Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

We have investigated the role of serine 40 (Ser-40) in tyrosine hydroxylase (TH) catalysis of basal and activated enzymes by protein kinase A (PKA)-mediated phosphorylation. Wild type and mutant TH were transiently and stably expressed in AtT-20 cells, and the enzymatic activities of the recombinant enzymes were analyzed. The specific enzymatic activity of transiently expressed TH mutants Ser-40-->leucine or-->tyrosine (Leu-40m or Tyr-40m) was higher than that of the wild type enzyme or of other mutants in which Ser-8, -19, and -31 were replaced by leucine. The kinetic studies carried out with the stably expressed TH show that the Km for the cofactor 6-methyltetrahydropterine is lower and the Ki for dopamine is higher when the enzymatic hydroxylation is catalyzed by the Leu-40m or Tyr-40m than by the wild type enzyme. The kinetic parameters and the pH profile of the enzymatic hydroxylation catalyzed by the Leu-40m or Tyr-40m are similar to the enzyme activated by PKA-mediated phosphorylation. We suggest that Ser-40 in TH exerts an inhibitory influence on the enzymatic activity, and its replacement with another amino acid by site-directed mutagenesis or its modification by phosphorylation leads to a change in conformation with an increased enzymatic activity. The importance of Ser-40 in the activation of TH by PKA-mediated phosphorylation was investigated by comparing the activation of the wild type enzyme with that of Leu-40m or Tyr-40m. The findings that the enzymatic activity is increased by PKA-mediated phosphorylation of the wild type enzyme, but not of the Leu-40m or Tyr-40m, demonstrate that phosphorylation at Ser-40 is essential for activation of TH by PKA. The findings that addition of ATP plus cAMP to homogenates from transfected AtT-20 cells stimulates the recombinant wild type TH activity indicate that these cells contain endogenous cAMP-dependent protein kinase.     

Enhancement of tyrosine hydroxylase phosphorylation and activity by glial cell line-derived neurotrophic factor

Although glial cell-line derived neurotrophic factor (GDNF) acts as a potent survival factor for dopaminergic neurons, it is not known whether GDNF can directly alter dopamine synthesis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis, and its activity is regulated by phosphorylation on three seryl residues: Ser-19, Ser-31, and Ser-40. Using a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuron cultures, the present study examined whether GDNF alters the phosphorylation of TH and whether these changes are accompanied by increased enzymatic activity. Exposure to GDNF did not alter the TH protein level in either neuroblastoma cells or in primary neurons. However, significant increases in the phosphorylation of Ser-31 and Ser-40 were detected within minutes of GDNF application in both cell types. Enhanced Ser-31 and Ser-40 phosphorylation was associated with increased TH activity but not dopamine synthesis in neuroblastoma cells, possibly because of the absence of l-aromatic amino acid decarboxylase activity in these cells. In contrast, increased phosphorylation of Ser-31 and Ser-40 was found to enhance dopamine synthesis in primary neurons. Pharmacological experiments show that Erk and protein kinase A phosphorylate Ser-31 and Ser-40, respectively, and that their inhibition blocked both TH phosphorylation and activity. Our results indicate that, in addition to its role as a survival factor for dopaminergic neurons, GDNF can directly increase dopamine synthesis.     

HIF-1α Activation by Intermittent Hypoxia Requires NADPH Oxidase Stimulation by Xanthine Oxidase

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O₂ saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O₂ for 30 sec) and normoxia (21% O₂ for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47^phox and p67^phox to the plasma membrane and their interaction with gp91^phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.     

Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing.

