Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect
of Protein Kinase and Phosphatase Inhibitors on Ser’9and Ser4°Phosphorylation
Carlos-Alberto Gonçalves, Amanda Hall, Alistair T. R. Sim, Stephen J. Bunn,*philip D. Marley, Tat B. Cheah, and Peter R. Dunkley
Neuroscience Group, Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences,University of Newcastle, Newcastle, New South Wales; and * Department of Pharmacology,
University of Melbourne, Parkville, Victoria, Australia
Abstract: The protein kinases and protein phosphatasesthat act on tyrosine hydroxylase in vivo have not beenestablished. Bovine adrenal chromaffin cells were perme-abilized with digitonin and incubated with [y-32P]ATP,inthe presence or absence of 10 p~MCa2~,1 jiM cyclicAMP, 1 jaM phorbol dibutyrate, orvarious kinase orphos-phatase inhibitors. Ca2~increased the phosphorylationof Ser19 and Ser40. Cyclic AMP, and phorbol dibutyratein the presence of Ca2~,increased the phosphorylationof only Ser40. Ser31 and Ser8 were not phosphorylated.The Ca2~-stimuIatedphosphorylation of Ser19 was in-completely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase 11(46% with KN93 and 68%with CaM-PKII 273—302), suggesting that another pro-tein kinase(s) was contributing to the phosphorylation ofthis site. The Ca2~-stimulated phosphorylation of Ser4°was reduced by specific inhibitors of protein kinase A(56% with H89 and 38% with PKAi 5—22 amide) andprotein kinase C (70% with Ro 31 -8220 and 54% withPKCi 19—31), suggesting that protein kinases A and Ccontributed to most of the phosphorylation of this site.Results with okadaic acid and microcystin suggested thatSer19 and Ser4°were dephosphorylated by PP2A. KeyWords: Adrenal chromaffin cells—Permeabilization——Tyrosine hydroxylase—Protein phosphorylation—Cal-cium—Protein kinases—Protein phosphatases.J. Neurochem. 69, 2387—2396 (1997).
Tyrosine hydroxylase (TH) is the rate-limiting en-zyme in catecholamine synthesis. Its activity is con-trolled by gene expression, by feedback inhibition ofcatecholamines, and by protein phosphorylation (Ku-mer and Vrana, 1996). Phosphorylation of TH in-creases its activity by relieving the inhibition due tocatecholamines and increasing the affinity of the hy-droxylase for its cofactor tetrahydrobiopterin (Daubneret al., 1992).
In vitroTH is able to be phosphorylated at four sites;Ser8 can be phosphorylated by a dimeric complex ofp34cdc2 and p58 cyclin A (Hall et al., 1992), Sert9can be phosphorylated by calciumlcalmodulin-stim-ulated protein kinase II (CaM-PKII) and mitogen-activated protein kinase-activated protein kinase 2(MAPKAP kinase 2) (Sutherland et al., 1993), Ser3’by two mitogen-activated protein kinases (MAPK Iand MAPK 2) (Haycock et al., 1992; Halloran andVulliet, 1994), and Ser4°by CaM-PKII (Sutherland etal., 1993), protein kinase A (PKA) (Daubner et al.,1992; Harada et al., 1996), protein kinase G (Roskoski
Received April 21, 1997; revised manuscript received July 18,1997; accepted July 24, 1997.
Address correspondence and reprint requests to Prof. P. Dunkleyat Discipline of Medical Biochemistry, University of Newcastle,Newcastle NSW 2308, Australia.
The present address of Dr. C.-A. Gonçalves is Departamento DcBioquimica, UFRGS, Porto Alegre, Brazil.
et al., 1987), protein kinase C (PKC) (Vulliet et al.,1985), and MAPKAP kinases 1 and 2 (Sutherland etal., 1993). Differences in moles of phosphate incorpo-rated permole of TH occur depending on which proteinkinase and incubation condition are used (Funakoshiet al., 1991; Sutherland et al., 1993). It is establishedthat phosphorylation of Ser40, or Ser31, alone increasesthe activity of TH (Sutherland et al., 1993). Phosphor-ylation of TH correlates with the activation of TH inthe presence of an activator protein (Yamauchi andFujisawa, 1981). However, if the enzyme is stabilizedwith other proteins, the activator is not required (Suth-erland et al., 1993), suggesting that the phosphoryla-tion of TH may destabilize the enzyme in situ. Ser4°and Sert9 are dephosphorylated primarily by proteinphosphatase 2A (PP2A), although protein phosphatase2C (PP2C) has some activity against Ser4°(Haavik etal., 1989; Berresheim and Kuhn, 1994). An endoge-nous Mn2~-stimulated protein phosphatase also de-phosphorylates TH (George et al., 1989).
All four sites on TH are phosphorylated in intactbovine adrenal chromaffin cells (BACCs). On depo-larization with acetylcholine, histamine, or high K ~the phosphorylation of Sert9, Ser31, and Ser4° is in-creased (Haycock, 1993; Bunn et al., 1995) and thereis a concomitant activation of TH (Tsutsui et al.,1994). The phosphorylation and TH activation arecompletely dependent on the rise in intracellular cal-cium (Ca2~).It is not certain which protein kinasesand/or protein phosphatases are responsible for theincreased phosphorylation of these three sites on THin the intact cells. PKA and PKC (Haycock, 1993),CaM-PKII (Yanagihara et al., 1994), MAPK (Hal-loran and Vulliet, 1994), and PP2A (Haavik et al.,1989) are all in BACCs, but it is yet to be determinedwhether MAPKAP kinase 1 and MAPKAP kinase 2are present. It is generally assumed that in BACCs,Sert9 is phosphorylated primarily by CaM-PKII, Ser3’by MAP kinase, and Ser4°by PKA and/or PKC, anddephosphorylation is catalyzed by PP2A (Haycock,1993).
