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• W2000954692 abstract "The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser19 and Ser40produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser19phosphorylation is less pronounced than that of the Ser40phosphorylation. The effect of Ser19 and Ser40phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser19 and Ser40phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser19 and Ser40 were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser40 is ∼2- to 3-fold higher if Ser19 is already phosphorylated. These results suggest that phosphorylation of Ser19 alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser40 to kinases. The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser19 and Ser40produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser19phosphorylation is less pronounced than that of the Ser40phosphorylation. The effect of Ser19 and Ser40phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser19 and Ser40phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser19 and Ser40 were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser40 is ∼2- to 3-fold higher if Ser19 is already phosphorylated. These results suggest that phosphorylation of Ser19 alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser40 to kinases. tyrosine hydroxylase calcium/calmodulin-dependent protein kinase mitogen-activated protein kinase cAMP-dependent protein kinase high pressure liquid chromatography tyrosine hydroxylase in which Ser40 is substituted with Ala single-phosphorylated TH dual-phosphorylated TH single-phosphorylated TH at Ser19 single-phosphorylated TH at Ser40 p70 ribosomal protein S6 kinase phosphoinositide-dependent protein kinase 4-morpholinepropanesulfonic acid Tyrosine hydroxylase (TH)1 (EC 1.14.16.2) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline (1Fitzpatrick P.F. Annu. Rev. Biochem. 1999; 68: 355-381Crossref PubMed Scopus (433) Google Scholar). TH activity is controlled by both short term and long term regulatory mechanisms (2Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar). Short term regulation of TH is accomplished by dynamic changes in the phosphorylation state of the enzyme (2Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar). Four phosphorylation sites have been identified (Ser8, Ser19, Ser31, and Ser40). Phosphorylation of only three of these serine residues (Ser19, Ser31, and Ser40) is regulated in vivo (3Haycock J.W. Haycock D.A. J. Biol. Chem. 1991; 266: 5650-5657Abstract Full Text PDF PubMed Google Scholar). In vitro experiments have shown that a number of protein kinases phosphorylate these residues (2Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar). The most likely physiological candidates for the phosphorylation of Ser19, Ser31, and Ser40 are CaMKII, MAPK, and PKA, respectively (1Fitzpatrick P.F. Annu. Rev. Biochem. 1999; 68: 355-381Crossref PubMed Scopus (433) Google Scholar). It is only the phosphorylation of Ser40that has a major effect on TH activity. Phosphorylation of TH by PKA produces a 2-fold decrease in the Km for the cofactor BH4, but quantitatively the major effect of phosphorylation of Ser40 by PKA is to abolish feedback inhibition by the catecholamines (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar, 5Ribeiro P. Wang Y. Citron B.A. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9593-9597Crossref PubMed Scopus (47) Google Scholar, 6Ramsey A.J. Fitzpatrick P.F. Biochemistry. 1998; 37: 8980-8986Crossref PubMed Scopus (75) Google Scholar). Phosphorylation of dopamine-bound TH by PKA at Ser40 can activate TH by up to 20-fold (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar). The roles of the two other sites are less clear. Phosphorylation of Ser31in vitro produces a modest (less than 2-fold) increase in TH activity (7Haycock J.W. Ahn N.G. Cobb M.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2365-2369Crossref PubMed Scopus (240) Google Scholar). Phosphorylation of Ser19 will not by itself increase TH activity. Phosphorylation of Ser19 by CaMKII will only increase TH activity in the presence of the 14-3-3 activator protein (8Yamauchi T. Nakata H. Fujisawa H. J. Biol. Chem. 1981; 256: 5404-5409Abstract Full Text PDF PubMed Google Scholar, 9Itagaki C. Isobe T. Taoka M. Natsume T. Nomura N. Horigome T. Omata S. Ichinose H. Nagatsu T. Greene L.A. Ichimura T. Biochemistry. 1999; 38: 15673-15680Crossref PubMed Scopus (81) Google Scholar), and again this results only in a modest (2-fold) increase in the activity. The precise role of the 14-3-3 protein in TH regulation is presently unclear (9Itagaki C. Isobe T. Taoka M. Natsume T. Nomura N. Horigome T. Omata S. Ichinose H. Nagatsu T. Greene L.A. Ichimura T. Biochemistry. 1999; 38: 15673-15680Crossref PubMed Scopus (81) Google Scholar, 10Kleppe R. Toska K. Haavik J. J. Neurochem. 