Journal of Neurochemistry, 2001, 79, 349±360 Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ Michael F. Salvatore,* Jack C. Waymire,² and John W. Haycock* *Department of Biochemistry and Molecular Biology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA ²Department of Neurobiology and Anatomy, University of Texas Houston Medical School, Houston, Texas, USA 1 Abstract elevated K (158 mM) for 5 min increased TH PS at each site Depolarizing stimuli increase catecholamine (CA) biosyn- in a Ca21-dependent manner. Pretreatment with PD98059 thesis, tyrosine hydroxylase (TH) activity, and TH phosphory- prevented elevated K1-stimulated increases in ERK phos- lation at Ser19, Ser31, and Ser40 in a Ca21-dependent phorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was manner. However, the identities of the protein kinases that not signi®cantly increased by forskolin, and elevated K1- phosphorylate TH under depolarizing conditions are not stimulated Ser40 PS was three- to ®ve-fold less than that in known. Furthermore, although increases in Ser31 or Ser40 PC12 cells. In both cell lines, CA biosynthesis was increased phosphorylation increase TH activity in vitro, the relative 1.5-fold after treatment with elevated K1 and was prevented in¯uence of phosphorylation at these sites on CA biosynthesis by pretreatment with PD98059. These results suggest that under depolarizing conditions is not known. We investigated ERK phosphorylates TH at Ser31 and that PKA phosphory- the participation of extracellular signal-regulated protein lates TH at Ser40 under depolarizing conditions. They also kinase (ERK) and cAMP-dependent protein kinase (PKA) in suggest that the increases in CA biosynthesis under depolar- elevated K1-stimulated TH phosphorylation in PC12 cells izing conditions are associated with the ERK-mediated using an ERK pathway inhibitor, PD98059, and PKA-de®cient increases in Ser31 PS. PC12 cells (A126-B1). In the same paradigm, we measured Keywords: blot immunolabeling, Ca21/calmodulin-dependent CA biosynthesis. TH phosphorylation stoichiometry (PS) was protein kinase II, cAMP-dependent protein kinase, extracel- determined by quantitative blot-immunolabeling using site- lular signal-regulated protein kinase, PC12, PD98059. and phosphorylation state-speci®c antibodies. Treatment with J. Neurochem. (2001) 79, 349±360. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in The identity of the protein kinases that phosphorylate TH catecholamine (CA) biosynthesis. Treatments that mimic under depolarizing conditions and the site or sites whose depolarization in situ increase CA biosynthesis, TH activity, 21 and phosphorylation of TH in a Ca -dependent manner Received June 20, 2001; revised manuscript received August 1, 2001; (Yanagihara et al. 1984; Waymire et al. 1988; Mitchell et al. accepted August 3, 2001. 1990; Haycock and Wakade 1992). Electrical stimulation of Address correspondence and reprint requests to Dr Michael F. the medial forebrain bundle also increases CA biosynthesis, Salvatore, Department of Anatomy and Neurobiology, University of TH activity, and phosphorylation at Ser19, Ser31, and Ser40 Kentucky Medical Center, Center for Sensor Technology, Davis Mills Building, 800 Rose Street, Lexington, KY 40536, USA. in the striatum (Haycock and Haycock 1991). Prolonged E-mail: [email protected]. electrical stimulation does not deplete cellular CA levels Abbreviations used: CA, catecholamine; CAMKII, Ca21/calmodulin- except in the presence of a TH inhibitor (Wakade 1988; dependent protein kinase II; CAMKIV, Ca21/calmodulin-dependent Wakade et al. 1988). Thus, depolarizing stimulation appears protein kinase IV; DMSO, dimethyl sulfoxide; ERK, extracellular to activate one or more Ca21-in¯ux-activated protein signal-regulated protein kinase 1 and 2; MEK, mitogen-activated protein kinase kinase; NGF, nerve growth factor; PKA, cAMP- kinases that phosphorylate TH at speci®c sites. The dependent protein kinase; PS, phosphorylation stoichiometry; SDS, phosphorylation of one or more of these sites increases sodium dodecyl sulfate; TH, tyrosine hydroxylase; tTH, total tyrosine TH activity and CA biosynthesis. hydroxylase. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 349±360 349 350 M. F. Salvatore et al. phosphorylation regulates TH activity and CA biosynthesis comparable to that observed in the nigrostriatal and are not known. Extracellular signal-regulated protein kinase mesolimbic systems (Salvatore et al. 2000). Thus PC12 (ERK) can be activated by depolarizing stimuli in a Ca21- cells are an appropriate model in which to study the dependent manner (Pavlovic-Surjancev et al. 1992; Frodin regulation of TH phosphorylation and its relationship to CA et al. 1995; Rosenblum et al. 2000) and is the only protein biosynthesis. We used two PC12 cell lines, one de®cient in kinase known to phosphorylate Ser31 both in vitro and the capacity to stimulate PKA activity (van Buskirk et al. in situ (Haycock et al. 1992; Sutherland et al. 1993; Thomas 1985; Ginty et al. 1991). The major objectives were to et al. 1997). Treatments that increase Ser31 phosphorylation determine the involvement of CAMKII, ERK, and/or PKA alone, such as nerve growth factor (NGF) treatment in depolarization-stimulated site-speci®c TH phosphoryla- (Haycock 1990), increase TH activity (Mitchell et al. 1990). tion and in CA biosynthesis. Ser31 phosphorylation may also increase CA biosynthesis under depolarizing conditions. In cells transfected with TH mutated at Ser40, treatment with elevated K1 increases CA Materials and methods biosynthesis in a manner comparable to that observed in Cell culture cells transfected with wild-type TH (Harada et al. 1996). PC12 and A126-B1 cells (van Buskirk et al. 1985) were propagated Increases in Ser19 phosphorylation do not appear to directly on 75-cm2 tissue culture ¯asks (Falcon, Franklin Lakes, NJ, in¯uence TH activity or CA biosynthesis in situ (Haycock USA) pretreated with 2 mg/cm2 human placental collagen (Sigma, 21 et al. 1998; Lindgren et al. 2000). Ca /calmodulin-dependent St Louis, USA; catalog no. C 7521). The growth medium was a protein kinase II (CAMKII) phosphorylates both Ser19 and 1 : 1 combination of Dulbecco's modi®ed Eagle medium/F12 Ser40 in vitro (Campbell et al. 1986; Funakoshi et al. 1991; (Gibco BRL, Gaithersburg, MD, USA; catalog no.10565±018) and Sutherland et al. 1993; Alterio et al. 1998) and is also acti- OPTI-MEM I reduced serum medium (Gibco BRL; catalog no. vated by depolarizing stimulation (Colbran 1992; Hanson and 51985±034) containing 5% heat-inactivated fetal bovine serum Schulman 1992). Although the site on TH phosphorylated by (Gibco BRL), 5% heat-inactivated donor horse serum (JRH Bio- sciences, Lenexa, KS, USA), and penicillin (25 U/mL)/strepto- CAMKII in situ is unknown, it has been long proposed that mycin (25 mg/mL) (Gibco BRL). For experiments, cells were CAMKII phosphorylation of TH is involved in depolariza- subcultured onto either 12- or 24-well tissue culture plates [Falcon tion-stimulated CA biosynthesis. A CAMKII inhibitor, KN- or Costar (Cambridge, MA, USA)], pretreated with 2 mg/cm2 93, has been shown to inhibit depolarization-stimulated human placental collagen, in the same media composition used for TH phosphorylation and CA biosynthesis (Sumi et al. 1991). propagation. Incubation conditions were 36±378C in a humidi®ed cAMP-dependent protein kinase (PKA), shown to phos- atmosphere of 6.0±6.5% CO2. phorylate Ser40 exclusively in vitro (Vulliet et al. 1980; Drugs and reagents Funakoshi et al. 1991; Sutherland et al. 1993), can also have Forskolin was purchased from Tocris (Baldwin, MO, USA). NGF its activity increased under depolarizing conditions (Shimizu (rat recombinant) was purchased from Sigma. KN-92 and KN-93 et al. 1970; Baizer and Weiner 1985; Keogh and Marley were purchased from Calbiochem (La Jolla, CA, USA) and 1991). Increases in Ser40 phosphorylation are associated Seikagaku Corporation (Tokyo, Japan). PD98059 was purchased with increases in TH activity and CA biosynthesis not only from Calbiochem. Dimethyl sulfoxide (DMSO) (cell culture in situ [in chromaf®n and PC12 cells (Haycock 1990; reagent grade) was purchased from Sigma (St Louis, MO, USA). Waymire et al. 1991)] and striatal slices (Lindgren et al. All drugs, with the exception of NGF, were dissolved into DMSO 2000), but in vivo as well (Lew et al. 1998). However, in to stock concentration and diluted accordingly into treatment addition to being phosphorylated by CAMKII and PKA, buffers. Ser40 is also phosphorylated in vitro by protein kinase C General experimental conditions (Albert et al. 1984; Funakoshi et al. 1991), protein kinase G Upon reaching at least 50% con¯uence, cells were washed with (Roskoski et al. 1987), and mitogen-activated protein kinase- phosphate-buffered saline (Gibco BRL) and pre-incubated (60± activated kinases 1 and 2 (Sutherland et al. 1993). Of these 90 min) in pH 7.3±7.4 buffer (124 mm NaCl, 25 mm NaHCO3, protein kinases, only PKA has been shown to directly 11 mm glucose, 10 mm HEPES, 4 mm KCl, 2 mm MgSO4,2mm phosphorylate Ser40 in situ (Haycock 1996b). These studies CaCl2, and 1.2 mm KH2PO4) used throughout the experimental indicate that the cellular mechanism by which depolariza- procedures. After pre-incubation, the buffer was aspirated and tion increases CA biosynthesis is by either (i) CAMKII- replaced