The problem with purified enzymes and in vitro sys-tems for studying TH phosphorylation is that the condi-tions used are generally optimal for particular kinasesor phosphatases and do not represent the normal invivo conditions. Intact cells are obviously optimal forthe study of TH phosphorylation, but they suffer thedisadvantage of not providing ready access to intracel-lular enzymes. Permeabilized BACCs have beenwidely used to investigate the secretion of catechol-amines (Sarafian et al., 1987; Wilson, 1990) and thesecells offer significant advantages for the study of THphosphorylation in that endogenous kinases and phos-phatases act on TH, the ionic environment can be moreclosely matched to that occurring in vivo, the effectorsof the enzyme and their subcellular locations can belargely retained, and membrane-impermeant activatorsand inhibitors can gain access to the endogenous ki-nases and phosphatases. Permeabilized BACCs have
been used to study TH phosphorylation. Niggli et al.(1984) used brief exposure to electrical fields to per-meabilize BACCs and found that TH was phosphory-lated by two systems, one activated by Ca2~and oneby cyclic AMP, both of which led to an increase inTH activity. Yanagihara et al. (1987) investigated therole of PKC in digitonin-permeabilized BACCs andfound that preincubation of cells with phorbol estersincreased TH activity and that this was not inhibitedby the calmodulin antagonists trifluoperazine (TFP)and N- (6-aminohexyl )-5-chloro- 1 -naphthalenesulfon-amide hydrochloride (W7). The Ca2~-stimulatedphosphorylation of TH was blocked completely by apeptide derived from CaM-PKII (CaM-PKII 291—317)but not by the PKC inhibitory peptide (PKCi 19—31)in digitonin-permeabilized BACCs (Terbush and Holz,1990). No previous study has investigated the siteson TH-phosphorylated in digitonin-permeabilizedBACCs.
The aim of this study was to use the permeabilizedBACCs to study the consequences of raising the levelsof cyclic AMP and Ca2~on the site-selective phos-phorylation of TH and, thus, to determine which en-dogenous protein kinases and phosphatases might beinvolved in the responses to receptor-mediated activa-tion of these cells. Selective peptide and nonpeptideinhibitors of CaM-PKII, PKC, PKA, and protein phos-phatase I (PPI) and PP2A, were used to modulate thephosphorylation of TH in the permeabilized BACCs.
(PIPES), phenol red, glutamate, sodium dodecyl sulfate(SDS), EGTA, and phorbol l2,13-dibutyrate (PDBu)were from Sigma. Okadaic acid, microcystin, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93),2- [N- (4- methoxybenzenesulfonyl) I amino - N- (4- chloro-cinnamyl ) -N-methylbenzylamine (KN92), and N- [2- (p -bro-mocinnamylamino ) ethyl] -5-isoquinolinesulfonamide (H89)were obtained from Sapphire Biosciences. 3-[l-[3-(Ami-dinothio)propyl]- 1H-indol-3-yl ]-3-( 1-methyl- IH-indo]-3-yl)maleimidemethane sulfonate (Ro 31-8220) was a giftfrom Roche Products (U.K.). ATP and cyclic AMP werefrom Boehringer. High-purity digitonin was obtained fromCalbiochem. [y-32P]ATP (spec. act. >3,000 Ci/mmol) wasfrom Amersham. N-Tosyl-L-phenylalanine chioromethyl ke-tone (TPCK)-treated trypsin was obtained from Worthing-ton Biochemical Corporation. The CaM-PKII peptide inhibi-tor (CaM-PKII 273-302; HRSTVASCMHRQETVDCLK-KFNARRKLKGA) was obtained from Prof. J. Rostas,Newcastle University. The PKC and PKA peptide inhibitorsPKCi 19-31 (RFARKGALRQKNV-NH
2) and PKAi 5-22amide (TTYADFIASGRTGRRNAI-NH2) were obtainedfrom Auspep (catalog nos. 2147 and 2022, respectively).Antibody to TH was obtained from Dr. J. Haycock (NewOrleans, LA, U.S.A.). The MaxChelator program for calcu-lation of free Ca
2~used was version 6.63. Other materialsused in this study were as described by Bunn et al. (1995).
All reagents were of analytical grade or tissue-culture gradefor cell culture.
Preparation and culture of adrenal chromaffincells
Bovine adrenal chromaffin cells were isolated from adre-nal glands by pronase and collagenase digestion and enrich-ment for chromaffin cells by density gradient centrifugationthrough 4% bovine serum albumin as described by Zerbeset al. (1997). Cells were plated onto 24-well culture platesat a density of 106 cells per well and maintained at 37°C(Bunn et al., 1995). Cultured cells were used for experimen-tation between days 3 and 12.
Cell permeabilization with digitoninCulture plates were removed from the incubator and
placed on a warming plate held at 37°C.Cells were washedtwice with 0.5 ml of washing buffer containing 140 mMNaC1, 5 mM KC1, 1 mM MgC12, 1.2 mM CaCl2, 6 mMglucose, 0.04 mM phenol red, and 10 mM HEPES adjustedto pH 7.4 with NaOH. The washing buffer was removedand 0.25 ml of permeabilization buffer, containing 15 jaMdigitonin, 140 mM potassium glutamate, 1 mM MgC12, 5mM EGTA, 6 mM glucose, 0.04 mM phenol red, and 20mM PIPES adjusted to pH 7.0 with HC1, was added to thecells for 5 mm. The permeabilization buffer was removedand 0.5 ml of Ca
2~’-free replacement buffer, containing 140mM potassium glutamate, 1 mM MgCl
2, 5 mM EGTA, 6mM glucose, 0.04 mM phenol red, and 20 mM PIPES ad-justed to pH 7.0 with HC1, was added to the cells.