2001; 77: 1097-1107Crossref PubMed Scopus (62) Google Scholar). It has been found that mutation of Ser19to leucine had no effect on the potassium-induced increase in TH activity in intact AtT-20 cells (11Haycock J.W. Lew J.Y. Garciaespana A. Lee K.Y. Harada K. Meller E. Goldstein M. J. Neurochem. 1998; 71: 1670-1675Crossref PubMed Scopus (60) Google Scholar). Phosphorylation alters enzyme activity by the introduction of a bulky group, which is also negatively charged. This will, in many cases, alter the conformation of the protein. In the case of TH, a decrease in the thermal stability of phosphorylated TH compared with non-phosphorylated TH has been interpreted in terms of a change of conformation in the phosphorylated form (12Lazar M.A. Truscott R.J. Raese J.D. Barchas J.D. J. Neurochem. 1981; 36: 677-682Crossref PubMed Scopus (26) Google Scholar). More recently, it has been shown that phosphorylation of Ser40 makes TH more sensitive to tryptic digestion, which indicates that a conformational change is likely to have occurred (13McCulloch R.I. Fitzpatrick P.F. Arch. Biochem. Biophys. 1999; 367: 143-145Crossref PubMed Scopus (37) Google Scholar). Unlike the above methods, gel filtration chromatography allows the extent of the conformational alteration to be determined. In the present work we used gel filtration chromatography to show that phosphorylation of both Ser40and Ser19 induces a substantial conformational change in TH. Recently, we developed a novel mass spectrometry method to examine the phosphorylation state of TH (14Graham M.E. Dickson P.W. Dunkley P.R. von Nagy-Felsobuki E.I. Anal. Biochem. 2000; 281: 98-104Crossref PubMed Scopus (9) Google Scholar). This method is unique in that it allows the number of phosphate groups on individual TH molecules to be measured. In this work, we used this methodology to show that phosphorylation of Ser19 increases the rate of phosphorylation of Ser40. Vent DNA polymerase was from New England Biolabs. T4 DNA ligase was from Roche Molecular Biochemicals and the restriction enzymes from Promega. Radiochemicals, heparin-Sepharose, and calmodulin-Sepharose were from Amersham Pharmacia Biotech. The oligonucleotides were synthesized by Bresatec Ltd. The catalytic subunit of PKA was from Sigma. HPLC-grade acetonitrile and AnalaR-grade formic acid, methanol, and chloroform were from BDH Laboratory Supplies (Merck Pty. Limited, Kilsyth, Australia). The concentration of purified TH was determined by A280 measurements, using an ε1% 280 value of 10.4 (15Haavik J. Andersson K.K. Petersson L. Flatmark T. Biochim. Biophys. Acta. 1988; 953: 142-156Crossref PubMed Scopus (74) Google Scholar) and an assumed molar mass of 56 kDa. Rat adrenal TH cDNA (16Quinsey N.S. Lenaghan C.M. Dickson P.W. J. Neurochem. 1996; 66: 908-914Crossref PubMed Scopus (25) Google Scholar) was cloned into pET-3a. A Lac inducible protein expression system was used that required isopropyl-1-thio-β-d-galactopyranoside induction for the production of the recombinant protein. The expression and the purification of the recombinant TH was based on the procedures described elsewhere (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar). However, the growth and induction ofEscherichia coli was at 32 °C rather than 37 °C. No exogenous iron was added to TH. Site-directed mutagenesis was used to replace serine 40 with alanine in TH ([S40A]TH) (17Dickson P.W. Jennings I.G. Cotton R.G.H. J. Biol. Chem. 1994; 269: 20369-20375Abstract Full Text PDF PubMed Google Scholar). The mutated cDNA was completely sequenced to verify that it contained only the desired amino acid substitution. Sequencing was performed by the Newcastle Biomolecular Research Facility. The [S40A]TH was expressed and purified using the protocol described for TH. When the [S40A]TH was phosphorylated in the presence of [γ-32P]ATP by PKA, no incorporation of radioactivity was observed. When the phosphorylation of [S40A]TH by CaMKII was analyzed by tryptic digestion and HPLC (18Bobrovskaya L. Cheah T.B. Bunn S.J. Dunkley P.R. J. Neurochem. 1998; 70: 2565-2573Crossref PubMed Scopus (33) Google Scholar), only a single peak corresponding to the position of the Ser19 peptide was detected. CaMKII was purified from rat brain by calmodulin-Sepharose chromatography (19Rostas J.A. Seccombe M. Weinberger R.P. J. Neurochem. 1988; 50: 945-953Crossref PubMed Scopus (44) Google Scholar). The major protein present in the fractions was CaMKII (>90%) as assessed by Coomassie Blue staining and Western blot using an anti-CaMKII antibody. CaMKII activity was assayed as described (20Lengyel I. Cammarota M. Brent V.A. Rostas J.A. J. Neurochem. 