Activation of Protein Kinase C by Purified Bovine Brain 14-3-3: Comparison with Tyrosine Hydroxylase Activation
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Journal ofNeurochemistryRaven Press, Ltd., New York 1994 International Society for Neurochemistry
Activation of Protein Kinase C by Purified Bovine Brain14-3-3 : Comparison with Tyrosine Hydroxylase Activation
M. Tanji, R. Horwitz, *G. Rosenfeld, and J . C. Waymire
Department ofNeurobiology and Anatomy and *Department of Pharmacology,University of Texas Medical School, Houston, Texas, U.S.A .
Abstract : In the course of the purification of 14-3-3 pro-tein (14-3-3) we found that 14-3-3 isolated from bovineforebrain activates protein kinase C (PKC), rather thanthe previously reported protein kinase C inhibitory activity(KCIP) . We have characterized the 14-3-3 activation ofPKC. The physical properties of purified PKC activatorare the same as those previously reported for 14-3-3 andKCIP ; i .e ., (1) it is composed of subunits of molecularweight 32,000, 30,000, and 29,000 ; (2) it is homogeneouswith respect to molecular weight, as judged by nativegradient-gel electrophoresis, with a molecular weight of53,000 ; and (3) it is composed of at least six isoformswhen analyzed by reverse-phase HPLC . The concentra-tion dependence of PKC activation by 14-3-3 is in thesame range as that shown previously for KCIP inhibitionof PKC, and as that required for 14-3-3 activation of tyro-sine hydroxylase ; a maximal stimulation of two- to three-fold occurs at 40-100 lug/ml . 14-3-3's activation of PKCis sensitive to a-chymotrypsin digestion but is not heatlabile . Activation is specific to PKC; at least two otherprotein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated . The activa-tion of PKC by 14-3-3 is independent of phosphatidylser-ine and calcium and, as such, is an alternative mechanismfor the activation of PKC that obviates its translocationto membranes . Key Words: Protein kinase C-14-3-3protein-Bovine forebrain-Inhibition-Tyrosine hy-droxylase-Kinase C inhibitory protein .J. Neurochem. 63, 1908-1916 (1994) .
14-3-3 is an acidic protein that is highly enrichedin brain tissue but also exists in a wide variety oftissues and organisms including keratinocytes (Lefferset al., 1993), Xenopus (Martens et al ., 1992), Dro-sophila melanogaster (Swanson and Ganguly, 1992),plants (Hirsch et al ., 1992; Wu et al ., 1992), and yeast(van Heusden et al ., 1992) . It is estimated to be atleast 1% of brain protein (Boston et al ., 1982) . In1987 another acidic brain protein, termed "activatorprotein," was shown to be highly related to 14-3-3(Ichimura et al ., 1987) . The activator protein was orig-inally described by Yamauchi et al . (1981) as an activa-tor of tyrosine hydroxylase and tryptophan hydroxy-
lase, the two rate-limiting enzymes in catecholamineand serotonin synthesis, respectively . These hydroxy-lases require the activator protein for increased activityinduced by their phosphorylation by calcium/calmodu-lin-dependent protein kinase 11 . The similarities be-tween 14-3-3 and the activator protein include subunitcomposition, molecular weight, amino acid content,immunological cross-reactivity, and most important,the capacity to activate these two hydroxylases (Ichi-mura et al ., 1987) . Further analysis of 14-3-3 hasshown that it is not a single protein, but instead afamily of seven to nine isoforms (Ichimura et al .,1988) . Amino acid sequencing of cyanogen bromidecleaved peptides of the isoforms and cloning of threeisoforms has shown each to have unique amino acidsequences but that each is highly homologous to theothers (Ichimura et al ., 1988) . Thus far, three novelbrain mRNAs for 14-3-3s have been cloned and se-quenced. The amino acid sequence of each isoformindicates the proteins have very acid C-terminal andneutral N-terminal regions . Acidic C-termini are hy-pothesized to activate phosphorylated hydroxylases byinteracting with the phosphorylated regions of theseenzymes to relieve constrained enzymatic activity(Ichimura et al ., 1988) .