TH phosphorylation using II32P] ATP
The incubation was started immediately after the additionof the Ca2~-free replacement buffer by the addition of 25jal ATP solution, providing finally 40 jaM ATP and 25 jiCi[y-32PIATP in the incubation. After 1 mm, or other timesas indicated in the figure legends, the buffer was removedand reactions were stopped by adding 110 jal of “SDS-stop”solution (4% SDS, 2 mM EDTA, 8% ~-mercaptoethanol,50 mM Tris, pH 6.8) to the cells.
When cyclic AMP was required, it was added to the Ca2~-free replacement buffer during the incubation only, and notto the permeabilization buffer, to give a final concentrationof 1 jaM. When Ca2~-containingreplacement buffer wasrequired, CaC1
2 was added to the Ca2~-freereplacement
buffer to give afreeCa2~concentration of 1—100 jaM (basedon the MaxChelator program for determining free Ca2~con-centrations, and confirmed by using a Ca2~electrode) beforethe pH was adjusted with KOH to pH 7.0. This buffer wasused in place of the Ca2~-free replacement buffer during theincubation only and it was not used during permeabilization.When calmodulin (25 jig/ml), PDBu (final concentrationduring incubation of 1 saM), KN93 or KN92 (10 jaM), Ro3 1-8220 (0.3 jaM), H89 (10 jaM), CaM-PKII 273—302(10—100 jiM), PKCi 19—31 (10—100 jaM), and PKAi 5—22amide (10—100 jaM) were used, they were added to boththe permeabilization and the appropriate replacementbuffer.When okadaic acid (0.1—5 jaM) or microcystin (0.1—5 jaM)was used, they were added to the incubation buffer only andnot to the permeabilization buffer.
Gel electrophoresisThe cells were scraped from each well, solubilized, and
applied for polyacrylamide gel electrophoresis (PAGE) asdescribed by Bunn et al. (1992). Gels were then either auto-radiographed or the proteins transferred to nitrocellulose
(Dunkley et al., 1996). Gels for autoradiographywere driedand exposed to autoradiographic film, and gels for HPLCand western blotting were equilibrated in transfer buffer andthe proteins were transferred to nitrocellulose, as describedby Jarvie and Dunkley (1995).
HPLC phosphopeptide analysisAfter transfer the nitrocellulose was stained with Ponceau
S (0.5% Ponceau S in 100 mM acetic acid), then destainedin 7% acetic acid and dried before being exposed to autora-diographic film. TH was located on the autoradiograph andregions containing the radioactive protein were cut out ofthe sheet. TH was eluted from the nitrocellulose by trypticdigestion as described by Van der Geer et al. (1993) andmodified as follows: Nitrocellulose pieces were incubatedtwice in 1 ml of eluting solution (0.02% TPCK-trypsin in50 mM NH
4HCO3 with 5 mM dithiothreitol), vacuum dry-ing the eluate after each elution, before digesting the pooledeluates with concentrated trypsin stock (0.05%). The HPLCprocedure for fractionation of TH phosphopeptides was asdescribed by Bunn et al. (1995).
Quantitation and analysis of dataAutoradiograms were quantitated by scanning with a Mo-
lecular Dynamics laser based scanning densitometer. THphosphorylation (percentage of control) was calculated bydetermining the extent of phosphorylation of TH and thensetting a value of 100% for the track on the gel consideredto be the control condition for that experiment. This wasnecessarybecause under basal conditions (+ EGTA, —
and — cyclic AMP) phosphorylation of TH was generallynot measurable (Fig. 1, track 1), and it was therefore notpossible to assess reliably increases in the phosphorylationof TH as percentages of basal phosphorylation. HPLC traces
FIG. 1. Calcium and cyclic AMP stimulate TH phosphorylationin permeabilized BACCs. a: Cells were permeabilized with 15jaM digitonin for 5 mm and incubated with [
32P]ATPin Ca2’-free replacement buffer (containing EGTA) for 1 mm (lane 1).Ca2~(10 jaM; lanes 2, 3, and 5), calmodulin (25 jag/mI; lanes 3and 5), and cyclic AMP (1 jiM; lanes 4 and 5) were added asdescribed in Materials and Methods. b: Cells were permeabilizedwith digitonin and incubated with [32P]ATP in Ca2~-free replace-ment buffer (containing EGTA) (lane 1). Ca2’ (0.7—10 jaM; lanes2—5) was added to the Ca2~-freereplacement buffer for the 1-mm incubation as indicated. The reaction was stopped with anSOS-stop solution, the proteins were fractionated by PAGE, andthe phosphoproteins were detected by autoradiography. Expo-sure was longer for (b) than for (a). The apparent molecularmasses of selected phosphoproteins, TH, and the MARCKS pro-tein are indicated.
of phosphopeptide peaks were analyzed by peak area deter-mination.