2001; 76: 149-154Crossref PubMed Scopus (9) Google Scholar). For PKA phosphorylation, TH was incubated with the catalytic subunit of PKA in a buffer containing 60 mm MOPS pH 7.2, 200 µmATP, and 90 mm MgCl2 at 30 °C. Non-phosphorylated TH control reactions contained every component except PKA. The units of PKA were as defined by the manufacturer. For CaMKII phosphorylation TH (or [S40A]TH) was incubated with CaMKII in a buffer containing 30 mm Tris, pH 7.5, 10 mmMgCl2, 200 µm ATP, 1.2 mmCaCl2, and 1.2 µm calmodulin. The reactions were incubated at 30 °C. Non-phosphorylated TH (or [S40A]TH) control reactions contained every component except CaMKII. One unit is defined as the amount of CaMKII that transfers 1.0 nmol of phosphate from ATP to autocamtide per min at pH 7.2 at 30 °C. TH was incubated in the presence of 50 mm Tris, pH 7.5, 5 mm GSH, and 200 µm dopamine for 30 min at 30 °C in a final volume of 100 µl. TH and [S40A]TH preparations were precipitated with 3 m(NH4)2SO4 and then centrifuged for 10 min at 4 °C at 16,000 × g. The pellet was resuspended in 50 mm Tris, pH 7.5. The resuspended pellet was centrifuged at 96,600 × g for 30 min prior to gel filtration to remove any particulate component. Samples were then analyzed using an Amersham Pharmacia Biotech SMART system. The TH samples were applied to a Superose 6 PC 3.2/30 column equilibrated with 50 mm Tris, pH 7.5, and 150 mm NaCl and chromatographed at 12 °C using a flow rate of 40 µl/min. The peak elution time was determined using the software package available with the instrument. The column was calibrated using aldolase, catalase, ferritin, and thyroglobulin as standards. To ensure that there was no change in the performance of the SMART system, four non-phosphorylated TH samples were run interspersed with the individual samples of each TH condition being tested. No change in the elution position of non-phosphorylated TH was found over a period of 6 months. After incubation of TH or [S40A]TH with or without kinases as described above, the reactions were halted using the chloroform/methanol/water precipitation method (21Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar). Chloroform/methanol/water precipitates of TH were dissolved in a 2% (v/v) formic acid solution to a final concentration of 2 µm and subjected to aspartic acid cleavage (14Graham M.E. Dickson P.W. Dunkley P.R. von Nagy-Felsobuki E.I. Anal. Biochem. 2000; 281: 98-104Crossref PubMed Scopus (9) Google Scholar). The sample preparation, liquid chromatography and electrospray ionization mass spectrometry (Platform II, Micromass, Altrincham, United Kingdom), and data processing have been described previously (14Graham M.E. Dickson P.W. Dunkley P.R. von Nagy-Felsobuki E.I. Anal. Biochem. 2000; 281: 98-104Crossref PubMed Scopus (9) Google Scholar). According to the Michaelis-Menten mechanism (22Michaelis L. Menten M.L. Biochem. Z. 1913; 49: 333-369Google Scholar), the phosphorylation of a substrate by a kinase reduces to a reaction that is zero order in the substrate on the assumption that the substrate concentration is far greater thanKm. The rate of phosphorylation is then dependent on the concentration of the kinase and independent of the equilibrium of binding and dissociation between the substrate and kinase. The phosphorylation of TH by CaMKII can be described as the following in Equation 1, TH→k′TH­P→k″TH­P2Equation 1 where the phosphorylation of TH (at either Ser19 or Ser40) produces single-phosphorylated TH (TH-P) and further phosphorylation produces dual-phosphorylated TH (TH-P2). The rate constants k′ and k“ describe the first and second phosphorylation steps, respectively. The three components, TH, TH-P, and TH-P2 may be easily quantified using mass spectrometry as demonstrated in Fig. 1. However, to determine the rate of phosphorylation of each site (Ser19 or Ser40), the production of TH-P2 must also be taken into account and it may occur via two different pathways as shown in Equation 2, TH→k1TH­P19→k3TH­P2 TH→k2TH­P40→k4TH­P2Equation 2 where k1 and k2are the rate constants for the formation of the single-phosphorylated species, TH phosphorylated at Ser19 (TH-P19) and TH phosphorylated at Ser40 (TH-P40), respectively. Here, k3 andk4 are the rate constants for the formation of TH-P2 by phosphorylation of TH-P19 and TH-P40, respectively. Equation 2 is sufficiently detailed to describe the possible influence of one phosphorylated site (Ser19 or Ser40) over the other. Particularly telling would be differences between the rate constants for single phosphorylation and double phosphorylation of each site (i.e. k1versus k4 and k2versus k3). The strategy for determining the four rate constants involved three different experiments and a total of three groups of phosphorylation reactions. The phosphorylation reactions were stopped at various times so that the mole fraction (χ) of product could be plotted against time, and the resulting curve was fitted to produce a rate constant. The equations used for curve