGMP-PDE5 inhibitors Sildenafil and Zaprinast have been demonstrated to potentiate the anti-platelet aggregatory effect of NO donors like sodium nitroprusside (SNP) in vitro but the mechanisms of Sildenafil's action on the secretory function of human platelets have not been analysed in detail. In the present paper, we show (1) that both compounds potentiate the SNP-induced increase in cGMP in human platelets concentration-dependently. (2) However, whereas Sildenafil plus SNP treatment only partially inhibits thrombin-induced release of serotonin, the less selective Zaprinast plus SNP cause a complete inhibition. (3) The inhibition mediated by Sildenafil plus SNP is limited to low compound concentrations at which cAMP levels are increased, probably due to cGMP-mediated inhibition of PDE3. (4) High concentrations of Sildenafil (plus SNP) neither affect cAMP levels, likely due to the activation of PDE2, nor inhibits the release of serotonin. Thus, increases in both cyclic nucleotides seem to control platelet function. (5) Accordingly, treatment with increasing concentrations of Sildenafil plus SNP and a selective PDE2 inhibitor, which by its own has no effect, induced a concentration-dependent increase in cAMP and complete inhibition of platelet activation. In summary, our data indicate that Sildenafil inhibits secretory function of human platelets at least in part due to the cGMP-mediated effects on intracellular cAMP and that entire inhibition of serotonin release from thrombin-activated platelets is controlled by both cyclic nucleotides.     

Identification of a prominent 85-kDa cAMP-dependent phosphoprotein associated with late G1 phase in mitogen-stimulated B lymphocytes

In order to elucidate late regulatory events which may be involved in the onset of S phase in B lymphocytes, we studied the effect of anti-Ig on phosphorylation of soluble proteins at late G1 phase. Stimulation of murine splenic B lymphocytes with anti-Ig and other mitogens for 18 h was found to be associated with a major increase in phosphorylation of an 85 kDa/pI approximately 5.3 cytosolic protein, conversely, stimulation of the cells with non-mitogenic stimuli did not induce the phosphorylation of pp85. The increase in phosphorylation of pp85 could not be detected after 30 min, was barely detectable after 6 h, but was very prominent after 18 h of stimulation with anti-Ig. Thus, the increase in phosphorylation of pp85 is not an early signal but is rather correlated with the late G1 phase. pp85 could not be detected in the nuclei of either control or stimulated cells. Stimulation of B cells for 30 min with forskolin induced the phosphorylation of pp85, while phorbol ester did not have any effect. The phosphorylation of pp85 was induced by the catalytic subunit of cAMP protein kinase. Comparison of the phosphopeptide map of pp85 phosphorylated by anti-Ig in intact cells to the phosphopeptide map phosphorylated by forskolin or by the catalytic subunit of cAMP protein kinase, showed a striking similarity indicating that cAMP protein kinase may be involved in phosphorylation of pp85 in mitogen-stimulated cells. An increase in intracellular cAMP levels at late G1 phase was found in B cells stimulated by mitogens. These results implicate an important role for cAMP-dependent phosphorylation events, specifically the phosphorylation of pp85/pI 5.3, at late G1 phase during the cell cycle.     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.