RESULTS
Protein phosphorylation in permeabilized BACCsWhen BACCs were permeabilized for 5 mm with
digitonin (15 ,iaM) and incubated for 1 mm with [y-32PIATP in a Ca2~-free buffer (containing EGTA),phosphorylation of a limited number of proteins wasseen (Fig. la, lane 1). Increasing the Ca2” concentra-tion to 10 ,aM increased the phosphorylation of a num-ber of proteins, including one of apparent molecularmass 60 kDa (Fig. la, lane 2). This protein was identi-fied as TH by its size, its immunoreactivity with anantibody against TH (not shown), its phosphorylationin the presence of various protein kinase activators,and the phosphorylation pattern of its tryptic peptides(see below). The phosphorylation of TH was first de-tected at 2 ,t.tM Ca2’’; the greatest increase in phosphor-ylation occurred at 2—10 jiM Ca2’ (Fig. lb). Furtherincreasing the concentration of Ca2’ indicated that THphosphorylation was maximum at 100 jiM (notshown). Calmodulin (25 jag/ml) increased the phos-phorylation of TH beyond that seen with Ca2~alone(Fig. la, lane 3). Cyclic AMP (1 jaM), in the absenceof Ca2’’, increased the phosphorylation of several pro-teins including TH (Fig. la, lane 4). The level of THphosphorylation was greater in the presence of cyclicAMP than in the presence of Ca2”. Addition of cyclicAMP and Ca2” together increased the phosphorylationof TH beyond that seen with either agent alone (Fig.la, lane 5).
Increasing the concentration of Mg2~from 1 to 10mM in the absence of Ca2’’ increased the phosphoryla-tion of TH under basal conditions (not shown), butthe specific Ca2’’ - and cyclic AMP-stimulated phos-phorylation of TH was not increased. As 1 mM Mg2~approximates the physiological concentration ofMg2’’, this level was used in all subsequent studies.
Increasing the time of incubation increased the phos-phorylation of TH under basal conditions over the first5 mm, but the specific Ca2’’- and cyclic AMP-stimu-lated phosphorylation of TH remained relatively con-stant (not shown). We therefore chose 1-mm incuba-tions for this study to minimize the loss of TH andother proteins from the permeabilized BACCs, whichoccurs with longer incubation times (Sarafian et al.,1987; Morgan and Burgoyne, 1992).
Other proteins showed changes in phosphorylationin the presence of Ca2’ (Fig. ib). The myristoylatedalanine-rich C kinase substrate (MARCKS) proteinwas identified in BACCs by its mobility, the diffusenature of the band, its phosphopeptide profile, its pat-tern of phosphorylation in the presence of Ca2’, PDBu,and inhibitors of PKC, as well as its solubility in aceticacid (Dunkley et al., 1996). A 50-kDa protein wasidentified in BACCs as the a subunit of CaM-PKII,based on its mobility, the increase in phosphorylation
observed in the presence of calmodulin and zinc, thepattern of phosphorylation in response to CaM-PKIIinhibitors, and its calmodulin-binding capacity (Buck-enham et al., 1995). Yanagihara et al. (1994) pre-viously found a 50-kDa CaM-PKII subunit in BACCs.
Sites on TH phosphorylated in response to Ca2”After separation of TH by PAGE and transfer to
nitrocellulose, the protein was digested with trypsinand the resulting peptides fractionated on an HPLCcolumn. The phosphopeptides were detected with anon-line radioactive detector. Cyclic AMP (1 jiM) in-creased the phosphorylation of only one peptide in THafter incubation with permeabilized BACCs for 1 mill(Fig. 2a). The mobility of this phosphopeptide corre-sponded exactly with the mobility of peak CC6 seenwhen TH was phosphorylated in intact cells with 32P,(Bunn et al., 1995). Peak CC6 corresponds to Scr4°in TH (Dunkley et al., 1996). TH was phosphorylatedon three peptides in the presence of 10 jiM Ca2’’ after1-mm incubation (Fig. 2a). The mobility of thesephosphopeptides corresponded exactly with the mobil-ity of peaks CC3, CC5, and CC6 seen when TH wasphosphorylated in intact cells with 32P~ (Bunn et al.,1995). Peaks CC3 and CC5 correspond to Sert9 in TH(Dunkley et al., 1996) and we found that they re-sponded in parallel to all the treatments described inthis study. Under the conditions used, cyclic AMP-stimulated TH phosphorylation was greater than thatseen with Ca21 (see, also, Fig. la). The ratio of Ser’°to Ser4°phosphorylation was found to be 2.1:1.0 after1-mm incubation in the presence of Ca2’ (Fig. 2b).
Increasing the time of incubation with [y-32P]ATPfrom 1 to 5 mm and at the same time increasing theconcentration of Ca2’’ from 10 to 100 jiM increasedthe overall phosphorylation of TH by 69%. HPLC anal-ysis of the TH phosphopeptides indicated that this waspredominantly due to an increase in Ser’9 phosphoryla-tion (Fig. 2a), such that the ratio of Ser’9 to Ser4°phosphorylation was increased to 5.7:1.0 (Fig. 2b).
Addition of PDBu (1 jaM) and Ca2~(10 jaM) for1 mm to activate PKC increased the phosphorylationof TH by 19% above that seen with Ca2” alone (Fig.2b). HPLC analysis of the TH phosphopeptides indi-cated that this was due to a 66% increase in Ser4°phosphorylation (Fig. 2a), such that the ratio of Ser’9to Ser4°phosphorylation was decreased to 0.78:1.0(Fig. 2b).
Effects of protein kinase inhibitors on THphosphorylation
To determine which protein kinase ( s) was responsi-ble for the phosphorylation of Ser19 and Scr4°in thepermeabilized BACCs, the effects of a series of inhibi-tors of protein kinases were determined.