Within the last 3 years three additional biologicalactivities of 14-3-3 have been noted . Exol, a proteinreported by Morgan and Burgoyne (1992a,b) to benecessary for catecholamine exocytosis in permeabil-ized bovine adrenal chromaffin cells, is homologousto 14-3-3 . Consistent with this observation, Wu et al .(1992) have shown that anti-14-3-3 inhibits catechola-
Received December 13, 1993 ; revised manuscript received Febru-ary 1, 1994; accepted March 21, 1994.
Address correspondence and reprint requests to Dr. J. C. Waymireat Department of Neurobiology and Anatomy, University of TexasMedical School, 6431 Fannin Street, Houston, TX 77225, U.S .A.
Abbreviations used: CaM kinase II, Ca21 /calmodulin-dependentprotein kinase II ; CAMP, cyclic AMP; KCIP, kinase C inhibitorprotein ; 6-MPH,, DL-6-methyl-5,6,7,8-tetrahydropterin ; PKC, pro-tein kinase C; SDS-PAGE, sodium dodecyl sulfate- polyacrylamidegel electrophoresis; TBS, Tris-buffered saline ; TCA, trichloroaceticacid .
14-3-3 PROTEIN ACTIVATES TYROSINE HYDROXYLASE AND PKC
mine secretion stimulated by adding back chromaffincell cytosolic proteins . Zupan et al . (1992) have re-ported the cloning and isolation of a protein with Ca2+ -activated phospholipase A activity from brain, whichhas a marked sequence similarity to 14-3-3 . Toker etal . (1992) have reported the isolation of a protein witha high degree of homology to 14-3-3s that is a novelinhibitor of protein kinase C (PKC) (Aitken et al .,1990 ; Toker et al ., 1992) and have termed this activity"PKC inhibitor protein" (KCIP) .
In the course of purifying 14-3-3 by the procedureof Toker et al . (1992) for use in studies of tyrosinehydroxylase regulation, we discovered another biologi-cal activity of this family of proteins . Instead of thePKC inhibitory activity reported previously joker etal ., 1992), our preparation activated PKC. Here wedescribe the purification of 14-3-3, the properties ofthe activation of PKC by 14-3-3, and compare theactivation of PKC with 14-3-3's activation of tyrosinehydroxylase. A preliminary report of this work hasappeared (Horwitz et al ., 1992) . While this work wasin progress, Isobe et al . (1992) reported that a rena-tured isoform of 14-3-3 also activates PKC.
MATERIALS AND METHODS
[y-32P]ATP (4,500 Ci/mmol) and '21I-protein A (30 Ci/g) were purchased from ICNBiomedicals, Inc. (Irvine, CA,U.S.A .) . L-[3,5-3H]Tyrosine (56 Ci/mmol) was from Am-ersham Co . (Arlington Heights, IL, U.S.A .) . Nitrocellulosemembrane (0.45 ym) was from Schleicher & Schuell, Inc.(Keene, NH, U.S.A .) . DEAE-Sephacel, phenyl-Sepharose4B, MonoQHR5/5, Superose-6 and 12, and molecular masscalibration kits were from Pharmacia LKB Biotechnology(Piscataway, NJ, U.S.A .) . Benzamidine hydrochloride hy-drate was from Aldrich (Milwaukee, WI, U.S .A .) and leu-peptin was from Chemicon (Temecula, CA, U.S .A .) . DL-6-Methyl-5,6,7,8-tetrahydropterin (6-MPH 4 ) was from Calbio-chem Co. (La Jolla, CA, U.S.A.) . L-Phosphatidylserine wasfrom Avanti Polar-Lipids, Inc. (Alabaster, AL, U.S.A.) . a-Chymotrypsin was purchased from AMRESCO (Solon, OH,U.S.A .) . Cellulose phosphate paper (P81) was from What-man (Maidstone, England). Other materials (analytical re-agent grade) were from Sigma (St. Louis, MO, U.S.A .) orBio-Rad (Richmond, CA, U.S .A .) .A synthetic peptide (Met-His-Arg-Gln-Glu-Thr-Val-Asp)
