Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline.     

The Sustained Phase of Tyrosine Hydroxylase Activation In vivo

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthetic pathway for catecholamine synthesis. Stress triggers an increase in TH activity, resulting in increased release of catecholamines from both neurons and the adrenal medulla. In response to stress three phases of TH activation have been identified (acute, sustained and chronic) and each phase has a unique mechanism. The acute and chronic phases have been studied in vivo in a number of animal models, but to date the sustained phase has only been characterised in vitro. We aimed to investigate the effects of dual exposure to lipopolysaccharide (LPS) in neonatal rats on TH protein, TH phosphorylation at serine residues 19, 31 and 40 and TH activity in the adrenal gland over the sustained phase. Wistar rats were administered LPS (0.05?mg/kg, intraperitoneal injection) or an equivolume of non-pyrogenic saline on days 3 and 5 postpartum. Adrenal glands were collected at 4, 24 and 48?h after the drug exposure on day 5. Neonatal LPS treatment resulted in increases in TH phosphorylation of Ser40 at 4 and 24?h, TH phosphorylation of Ser31 at 24?h, TH activity at 4 and 24?h and TH protein at 48?h. We therefore have provided evidence for the first time that TH phosphorylation at Ser31 and Ser40 occurs for up to 24?h in vivo and leads to TH activation independent of TH protein synthesis, suggesting that the sustained phase of TH activation occurs in vivo.     

Differential expression of the B'beta regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Regulation of basal expression of catecholamine-synthesizing enzyme genes by PACAP.

We have previously reported that the cAMP/protein kinase A (PKA) pathway is important in the gene regulation of both induction and basal expressions of the catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to activate the intracellular cAMP/PKA pathway. In the present study, using primary cultured bovine adrenal medullary cells, we determined whether the basal activity of the PACAP receptor might play a role in the maintenance of the basal expression of these enzyme genes via the cAMP/PKA pathway. The potent PACAP receptor antagonist PACAP (6-38) caused a reduction of TH and DBH mRNA levels in a dose dependent manner as well as their enzyme activities and TH protein level. The effects of PACAP (6-38) and the PKA inhibitor H-89 exhibited generally similar trends, and were not additive in the reduction of TH and DBH gene expression and activities, suggesting that they take a common intracellular signaling pathway. The antagonist also caused decreases in the intracellular norepinephrine and epinephrine levels similar to the effect of H-89. Taken together, the data suggests that PACAP is involved in the regulation of maintenance of the catecholamine synthesizing enzymes TH and DBH by utilizing the cAMP/PKA pathway.     

Activation and Multiple-Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands

Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 microM tetrahydrobiopterin. Nicotine (3 microM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 microM, 3 min) increased Ser40 phosphorylation, and muscarine (100 microM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.     

Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca(2+)-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K(+)-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K(+) (+ 58 mM) for 5 min increased TH PS at each site in a Ca(2+)-dependent manner. Pretreatment with PD98059 prevented elevated K(+)-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K(+)-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K(+) and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.     

Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N-terminal kinases (JNK) on the histamine-induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type-1 (H1) receptor-dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor-dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine-induced TH phosphorylation, we generated phospho-specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine-induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine-induced TH phosphorylation. Our data support the hypothesis that the up-regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.     

Neurobiological consequences of acute footshock stress: effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla

Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40‐min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone, corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, ventral tegmental area and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31 phosphorylation than the mPFC, presumably because of their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma adrenocorticotropic hormone at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal‐regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min. TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, pre‐synaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the hypothalamic‐pituitary‐adrenal axis.

     

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28-38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 microM PACAP(6-38), a PAC(1) receptor antagonist or 10 microM PD98059, a MAPKK inhibitor. These results suggest that PAC(1) receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6-38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC(1) and VPAC(1) receptors regulate VEGF expression in lung cancer cells.     

Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.     

Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum

Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000 μM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000 μM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000 μM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8 h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model.     

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Activation of tyrosine hydroxylase by intermittent hypoxia: involvement of serine phosphorylation.

Regulation of tyrosine hydroxylase (TH) by intermittent hypoxia (IH) was investigated in rat pheochromocytoma 12 (PC-12) cells by exposing them to alternating cycles of hypoxia (1% O2, 15 s) and normoxia (21% O2, 3 min) for up to 60 cycles; controls were exposed to normoxia for a similar duration. IH exposure increased dopamine content and TH activity by approximately 42 and approximately 56%, respectively. Immunoblot analysis revealed that comparable levels of TH protein were expressed in normoxic and IH cells. Removal of TH-bound catecholamines and in vitro phosphorylation of TH in cell-free extracts by the catalytic subunit of protein kinase A (PKA) increased TH activity in normoxic but not in IH cells, suggesting possible induction of TH phosphorylation and removal of endogenous inhibition of TH by IH. To assess the role of serine phosphorylation in IH-induced TH activation, TH immunoprecipitates and extracts derived from normoxic and IH cells were probed with anti-phosphoserine and anti-phospho-TH (Ser-40) antibody, respectively. Compared with normoxic cells, total serine and Ser-40-specific phosphorylation of TH were increased in IH cells. IH-induced activation of TH and the increase in total serine and Ser-40-specific phosphorylation of TH were inhibited by Ca2+/calmodulin-dependent protein kinase (CaMK) and PKA-specific inhibitors but not by inhibitors of the extracellular signal-regulated protein kinase pathway, suggesting that IH activates TH in PC-12 cells via phosphorylation of serine residues including Ser-40, in part, by CaMK and PKA. Our results also suggest that IH-induced phosphorylation of TH facilitates the removal of endogenous inhibition of TH, leading to increased synthesis of dopamine.     

A predominant role for inhibition of the adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid receptor 1 in N1E-115 neuroblastoma cells

Cannabinoids activate several members of the mitogen-activated protein kinase superfamily including p44 and p42 extracellular signal-regulated kinase (ERK). We used N1E-115 neuroblastoma cells and the cannabinoid receptor agonist WIN 55,212-2 (WIN) to examine the signal transduction pathways leading to the activation of ERK. ERK phosphorylation (activation) was measured by Western blot. The EC50 for stimulation of ERK phosphorylation was 10 nm, and this effect was blocked by pertussis toxin and the CB1 (cannabinoid) receptor antagonist SR141716A. The MEK inhibitors PD 98059 and U0126 blocked ERK phosphorylation, as did the adenylate cyclase activator forskolin. The phosphatidylinositol (PI) 3-kinase inhibitor LY 294002 and the Src kinase inhibitor PP2 partially occluded the response but also decreased basal levels of phospho-ERK. The PI 3-kinase and Src pathways are known to promote cell survival in many systems; therefore, MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) conversion was used to examine the effects of these inhibitors on cellular viability. LY 294002 decreased the number of viable cells after 18 h of treatment; therefore, the inhibition of ERK by this inhibitor is probably because of cytotoxicity. Forskolin blocked ERK phosphorylation with an EC50 of <3 microm, and the protein kinase A (PKA) inhibitor H-89 enhanced ERK phosphorylation. c-Raf phosphorylation at an inhibitory PKA-regulated site (Ser259) was also reduced by WIN. This is probably due to constitutive phosphatase activity because WIN did not directly stimulate PP1 or PP2A activity when measured using 6,8-difluoro-4-methylumbelliferyl phosphate as a fluorogenic substrate. These data implicate the inhibition of PKA as the predominant pathway for ERK activation by CB1 receptors in N1E-115 cells. PI 3-kinase and Src appear to contribute to ERK activation by maintaining activation of kinases, which prime the pathway and maintain cellular viability.     

Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras

Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.     

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.