The CaM-PKII inhibitor KN93 (Marley and Thom-son, 1996a) also decreased the Ca2’ -stimulated phos-phorylation of TH in a concentration-dependent man-ner with an inhibition of 46% at 50 jaM (Fig. 3),reducing the ratio of Ser’9 to Ser4°peptide phosphory-
FIG. 3. The effect of KN93 on Ca2 -stimulated TH phosphoryla-
tion in permeabilized BACCs. Cells were permeabilized with dig-itonin and incubated with [32P]ATP in Ca2*~containingreplace-ment buffer for 1 mm. KN93, or its inactive analogue KN92,was added as described in Materials and Methods at increasingconcentrations (10—50 jiM). Reaction was stopped with SOS,the proteins were fractionated by PAGE, and TH was detectedby autoradiography. The level of TH phosphorylation was deter-mined by densitometry and is expressed as a percentage of thecontrol incubation containing no inhibitor (set at 100%), whichwas run with each experiment (n = 5; **p < 0.001).
FIG. 2. TH phosphopeptide profiles after fractionation by HPLC.a: BACCs were permeabilized with digitonin and incubated with[32P]ATP in Ca2’~-free replacement buffer for 1 or 5 mm. Ca2*(10 jiM), cyclic AMP (1 jaM), or Ca2~(10 jaM) plus POBu (1pM) wasadded as described in Materials and Methods. Reactionwas stopped with an SOS-stop solution and the proteins werefractionated by PAGE. TH was transferred to nitrocellulose anddetected by autoradiography. The TH band was cut out, digestedwith trypsin, and the resulting phosphopeptides were fraction-ated by HPLC chromatography on a C18 reverse-phase column,using a0—15% acetonitrile gradient. Thephosphopeptides weredetected and quantitated by using an on-line radioisotope detec-tor. The Ca2~(5 mm) profile used a different detector settingthan the other three profiles, and there was no change in Ser4°phosphorylation from that seen at 1 mm whereas Ser19 phos-phorylation was markedly increased. b: Quantitation of the re-sults such as those shown in (a) provide information on therelative phosphorylation of 5er19 and 5er40. These data werecalculated by first determining the total phosphorylation of 5er19plus Ser4°in TH in the presence of Ca2~and setting this sumto equal 100%. The proportion of this total phosphorylation onSer19 and Ser4°was then represented by the gray and blackareas, respectively. Similar calculations were undertaken for theother incubation conditions with the total phosphorylation ofSer19 plus Ser4°in TH being set relative to that for Ca2~alone.
lation to 1.1: I .0. The inactive analogue of KN93,KN92 (Marley and Thomson, 1996a), had no effectup to 50 jiM (Fig. 3). A selective peptide inhibitor ofCaM-PKII (CaM-PKII 273—302; Dunkley, 1991) wasadded to the permeabilized BACCs and inhibition ofTH occurred in a concentration-dependent mannerwith inhibitions of 32% at 10 ,uM and 68% at 50 jaM.This was due to a decrease in Ser’9 phosphorylation(Fig. 4a), and the ratio of 5cr’9 to Scr4°phosphoryla-tion was significantly decreased, from 2.1:1.0 for theCa2~control to 0.8:1.0 with the peptide inhibitor (Fig.4b). There was no effect of CaM-PKII 273—302, orKN-93, up to 50 jaM on the phosphorylation of THinduced by either cyclic AMP or PDBu (not shown).
TH and the MARCKS protein, which is known tobe phosphorylated by PKC in vitro (Dunkley et a!.,1996), showed increased phosphorylation on additionof PDBu (Fig. 5; lanes I and 3). As noted above, theincrease mn TH phosphorylation was due to an increasein the phosphorylation of Scr4°(Fig. 2a). PKC inhibi-tors were therefore tested on the Ca2~- and PDBu-stimulated phosphorylation of TH. Ro 31-8220 (Mar-ley and Thomson, 1996b) at 0.3 jaM completelyinhibited the PDBu-stimulated increase in phosphory-lation of MARCKS protein and TH (Fig. 5; lane 4)but did not inhibit the phosphorylation of TH in re-sponse to cyclic AMP (not shown). HPLC analysis ofthe TH-phosphorylated peptides indicated that Ro 31-8220 inhibited the Ca2~-stimulated increase in Ser4°phosphorylation by 70% and not the phosphorylationof Ser’9, leading to a Ser’9-to-Ser4°phosphorylationratio of 7.5:1.0 (Fig. 4b). PKCi 19—31 (50 jiM), apeptide inhibitor of PKC (Terbush and Holz, 1990),also completely blocked the PDBu-stimulated phos-phorylation of TH and the MARCKS protein, as wellas inhibiting the Ca2’ -stimulated phosphorylation ofSer4°on TH by 54% (not shown).
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2392 C-A. GON~ALVES ET AL.
FIG. 5. The effect of phorbol ester andthe PKC inhibitor Ro 31-8220 on TH phosphorylation in permeabilized BACCs. Cells werepermeabilized with digitonmn and incubated with [
32P]ATP inCa2~-containing replacement buffer for 1 mm. POBu (1 jaM) orRo 31-8220 was added as described in Materials and Methods.The reaction was stopped with an SOS-stop solution, the pro-teins were fractionated by PAGE, and TH phosphorylation wasdetected by autoradiography.
FIG. 4. The effect of selective protein kinase inhibitors on THpeptide profiles. a: BACCs were permeabilized with digitoninand incubated with [32P]ATP in Ca2~-containing replacementbuffer for 1 mm. The peptide inhibitor of CaM-PKll (CaM-PKII273—302; 50 jaM), the nonpeptide inhibitor of PKA (H89; 10jaM), and the nonpeptide inhibitor of PKC (Ro 31 -8220, 0.3 jaM)were added as described in Materials and Methods. Reactionwas stopped with an SOS-stop solution and the proteins werefractionated by PAGE. TH was transferred to nitrocellulose bywestern transfer and detected by autoradiography. The TH bandwas cut out, digested with trypsin, and the resulting phospho-peptides were fractionated by HPLC chromatography on a C18reverse-phase column, using a 0—15% acetonitrile gradient. Thephosphopeptides were detected and quantitated by using an on-line radioisotope detector. b: Quantitation of the results shown in(a) provides information on the relative phosphorylation of Ser19and Ser40. These data were calculated by first determining thetotal phosphorylation of Ser19 plus Ser4°in TH in the presenceof Ca~and setting this sum to equal 100%. The proportion ofthis total phosphorylation in Ser19 and Ser4°was then repre-sented by the gray and black areas, respectively. Similar calcula-tions were undertaken for the other incubation conditions withthe total phosphorylation of Ser19 plus Ser4°in TH being setrelative to that for Ca2~alone.