fitting are based on a consecutive reaction model (23Harcourt A.V. Esson W. Philos. Trans. R. Soc. Lond-Biol. Sci. 1866; 156: 193-220Crossref Google Scholar) (see Equation 1). This model was further elaborated to accommodate the two simultaneous consecutive reactions of Equation2. The rate equations describing the concentration of TH, TH-P19, and TH-P40 have the form shown in Equation 3. [TH]=[TH]0e−(k1+k2)tEquation 3 [TH-P19]=k1k3−k1(e−k1t−e−k3t)[TH]0Equation 4 [TH-P40]=k2k4−k2(e−k2t−e−k4t)[TH]0Equation 5 Curve fitting of the experimental data was done using Mathematica version 4.0.2.0. Mass spectrometry methods for the quantification of incorporated phosphate have only recently been developed (24Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6591-6596Crossref PubMed Scopus (944) Google Scholar, 25Weckwerth W. Willmitzer L. Fiehn O. Rapid Commun. Mass Spectrom. 2000; 14: 1677-1681Crossref PubMed Scopus (102) Google Scholar). The advantage to using mass spectrometry over other techniques is the ability to detect non-phosphorylated and phosphorylated proteins simultaneously. However, direct comparison of the amount of non-phosphorylated to phosphorylated protein is difficult because the efficiencies of ionization and detection for the non-phosphorylated versus phosphorylated protein may differ (26Poulter L. Ang S.G. Williams D.H. Cohen P. Biochim. Biophys. Acta. 1987; 929: 296-301Crossref PubMed Scopus (18) Google Scholar, 27Craig A.G. Engstrom A. Bennich H. Hoffmann-Posorske E. Meyer H.E. Biol. Mass Spectrom. 1991; 20: 565-574Crossref PubMed Scopus (12) Google Scholar). Recently, a novel mass spectrometric method was developed in our laboratory to quantify the extent of phosphorylation of TH (14Graham M.E. Dickson P.W. Dunkley P.R. von Nagy-Felsobuki E.I. Anal. Biochem. 2000; 281: 98-104Crossref PubMed Scopus (9) Google Scholar). This method utilizes an 8-kDa N-terminal fragment that is released after formic acid cleavage of TH. This 8-kDa fragment contains all the phosphorylation sites in TH. It is also an ideal-sized fragment for ensuring that the efficiency of detection is the same when the fragment is phosphorylated. The advantage of this method is that the proportion of nonphosphorylated TH can be directly compared with the amount of single- or multiple-phosphorylated TH (14Graham M.E. Dickson P.W. Dunkley P.R. von Nagy-Felsobuki E.I. Anal. Biochem. 2000; 281: 98-104Crossref PubMed Scopus (9) Google Scholar). Electrospray ionization mass spectrometry was used to analyze recombinant TH phosphorylated by PKA, a control TH sample (no PKA), TH phosphorylated by CaMKII, [S40A]TH phosphorylated by CaMKII, and a control [S40A]TH sample (no CaMKII). The spectra of the 8-kDa moieties are shown in Fig. 1. The TH and [S40A]TH control samples are shown in Fig. 1, A andB, respectively. These non-phosphorylated samples were determined to have molar masses of 8146.7 and 8129.0 Da, respectively, which agree well (i.e. within the resolution of the instrument) with the theoretical molar masses of 8144.0 and 8128.0 Da, respectively. Analysis of the [S40A]TH mutant by this method shows that the molar mass is consistent with a substitution of alanine for serine at position 40. This provides an independent confirmation of the identity of the mutant TH. Phosphorylation of TH by PKA (Fig. 1D) led to the expected increase in size of the N-terminal cleavage product because of the addition of a phosphate group (80 Da) to Ser40. The molar mass was determined to be 8225.6 Da, which agrees well with the theoretical molar mass of 8224.0 Da. This fragment is 78.9 Da larger than the non-phosphorylated TH cleavage product. Moreover, it can be seen that, within the detection limits of the instrument, there is no non-phosphorylated TH remaining. When TH is phosphorylated with CaMKII (Fig. 1E), the dual-phosphorylated form is predominant. At 8305.4 Da, it appears 158.7 Da greater in mass than the non-phosphorylated form. No non-phosphorylated TH was detected, and only a very small amount of single-phosphorylated TH was evident in the spectrum. Phosphorylation of [S40A]TH by CaMKII shows that a single phosphate group is incorporated (Ser19) and that there is no non-phosphorylated form remaining (Fig. 1C). The phosphorylated [S40A]TH cleavage product is 79.7 Da larger at a molar mass of 8208.7 Da (which agrees well with the theoretical molar mass of 8208.0 Da). Gel filtration separates molecules on the basis of both size and shape. If the size of a given protein does not change, any changes in elution position must be due to changes in the shape of the molecule. TH is a tetramer of identical subunits, and it is known that even if the first 155 residues are deleted, the molecule is still a tetramer (28Goodwill K.E. Sabatier C. Marks C. Raag R. Fitzpatrick P.F. Stevens R.C. Nat. Struc. Biol. 