The single human tyrosine hydroxylase (TH) gene generates four different mRNA species through alternative splicing events. TH-1 and TH-2 mRNAs are expressed mostly in the brain. We have produced large amounts of the corresponding proteins in Escherichia coli to analyze their respective molecular characteristics. The polypeptides have molecular weights similar to those of TH expressed in Xenopus oocytes and react with antibodies to TH. The two isoforms were purified with a purity of 90% using a three-step procedure. The phosphorylation sites have been determined in the two isoforms after labeling with [gamma-32P]ATP in the presence of cAMP-dependent protein kinase (PKA) or calmodulin-dependent protein kinase II (CaM-PK II). In both isoforms, Ser-40 was found to be phosphorylated by PKA, and Ser-19 and Ser-40 were found to be phosphorylated by CaM-PK II. The putative phosphorylation site generated by alternative splicing (Ser-31) was phosphorylated specifically by CaM-PK II in TH-2 only. The kinetic properties of the two isoforms in the presence of various concentrations of the substrate (tyrosine) and of the natural cofactor [6R)-tetrahydrobiopterin) were also analyzed. TH produced in E. coli is unphosphorylated but nevertheless active. At 50 microM tyrosine and 300 microM (6R)-tetrahydrobiopterin, the specific activities of TH-1 and TH-2 are 1300 and 620 nmol of dihydroxyphenylalanine/min/mg, respectively. Phosphorylation of TH-1 and TH-2 by PKA activates both isoenzymes as shown by the increase in the affinity for the cofactor. No changes in kinetic parameters of the isoenzymes were observed after phosphorylation by CaM-PK II. Dopamine was found to inhibit both TH isoenzymes to the same extent as shown by their similar Ki values for dopamine. These values were increased after phosphorylation of each enzyme by PKA. Unlike TH-1, phosphorylation of TH-2 by CaM-PK II resulted in an increase of the Ki value for dopamine. This property may be related to the presence of the additional phosphorylated residue in TH-2 isoform.     

PACAP stimulates the sustained phosphorylation of tyrosine hydroxylase at serine 40

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is controlled by PACAP, acutely by phosphorylation at Ser40 and chronically by protein synthesis. Using bovine adrenal chromaffin cells we found that PACAP, acting via the continuous activation of PACAP 1 receptors, sustained the phosphorylation of TH at Ser40 and led to TH activation for up to 24 h in the absence of TH protein synthesis. The sustained phosphorylation of TH at Ser40 was not mediated by hierarchical phosphorylation of TH at either Ser19 or Ser31. PACAP caused sustained activation of PKA, but did not sustain activation of other protein kinases including ERK, p38 kinase, PKC, MAPKAPK2 and MSK1. The PKA inhibitor H89 substantially inhibited the acute and the sustained phosphorylation of TH mediated by PACAP. PACAP also inhibited the activity of PP2A and PP2C at 24 h. PACAP therefore sustained TH phosphorylation at Ser40 for 24 h by sustaining the activation of PKA and causing inactivation of Ser40 phosphatases. The PKA activator 8-CPT-6Phe-cAMP also caused sustained phosphorylation of TH at Ser40 that was inhibited by the PKA inhibitor H89. Using cyclic AMP agonist pairs we found that sustained phosphorylation of TH was due to both the RI and the RII isotypes of PKA. The sustained activation of TH that occurred as a result of TH phosphorylation at Ser40 could maintain the synthesis of catecholamines without the need for further stimulus of the adrenal cells or increased TH protein synthesis.     

Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 --> Gly (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-zeta and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.     

Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin-dependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for approximately 90% and approximately 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+ (1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.     

Intermittent Hypoxia-Induced NF-κB and HO-1 Regulation in Human Endothelial EA.hy926 Cells

Intermittent hypoxia (IH) is a hallmark feature in obstructive sleep apnea (OSA) which is increasingly recognized as an independent risk factor for atherosclerosis. Oxidative stress, inflammation, and cell apoptosis are major pathological events initiating or accelerating atherogenesis. This study addressed whether IH would affect these proatherogenic factors in endothelial cells and the mechanistic pathways involved. EA.hy926 cells were exposed to intermittent normoxia or IH for different numbers of cycles (32, 64, or 96). IH exposure time-dependently raised cellular GSSG/GSH ratio, increased production of IL-6 and IL-8, and accelerated cell apoptosis and death, concurrent with activation of NF-κB and inhibition of Nrf2/HO-1 pathways. At 64 cycles, inhibition of NF-κB attenuated IH-induced cellular oxidative stress and accumulation of inflammatory cytokines in cell culture medium but aggravated IH-induced cell apoptosis, while stimulation of HO-1 suppressed IH-induced cellular oxidative stress and cell apoptosis without affecting accumulation of inflammatory cytokines in cell culture medium. We demonstrated that early stage of exposure to IH-induced oxidative and inflammatory stresses leading to acceleration of cell apoptosis via NF-κB and Nrf2/HO-1 pathways in endothelial cells, suggesting the potential mechanisms for IH-induced vascular pathogenesis, in resemblance to OSA.     

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.     