H89 is a selective PKA inhibitor (Chijiwa et al.,1990) and it inhibited completely the cyclic AMP-stimulated phosphorylation of TH at 10 jaM, but it hadno effect on the PDBu-stimulated TH phosphorylation(not shown). There was also an inhibition of Ca2~-stimulated TH phosphorylation by H89, due to a 56%decrease in Scr4°phosphorylation and no decrease in5cr’9 phosphorylation (Fig. 4a), leading to a Ser’9-to-Scr4°phosphorylation ratio of 8.2:1.0 (Fig. 4b). PKAi5—22 amide (50 jaM), a selective PKA inhibitory pep-tide, blocked the 1 pM cyclic AMP-stimulated increasein TH phosphorylation, and inhibited the Ca2’-stimu-lated phosphorylation of Scr4°by 38% (not shown).
Effects of protein phosphatase inhibitors on THphosphorylation
To determine which protein phosphatase(s) was re-sponsible for the dephosphorylation of 5cr’9 and Ser4°in the permeabilized BACCs, the effects of two inhibi-tors of protein phosphatases were determined.
Incubation of permeabilized BACCs with [y-32P]-ATP and 50 jaM Ca2’ for 1 mm in the presence ofokadaic acid, an inhibitor of PP2A (Haystead et al.,1989), increased the total phosphorylation of TH in aconcentration-dependent manner to 240% above thatseen with Ca2~alone (Fig. 6a). HPLC analysis ofthe TH phosphopeptides gave a Ser’9-to-Ser4°ratio of7.3:1.0, indicating that the major increase in phosphor-ylation was on Ser’9. Microcystin, an inhibitor ofPP2A and PPI (Mackintosh et al., 1990), similarlyincreased the phosphorylation of TH to 208% abovethat seen with Ca2~alone (Fig. 6a). Increasing theperiod of incubation from I to 5 mm in the presence
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TYROSINE HYDROXYLASE PHOSPHOR YLATION 2393
FIG. 6. The effect of protein phosphatase inhibitors on TH phos-phorylation in permeabilized BACCs.a: Cells were permeabilizedwith digitonmn and incubated with [
32P]ATP in Ca2~-contamningreplacement buffer for 1 mm. Okadaic acid (0.1—5 jaM) or micro-cystin (0.1—5 pM) was added to the replacement buffer for theincubation as indicated. b: Cells were permeabilized with digito-nm and incubated with [32P]ATP in Ca2~-free replacement buffercontaining cyclic AMP (1 pM) for 1 mm. Okadaic acid (0.1—5pM) or microcystin (0.1—5 pM) was added to the replacementbuffer for the incubation as indicated. Reaction was stoppedwith an SOS-stop solution, the proteins were fractionated byPAGE, and TH phosphorylation was detected by autoradiogra-phy. The level of TH was determined by densitometry (n = 4).
of microcystin also increased the level of TH phos-phorylation by 160%. Analysis of the TH phosphopep-tide profiles indicated that only Ser’9 and Ser4°werephosphorylated and the ratio of Ser’9 to Ser4°phos-phorylation was 6.7:1.0 due to an increase in Ser’9phosphorylation and no change in Ser4°phosphoryla-tion.
Okadaic acid and microcystin stimulated an increasein TH phosphorylation after incubation of permeabil-ized BACCs with 1 jaM cyclic AMP for I mm (Fig.6b). Only Scr4°was phosphorylated under these condi-tions.
DISCUSSION
In intact BACCs the phosphorylation of Ser’9,~ and 5cr4°are all increased in response to depo-larizing stimuli and this increase is dependent on a risein intracellular Ca2”, either from outside the cell suchas with nicotine (Haycock, 1993), or partially from
intracellular stores such as with histamine (Bunn etal., 1995). There is a concomitant Ca2’-stimulatedincrease in TH activity (Marley et al., 1995). Ser’°isthe first and most prominent site labeled on depolariza-tion and its phosphorylation is maximal after 1—3 mm,followed by Ser40, which is maximal after 3 mm butis never as prominent as Ser’9 (Haycock, 1993; Bunnet al., 1995). Depolarization also increases the phos-phorylation of Ser3t, but this response is generally lessprominent than Scr4°and is delayed, being maximalonly after 5—10 mm (Haycock, 1993; Bunn et al.,1995). The aim of this study was to determine whichendogenous protein kinases and phosphatases are in-volved in the phosphorylation and dephosphorylationof Ser’9, Ser3’, and Ser4°in situ in response to a risein intracellular Ca2’.