1997; 4: 578-585Crossref PubMed Scopus (269) Google Scholar, 29Daubner S.C. Lohse D.L. Fitzpatrick P.F. Protein Sci. 1993; 2: 1452-1460Crossref PubMed Scopus (70) Google Scholar). Therefore, it is unlikely that phosphorylation events in the first 40 residues would affect tetramer formation. The gel filtration elution profiles of phosphorylated TH and dopamine-bound TH are shown in Fig. 2. Phosphorylation by PKA produces a substantial decrease in the elution time of TH (Fig. 2A). This change is highly significant (Table I). TH phosphorylated by PKA migrated with an elution time that is consistent with an increase in size of 14%. This apparent increase in the size of TH could be due to either the phosphorylated molecule adopting a more open conformation or to the tetramer weakly self-associating. If the tetramer was weakly self-associating, we would expect to see a substantial peak broadening because of the interconversion between the TH tetramer and higher order complexes of the TH tetramer during the chromatographic process. As can be seen in Fig. 2A, this is clearly not the case. Therefore, we conclude that the observed shift in elution position of TH is due to a substantial opening of the molecule.Table IEffect of phosphorylation of Ser19 and Ser40 and of dopamine binding on the elution time of TH or [S40A]TH after gel filtration chromatographyEnzymeElution timenStudent's ttest p valueminTH37.70 ± 0.028[S40A]TH37.53 ± 0.019<<0.0011-aWhen compared to TH.TH + PKA36.86 ± 0.018<<0.0011-aWhen compared to TH.<<0.0011-bWhen compared to TH + CKII.TH + CaMKII36.71 ± 0.028<<0.0011-aWhen compared to TH.[S40A]TH + CaMKII37.22 ± 0.038<<0.0011-cWhen compared to [S40A]TH.TH + Dopamine37.93 ± 0.028<<0.0011-aWhen compared to TH.12 µm TH was phosphorylated to full stoichiometry with 186 units/ml PKA or 25 units/ml CaMKII or was incubated with 200 µm dopamine and processed as described under “Experimental Procedures.” 12 µm [S40A]TH was phosphorylated to full stoichiometry with 25 units/ml CaMKII and processed as described under “Experimental Procedures.” The samples were chromatographed in a Superose 6 gel filtration column, and the peak elution times were determined. n = number of individual experiments performed. Values are shown as means ± S.E.1-a When compared to TH.1-b When compared to TH + CKII.1-c When compared to [S40A]TH. Open table in a new tab 12 µm TH was phosphorylated to full stoichiometry with 186 units/ml PKA or 25 units/ml CaMKII or was incubated with 200 µm dopamine and processed as described under “Experimental Procedures.” 12 µm [S40A]TH was phosphorylated to full stoichiometry with 25 units/ml CaMKII and processed as described under “Experimental Procedures.” The samples were chromatographed in a Superose 6 gel filtration column, and the peak elution times were determined. n = number of individual experiments performed. Values are shown as means ± S.E. CaMKII phosphorylates both Ser19 and Ser40 on TH. To examine the effect of phosphorylation of Ser19 alone on the shape of TH, we generated a mutant [S40A]TH. This mutant has biochemical properties similar to that of TH (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar). When analyzed by gel filtration, the [S40A]TH had a small but significant change in elution position when compared with TH (Table I). Therefore, the CaMKII phosphorylated [S40A]TH was compared with the unphosphorylated mutant instead of TH. When [S40A]TH was phosphorylated at Ser19, it eluted earlier than the control [S40A]TH (Fig. 2B). The results in Table I show that the decrease in the elution position of the CaMKII-phosphorylated [S40A]TH compared with the non-phosphorylated control is highly significant. The conformational change because of the phosphorylation of Ser19 is smaller than that observed with Ser40. In this case, the change is equivalent to an increase in size of TH of ∼5%. When TH was phosphorylated by CaMKII (at both Ser19 and Ser40), there was a substantial change in the elution time when compared with non-phosphorylated TH. This change in elution time is equivalent to an increase in size of TH of ∼16%. In addition, the CaMKII-phosphorylated TH eluted earlier than the PKA (Ser40)-phosphorylated TH (Figure 2A). This change is small but nevertheless highly significant (see Table I). Dopamine and the other catecholamines inhibit TH and this inhibition is relieved by phosphorylation at Ser40 (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar). When dopamine-bound TH was analyzed, there was a small but highly significant increase in the elution time compared with TH (Table I). In this case the change is equivalent to a decrease in the size of TH of ∼4%. The kinetic model used to derive the rate constants is detailed under “Experimental Procedures.” Initially, TH was phosphorylated by CaMKII. A plot of the mole fraction of TH (χTH) versus time is shown in Fig. 3A. A curve can be fitted to the data to determine k′ (see Equation 1). The equation used in curve fitting is a simpler version of Equation 3 in that it can now be shown that k′ = k1+ k2. The fitted curve yielded the equation χTH = e−0.288 ± 0.017t with an R2 of 0.962. Therefore, k′ is equal to 0.288 ± 0.017 min−1. In a further experiment, [S40A]TH was phosphorylated by CaMKII. The mole fraction of [S40A]TH (χTHSer40Ala) plotted against time is shown in Fig. 3B. The absence of the Ser40 simplifies the description of the kinetic model so that the only rate constant in the rate equation isk1 because k2 has been eliminated by use of the mutant (see Equation 2). Hence,k1 can be determined by curve fitting with an exponential decay-type equation as in Equation 3. The equation χTHS40A =e−0.122 ± 0.009t is found to fit the consumption of [S40A]TH with anR2 of 0.929. Therefore,k1 is equal to 0.122 ± 0.009 min−1. Because k′ = k1 +k2, it may be inferred thatk2 is equal to 0.166 ± 0.026 min−1. In a final experiment, TH was first phosphorylated with PKA to a point where ∼100% of the TH was converted to TH-P40. CaMKII was then added to the reaction, and the disappearance of TH-P was determined. The plot of χTH-Pversus time for the CaMKII phosphorylation, which followed PKA phosphorylation, is shown in Fig. 3C. It is assumed that the PKA, present during the CaMKII phosphorylation, did not interfere with the binding of TH and CaMKII. There is no evidence in the literature that these kinases interact. Because the Ser40 sites are stoichiometrically phosphorylated, the only site available for phosphorylation by CaMKII is Ser19. The rate of consumption of TH-P40 in this secondary phosphorylation by CaMKII is described by an exponential decay because k2 is eliminated. The fit yielded the equation χTH-P = e−0.101 ± 0.004t with an R2 of 0.978. Therefore, k4 is equal to 0.101 ± 0.004 min−1. The remaining rate constant, k3, can be calculated by curve fitting the mole fraction of TH-P (χTH-P) versus time for the phosphorylation of TH by CaMKII (the initial experiment). Equations 4 and 5 were combined to yield an expression in terms of [Th-P] = [TH-P19] + [TH-P40]. The measured values fork1, k2 andk4 were substituted, leavingk3 as the only unknown in the curve fit. Fig.3D shows the curve fit for the χTH-Pversus time, which was used to determinek3. The fit produced Equation 6, χTH­P=0.326e−0.122te−0.497±0.082t−2.55e−0.166t−e−0.101tEquation 6 with an R2 of 0.871. Therefore,k3 = 0.497 ± 0.082 min−1. The determined rate constants are shown in TableII.Table IIRate constants for the phosphorylation of TH by CaMKIIRate constantMeasurement ± S.E.R2min−1k′0.288 ± 0.0170.962k10.122 ± 0.0090.929k20.166 ± 0.0262-aInferred result fromk′ and k1.k30.497 ± 0.0820.871k40.101 ± 0.0040.978The rate constants are for the reactions shown in Equations 1 and 2. The constants were determined by curve fitting mass spectral data from three separate phosphorylation experiments (see Fig. 3 and text).2-a Inferred result fromk′ and k1. Open table in a new tab The rate constants are for the reactions shown in Equations 1 and 2. The constants were determined by curve fitting mass spectral data from three separate phosphorylation experiments (see Fig. 3 and text). Although the measurements have shown that k2 >k1, because of the uncertainty in the measurement of k1, it can only be concluded that the two rate constants are comparable in magnitude (i.e. k1 ∼k2). Similarly, it appears thatk1 > k4, but it can only be concluded that the two rate constants are comparable in magnitude (i.e. k1 ∼k4). However, it is clear thatk3 > k1,k2, and k4. The significance of k3 > k2will be discussed in greater detail below. Nevertheless, given the precision of these results, it can be concluded thatk3 is at least 2- to 3-fold larger thank2. Phosphorylation of proteins is one of the key elements involved in short term regulatory mechanisms in the cell. The addition of a phosphate group to a protein could act in a number of ways. The phosphate group could simply block the entry of a substrate into a binding pocket with no change in the structure of the protein. Alternatively, it could induce a subtle change in the structure of the protein to alter the substrate binding site but not change the overall shape of the molecule (e.g. the activation of MAPK by phosphorylation) (30Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). Finally, phosphorylation could produce dramatic changes in the overall shape of proteins that would abolish the substrate binding site. It has been previously shown that phosphorylation of TH at Ser40 makes the molecule more sensitive to tryptic digestion (13McCulloch R.I. Fitzpatrick P.F. Arch. Biochem. Biophys. 