Cyclin-dependent kinase 5 phosphorylates serine 31 of tyrosine hydroxylase and regulates its stability

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its activity is regulated by phosphorylation in the N-terminal regulatory domain. The proline-directed serine/threonine kinase cyclin-dependent kinase 5 (cdk5) plays an important role in diverse neuronal processes. In the present study, we identify TH as a novel substrate of cdk5. We show that cdk5 phosphorylates TH at serine 31 and that this phosphorylation is associated with an increase in total TH activity. In transgenic mice with increased cdk5 activity, the immunoreactivity for phosphorylated TH at Ser-31 is enhanced in neurons of the substantia nigra, a brain region enriched with TH-positive neurons. In addition, we demonstrate that co-expression of cdk5 and its regulatory activator p35 with TH increases the stability of TH. Consistent with these findings, TH protein levels are reduced in cdk5 knock-out mice. Importantly, the TH activity and protein turnover of the phosphorylation-defective mutant TH S31A was not altered by cdk5 activity. Taken together, these data suggest that cdk5 phosphorylation of TH is an important regulator of TH activity through stabilization of TH protein levels.     

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.     

Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon beta-adrenergic stimulation in normal and failing hearts

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.     

Regulation of microtubule dynamics by Ca2+/calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18.

Oncoprotein 18 (Op18; also termed p19, 19K, p18, prosolin, and stathmin) is a regulator of microtubule (MT) dynamics and is phosphorylated by multiple kinase systems on four Ser residues. In addition to cell cycle-regulated phosphorylation, external signals induce phosphorylation of Op18 on Ser-25 by the mitogen-activated protein kinase and on Ser-16 by the Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr). Here we show that induced expression of a constitutively active mutant of CaMK IV/Gr results in phosphorylation of Op18 on Ser-16. In parallel, we also observed partial degradation of Op18 and a rapid increase of total cellular MTs. These results suggest a link between CaMK IV/Gr, Op18, and MT dynamics. To explore such a putative link, we optimized a genetic system that allowed conditional coexpression of a series of CaMK IV/Gr and Op18 derivatives. The result shows that CaMK IV/Gr can suppress the MT-regulating activity of Op18 by phosphorylation on Ser-16. In line with these results, by employing a chemical cross-linking protocol, it was shown that phosphorylation of Ser-16 is involved in weakening of the interactions between Op18 and tubulin. Taken together, these data suggest that the mechanism of CaMK IV/Gr-mediated suppression of Op18 activity involves both partial degradation of Op18 and direct modulation of the MT-destabilizing activity of this protein. These results show that Op18 phosphorylation by CaMK IV/Gr may couple alterations of MT dynamics in response to external signals that involve Ca2+.     

Post-translational modification of glutamic acid decarboxylase 67 by intermittent hypoxia: evidence for the involvement of dopamine D1 receptor signaling

Intermittent hypoxia (IH) associated with sleep apnea leads to cardio-respiratory morbidities. Previous studies have shown that IH alters the synthesis of neurotransmitters including catecholamines and neuropeptides in brainstem regions associated with regulation of cardio-respiratory functions. GABA, a major inhibitory neurotransmitter in the CNS, has been implicated in cardio-respiratory control. GABA synthesis is primarily catalyzed by glutamic acid decarboxylase (GAD). In this study, we tested the hypothesis that IH like its effect on other transmitters also alters GABA synthesis. The impact of IH on GABA synthesis was investigated in pheochromocytoma 12 cells, a neuronal cell line which is known to express active form of GAD67 in the cytosolic fraction and also assessed the underlying mechanisms contributing to IH-evoked response. Exposure of cell cultures to IH decreased GAD67 activity and GABA level. IH-evoked decrease in GAD67 activity was caused by increased cAMP - protein kinase A (PKA) - dependent phosphorylation of GAD67, but not as a result of changes in either GAD67 mRNA or protein expression. PKA inhibitor restored GAD67 activity and GABA levels in IH treated cells. Pheochromocytoma 12 cells express dopamine 1 receptor (D1R), a G-protein coupled receptor whose activation increased adenylyl cyclase activity. Treatment with either D1R antagonist or adenylyl cyclase inhibitor reversed IH-evoked GAD67 inhibition. Silencing D1R expression with siRNA reversed cAMP elevation and GAD67 inhibition by IH. These results provide evidence for the role of D1R-cAMP-PKA signaling in IH-mediated inhibition of GAD67 via protein phosphorylation resulting in down-regulation of GABA synthesis.     

Functional consequence of protein kinase A-dependent phosphorylation of the cardiac ryanodine receptor: sensitization of store overload-induced Ca2+ release

The phosphorylation of the cardiac Ca(2+)-release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.