We used digitonin-permeabilized BACCs for thesestudies. No phosphorylation of TH occurred in cellsincubated with [y- 32P I ATP that had not been perme-abilized. The conditions chosen for permeabilizationwere 15 jaM digitonin for 5 mm. These conditionspermeabilized ‘=70% of the BACCs and retained>65% of the TH within the permeabilized cells (T.B. Cheah et al., unpublished data); we found that useof higher concentrations of digitonin and/or longertimes of incubation led to greater permeabilization, butalso to a significantly greater loss of TH and proteinkinases from the permeabilized cells. It is establishedthat a loss of Ca2~-stimulated secretion of catechol-amines and intracellular protein such as calmodulinand PKA also occurs on extended permeabilizationfor 20—30 mm (Sarafian et a!., 1987; Morgan andBurgoyne, 1992). We used incubation times with ATPof only 1—5 mm in an attempt to further minimize anychanges to the intracellular environment induced bypermeabilization. However, Ser8 and Ser3’ were notobserved to be phosphorylated in permeabilizedBACCs under any of the conditions used. Clearly, dig-itonin permeabilization has an effect on the relationbetween certain protein kinases and TH. It is also likelythat the levels of catecholamines that bind to TH andinhibit its activity are altered by digitonin permeabili-zation.
We found that addition of 10pMCa2’’ to permeabil-ized BACCs increased the phosphorylation of TH andin other studies approximately doubled the activity ofTH after 1 mm (T. B. Cheah et al., unpublished data).The phosphorylation of both 5cr’9 and Ser4°was in-creased with the ratio being 2.1:1.0 after 1 mm. Thisis essentially the same ratio found when intact BACCsare stimulated with nicotine for 1 mm (1.9:1.0) (Bunnet al., 1995). Addition of 1 jaM cyclic AMP to perme-abilized BACCs increased the phosphorylation of THon Ser4°only and in other studies was found to doubleits activity after I mm (T. B. Cheah et al., unpublisheddata).The phosphorylation of Ser3t
It is known that TH is phosphorylated on Ser3’ inintact PCI2 cells by MAPK (Haycock et al., 1992).
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MAPK is activated by phorbol ester stimulation ofPKC and by depolarization-induced influxes of extra-cellular Ca2~(PavlovIc-Surjanèev et al., 1992; Rosenet al., 1994), and both these treatments increase 5cr3’phosphorylation (Haycock, 1993). We initially con-firmed the minimal loss of PKC from the permeabil-ized BACCs during the 1 mm of permeabilization(T. B. Cheah et al., unpublished data), using both anantibody to PKC and determination of PKC activityby using a specific PKC substrate assay (Jarvie andDunkley, 1995). However, attempts to induce thephosphorylation of Ser3’ in the permeabilized cellswere unsuccessful. Thus, activation of PKC by addi-tion of PDBu increased the phosphory!ation of theMARCKS protein (Fig. 5), a known PKC substratein BACCs (Dunkley et al., 1996), but had no effecton Ser3’ phosphorylation. Increasing the concentrationof Mg2~generally increases the activity of PKC andother Ca2’ -activated protein kinases (Dunkley, 1991),but this also had no effect on Ser3’ phosphorylation.Increasing the time of incubation and/or addition ofprotein phosphatase inhibitors increased Ser’9 andScr4°phosphorylation but did not lead to Ser3’ phos-phorylation. We have established, using 32P labelingof the BACCs, that Ser3’ is phosphorylated (Bunn etal., 1995; Dunkley et al., 1996). Prelabeling BACCswith 32P~followed by digitonin permeabilization indi-cated that Ser3’ was still phosphorylated in the 30%of cells that had not been permeabilized by digitonin,so that the cells had the capacity to phosphorylate Ser3’before permeabilization. Addition of Ca2~to the high-K~buffer increased 5cr3’ phosphorylation in these in-tact cells, due to the opening of voltage-sensitive Ca2~channels by the high-K’~depolarization. It is clear fromthese studies that permeabilization of the BACCs in-hibits the phosphorylation of Ser3’ by MAPK. Thereasons for this are unknown but may relate to thedisruption of the physical associations of the kinasecascade that couples MAPK to its activators and/orTH, or an inactivation of the kinase, perhaps due todephosphorylation and/or protease activity.
The phosphorylation of Scr’9It has been suggested that Ser’9 is phosphorylated
by CaM-PKII in intact cells (Haycock, 1993; Tsutsuiet al., 1994). The evidence for this is that the phosphor-ylation of Ser’9 is dependent on Ca2’~(Funakoshi etal., 1991; Haycock, 1993; Bunn et al., 1995), thatCaM-PKII is present in BACCs (Yanagihara et al.,1994) and can phosphorylate Ser’9 in vitro (Yamauchiand Fujisawa, 1981; Sutherland et a!., 1993), and thatactivation of CaM-PKII occurs when the level of intra-cellular Ca2”’ is increased (Tsutsui et al., 1994). How-ever, Ser’9 can also be phosphorylated by MAPKAPkinase 2 in vitro (Sutherland et al., 1993) and otherunidentified kinases could also be phosphorylating thissite. It is possible that MAPKAP kinase 2 is inactivein the permeabilized cells, if it is present in BACCs,as there was no phosphorylation of 5cr3’ by the MAPK
that activates MAPKAP kinase 2. We used inhibitorsof CaM-PKII to determine the extent to which CaM-PKII contributes to the phosphorylation of TH in thepermeabilized cells. A selective peptide inhibitor,CaM-PKII 273—302, and KN93 both inhibited thephosphorylation of 5cr’9. Under the conditions used,CaM-PKI I autophosphorylation was completelyblocked and the activity of CaM-PKII against exoge-nous peptide substrates was also completely inhibited(Buckenham et al., 1995). There was no inhibition of5cr4°phosphorylation with these inhibitors and theydid not inhibit either the PDBu- or cyclic AMP-stimu-lated phosphorylation of TH. These data suggest thatthe phosphorylation of Ser’9 was due in part to CaM-PKII and that Scr4° is not phosphorylated primarilyby this kinase. They also indicate that another proteinkinase(s) is likely to contribute to the phosphorylationof TH at 5cr’9. We found (T. B. Cheah et al., unpub-lished data) that the nonspecific calmodulin kinase in-hibitor W7 completely blocked the effects of Ca2” onSer’9 phosphorylation and this suggests that the otherkinase(s) is calmodulin stimulated.