1999; 367: 143-145Crossref PubMed Scopus (37) Google Scholar). The increased accessibility of the trypsin-sensitive site suggests that phosphorylation does alter the structure of TH. We have presented here, for the first time, direct evidence showing that phosphorylation of Ser40 induces a major conformational change in TH such that it adopts a much more open structure. In addition, we have shown that phosphorylation of TH at Ser19induces a smaller but very significant opening of TH. The simplest model that could account for these changes would be one in which the N terminus of TH forms a hinged regulatory arm with the hinge located somewhere C-terminal to Ser40. Such a model would explain why phosphorylation of Ser40, which would be closer to the hinge, would produce a more dramatic effect on shape than phosphorylation of Ser19, which would be further away from the hinge. Under this model we would have expected that phosphorylation of Ser40 alone would produce the maximal opening of TH, and therefore the elution time of the dual-phosphorylated TH would be the same as that obtained for TH phosphorylated only at Ser40. Our data show that the dual-phosphorylated TH adopts a more open conformation than TH phosphorylated at Ser40 alone; thus the effect of phosphorylation of Ser19 and Ser40 is additive. The data are not consistent with a simple model of a hinged regulatory arm. Previous work has shown that binding of dopamine renders TH less sensitive to tryptic digestion (13McCulloch R.I. Fitzpatrick P.F. Arch. Biochem. Biophys. 1999; 367: 143-145Crossref PubMed Scopus (37) Google Scholar). This could be due to a dopamine-induced conformational change in TH, which would make it less accessible to trypsin, or it could simply be that dopamine sterically hinders access of trypsin to the active site. We have shown here that dopamine binding produces a small but significant contraction of the TH molecule. It is more difficult to phosphorylate TH with PKA when it is bound with dopamine (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar). The compaction of TH produced by dopamine binding as shown in this report probably plays an important role in reducing the accessibility of Ser40 to PKA. There is a large body of evidence confirming that in vitrophosphorylation of TH at Ser40 by PKA is associated with a strong activation of the enzyme (2Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar). This also occurs in intact cells. Treatment of bovine adrenal chromaffin cells with forskolin produces an increase in the phosphorylation of Ser40 with a concomitant increase in TH activity (31Cheah T.B. Bobrovskaya L. Goncalves C.A. Hall A. Elliot R. Lengyel I. Bunn S.J. Marley P.D. Dunkley P.R. J. Neurosci. Methods. 1999; 87: 167-174Crossref PubMed Scopus (21) Google Scholar). The effect of PKA phosphorylation of Ser40 is to decrease the binding of the catecholamines that inhibit TH. Direct evidence for a significant conformational change in TH shown here is likely to be a critical part of this reduction in catecholamine binding. The role of Ser19 phosphorylation in TH activation has been rather more controversial. Although CaMKII can phosphorylate both Ser19 and Ser40in vitro, it cannot activate TH by itself (8Yamauchi T. Nakata H. Fujisawa H. J. Biol. Chem. 1981; 256: 5404-5409Abstract Full Text PDF PubMed Google Scholar). Evidence from intact cells also indicates that phosphorylation of Ser19 does not directly activate TH. Treatment of bovine adrenal chromaffin cells with anisomycin produced a significant increase in the phosphorylation of Ser19 but not of Ser31 or Ser40(32Bobrovskaya L. Odell A. Leal R.B. Dunkley P.R. J. Neurochem. 2001; 78: 490-498Crossref PubMed Scopus (37) Google Scholar). Under these conditions there was no increase in TH activity. 2L. Bobrovskaya and P. R. Dunkley, unpublished results. Further, it has been shown that abolishing Ser19 phosphorylation has no effect on the final activation state of TH in response to potassium-induced depolarization in vivo (11Haycock J.W. Lew J.Y. Garciaespana A. Lee K.Y. Harada K. Meller E. Goldstein M. J. Neurochem. 1998; 71: 1670-1675Crossref PubMed Scopus (60) Google Scholar). These authors suggested that Ser19 phosphorylation might indirectly affect TH activity by facilitating phosphorylation at other sites that directly influence TH activity. If this were correct, we would expect phosphorylation of Ser19 to precede the phosphorylation of the other sites. It has been shown that phosphorylation of Ser19 precedes the phosphorylation of Ser40 in response to nicotine (33Bunn S.J. Sim A.T. Herd L.M. Austin L.M. Dunkley P.R. J. Neurochem. 1995; 64: 1370-1378Crossref PubMed Scopus (34) Google Scholar), histamine (33Bunn S.J. Sim A.T. Herd L.M. Austin L.M. Dunkley P.R. J. Neurochem. 1995; 64: 1370-1378Crossref PubMed Scopus (34) Google Scholar), angiotensin II (32Bobrovskaya L. Odell A. Leal R.B. Dunkley P.R. J. Neurochem. 