Terbush and Holz (1990) found that CaM-PKII291—317 largely abolished the Ca2+ -stimulated phos-phorylation of TH, but the PKC inhibitory peptide PKC19—31 was ineffective. This differs from our findings.We found that a shorter CaM-PKII inhibitory peptidewas not as effective at blocking calcium-stimulated THphosphorylation (only 68% at 50 pM) and that thePKCi 19—31 peptide partially inhibited Scr4° phos-phorylation. Terbush and Holz (1990) determined totalTH phosphory!ation and not the phosphorylation ofindividual sites as we did. We found here that highconcentrations of CaM-PKII inhibitory peptide werenonspecific and blocked phosphorylation of TH byPKC and PKA as well as that by CaM-PKII, and thismay have contributed to the effectiveness of theirCaM-PKII inhibition. However, it is also possible thattheir longer peptide, which contains a calmodulin-binding domain, interfered with the availability of cal-modulin, whereas our peptide, which lacked the cal-modulin-binding domain, did not. This would be con-sistent with our W7 data, which suggested that Ser’9was phosphorylated by a calmodulin-dependent kinase.The lack of effect of PKCi 19—31 seen by Terbushand Holz (1990) was presumably because most phos-phorylation of TH was on Ser’9 and this was not alteredby the PKC inhibitor, and the effect on the minor5cr4°site went undetected.
Phosphorylated Ser’9 was dephosphorylated by pro-tein phosphatases that can be inhibited by okadaic acidand microcystin. This suggests that PP2A is the majorphosphatase acting on Ser’9, and this is entirely consis-tent with previous studies (Haavik et al., 1989; Berres-heim and Kuhn, 1994).
The phosphorylation of Ser4°Many protein kinases can phosphorylate 5cr4°in
vitro. It was found that PKA was able to phosphorylate
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TYROSINE HYDROXYLASE PHOSPHORYL,4TION 2395
5cr4°in response to both cyclic AMP and Ca2’’. Thecyclic AMP-stimulated phosphorylation was reducedby H89 (Fig. 4) and the selective peptide inhibitor ofPKA, PKAi 5—22 amide. These inhibitors also de-creased significantly the Ca2’’ -stimulated phosphoryla-tion of Ser40. This suggests that addition of Ca2”’ mayactivate PKA, perhaps by activating adenylyl cyclaseand, hence, PKA (Keogh and Marley, 1991; Andersonet al., 1992), leading to the phosphorylation of Ser40.It was also found that PKC was able to phosphorylate5cr4°in response to both PDBu and Ca2’’. The PDBu-stimulated phosphorylation was blocked completely bythe selective PKC inhibitor Ro 3 1-8220 and the selec-tive peptide inhibitor of PKC, PKCi 19—31. Theseinhibitors also decreased significantly the Ca2” -stimu-lated phosphorylation of Ser40. Yanagihara et al.(1987) found that preincubation of cells with the phor-bol ester I 2-O-tetradecanoylphorbol 13-acetate in-creased TH activity and this was not inhibited with thecalmodulin antagonists TFP and W7. This result canbe explained by the phosphorylation of Scr4°by PKC.Addition of both H89 and Ro 31-8220 was unable tocompletely block the phosphorylation of Ser40, and~l0% always remained, regardless of the concentra-tions of inhibitors used. Clearly, phosphorylation ofScr4°is mediated primarily by both PKA and PKC inpermeabilized BACCs.
Phosphorylation of 5cr4°was increased by okadaicacid and microcystin. This confirms previous studiessuggesting that PP2A is the primary protein phospha-tase involved in dephosphorylating Scr4°in BACCs(Haavik et al., 1989; Berresheim and Kuhn, 1994). Itis noteworthy that okadaic acid increased Scr4°phos-phorylation by ~200% in the presence of cyclic AMP,but with Ca21 there was primarily an increase in Ser’9phosphorylation with hardly any change in Scr4°phos-phorylation, suggesting an interaction between thephosphorylation and dephosphorylation of Ser’9 andSer40. There are several possible explanations for thisresult. Perhaps phosphorylation of Ser’9 on TH inhibitsScr4°dephosphorylation directly, perhaps the phospha-tase inhibitors are affecting the activity of protein ki-nases differentially, as it is established that CaM-PKIIand PKC are autophosphorylated, or perhaps some ef-fector of TH or the protein kinases is modulated byincreased phosphorylation in the presence of the phos-phatase inhibitors.
Permeabilized BACCs have been used extensivelyto investigate the secretion of catecholamines and acti-vation of TH activity. We have now established thatthey offer a system to study the endogenous proteinkinases and phosphatases that phosphorylate and de-phosphorylate TH. We have found that the Ca21’-de-pendent phosphorylation of TH in BACCs is a consid-erably more complex process than suggested by previ-ous studies. The present data suggest that a significantportion of the phosphorylation of Ser’9 may be medi-ated by a kinase other than CaM-PKII, that CaM-PKIIdoes not play a major role in phosphorylation of Ser40,
and that PKA and PKC mediate most phosphorylationof Ser40. We haverecently used permeabilized BACCsto determine the phosphorylation of TH and its activityin the same cells (T. B. Cheah et al., unpublished data)and this system can now be used to determine therespective roles of Ser’9 and Scr4°in activation of THin situ.
Acknowledgment: This study was supported by grant950325 from the NH & MRC (Australia) and an award toC-AG. from CNPq (Brazil).
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