2001; 78: 490-498Crossref PubMed Scopus (37) Google Scholar), and potassium-induced depolarization (11Haycock J.W. Lew J.Y. Garciaespana A. Lee K.Y. Harada K. Meller E. Goldstein M. J. Neurochem. 1998; 71: 1670-1675Crossref PubMed Scopus (60) Google Scholar). All these agents activate TH. We have shown here that Ser19 phosphorylation induces a conformational change in TH. This conformational change could increase the accessibility of Ser40 to kinases. If phosphorylation of Ser19 were a prerequisite for the rapid phosphorylation of Ser40, this would provide an explanation for the fact that in vivo Ser19 phosphorylation precedes the phosphorylation of Ser40 (see above). To obtain direct evidence that phosphorylation at Ser19 could alter the rate of phosphorylation of Ser40, we developed a kinetic analysis of Ser19 and Ser40 phosphorylation. This analysis was dependant on our ability to simultaneously quantify whether individual molecules had zero, one, or two phosphate groups incorporated. Electrospray ionization mass spectrometry is the only direct methodology capable of this type of measurement. The results clearly show that initial phosphorylation of Ser19significantly increased the rate of phosphorylation of Ser40. This is consistent with a model in which phosphorylation of Ser19 alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser40 to kinases. It is clear that phosphorylation of TH at Ser40 is closely associated with TH activation. Hence, it is likely that the phosphorylation of Ser19 indirectly increases the rate of activation of TH. The data presented in this paper suggest that there is a hierarchical structure in TH phosphorylation. This interdependence of phosphorylation sites has been found in a number of systems. The best known is the phosphorylation of glycogen synthase by casein kinase II and glycogen synthase kinase 3. Casein kinase II has to phosphorylate glycogen synthase at one site before glycogen synthase kinase 3 can then phosphorylate four further serine residues in a clearly defined order. This sequential phosphorylation of glycogen synthase is achieved by the fact that each phosphorylation event produces a new kinase recognition sequence (34Fiol C.J. Wang A. Roeske R.W. Roach P.J. J. Biol. Chem. 1990; 265: 6061-6065Abstract Full Text PDF PubMed Google Scholar, 35Fiol C.J. Mahrenholz A.M. Wang Y. Roeske R.W. Roach P.J. J. Biol. Chem. 1987; 262: 14042-14048Abstract Full Text PDF PubMed Google Scholar). This mechanism is quite different from the one we are proposing for TH in which phosphorylation at one site induces a conformational change that allows increased access of a kinase to a second site. A situation much more akin to that for TH can be seen with the p70 ribosomal protein S6 kinase, p70s6k (36Dennis P.B. Pullen N. Pearson R.B. Kozma S.C. Thomas G. J. Biol. Chem. 1998; 273: 14845-14852Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 37Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar). It was proposed that phosphorylation of three sites within the autoinhibitory domain of p70s6k alters the conformation of p70s6k to allow the phosphorylation of Thr389. Phosphorylation of Thr389, in turn, further alters the conformation of p70s6k to allow the phosphoinositide-dependent protein kinase, PDK1, to phosphorylate and activate p70s6k. PDK1 can also activate protein kinase B. It was suggested that even though p70s6k and protein kinase B are both activated by PDK1, they could be differentially regulated, as different pathways control the accessibility of PDK1 to each kinase (36Dennis P.B. Pullen N. Pearson R.B. Kozma S.C. Thomas G. J. Biol. Chem. 1998; 273: 14845-14852Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In the case of TH it has been shown that, at least in vitro, six kinases can phosphorylate Ser40 (2Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar). At first glance, this suggests that TH could be activated through a large number of pathways that may not be relevant to increased catecholamine synthesis and secretion. The hierarchical TH phosphorylation described here suggests that additional signaling pathways would need to be activated to increase TH activity. This would provide additional specificity so that TH would only be activated under appropriate conditions. We thank Dr. Martin Cammarota for assistance with the characterization of the CaMKII preparations and for helpful comments and Amanda Hall-Griffin and Dr. Alistair Sim for use of the SMART system. We also thank Dr Geoffrey J. Howlett for helpful discussions." @default.
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• W2000954692 title "Phosphorylation of Ser19 Alters the Conformation of Tyrosine Hydroxylase to Increase the Rate of Phosphorylation of Ser40" @default.
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