used to assay Cat+ /calmodulin-dependent protein kinase II(CaM kinase II) was generously provided by Dr . P. T. Kelly(Department of Neurobiology and Anatomy, University ofTexas Medical School, Houston, TX, U.S.A .) . Calmodulinand synapsin I were purified from bovine forebrain as de-scribed by Gopalakrishna and Anderson (1982) and Bahlerand Greengard (1987), respectively . The catalytic subunit ofcyclic AMP (cAMP)-dependent protein kinase was purifiedfrom bovine heart as described (Okuno and Fujisawa, 1990) .PKC was purified from bovine or rat forebrain by a modifi-cation of the procedure of Walsh et al . (1984) . DEAE-Sephacel anion-exchange chromatography and Superose-6HR10/30 size-exclusion column chromatography steps wereinserted before phenyl-Sepharose hydrophobic interactionchromatography and the last step used MonoQcolumn chro-matography instead of DEAE-Sephacel chromatography .
PKC was not separated into separate isozymes . All datapresented are from bovine forebrain PKC unless otherwisestated . CaM kinase II was prepared from rat or bovine fore-brain as described by McGuinness et al. (1983) . Tyrosinehydroxylase was purified from bovine adrenal medullae bya procedure to be published elsewhere (M . Tanji and J. C.Waymire) . The antibody to 14-3-3 was generated by Dr .Gary Rosenfeld. Its specificity was confirmed by using theantibody to screen a rat brain cDNA expression library . Puri-fication of the antibody-positive plaques and a 1 .9-kb cDNAinsert yielded the deduced amino acid sequence of 14-3-3~(Aitken et al ., 1990) .
Immunoreactive assay of 14-3-3One microliter of sample was spotted on nitrocellulose
membrane . After spotting, membranes were washed withTris-buffered saline (TBS) buffer (8 mM Tris-Cl, pH 7 .5,containing 0.15 M NaCl), dried on a filter paper, and sub-merged in TBST buffer (TBS buffer containing 0.05%Tween 20). The membrane was blocked with 1 % bovineserum albumin in TBST buffer for 30 min and incubatedwith a 1 :2,000 dilution ofprimary rabbit polyclonal antibodyagainst 14-3-3 in TBST buffer . After washing the membranewith four changes of TBST buffer for 20 min, the membranewas incubated with a 1 :7,500 dilution of second antibodyand 121I-protein A (0.2 yCi/ml) in TBST for 30 min. Themembrane was washed with five changes of TBST bufferfor 25 min and dried. The radioactivity of each spot wascounted in an LKB Model 1270 y-counter. All procedureswere performed at room temperature. The observed radioac-tivity was proportional to the amount of spotted protein,between 0 and 130 leg of protein per spot.
Purification of 14-3-3Bovine forebrain was obtained fresh from the local slaugh-
terhouse, frozen immediately in liquid nitrogen, and kept at-80C until used . All procedures were performed at 4C oron ice if not described otherwise. Frozen brains (50 g) werepulverized on dry ice and -10 g was homogenized in 200ml of buffer A [20 mM Tris-Cl, pH 7.5, containing 2 mMEDTA, 10 mM EGTA, 1 mM dithiothreitol, and 8 .55%sucrose (wt/vol) to which protease inhibitors had beenadded immediately before use (10 yg/ml soybean trypsininhibitor, 75 l-tg/ml phenylmethylsulfonyl fluoride, 10 mMbenzamidine, 10 Fig/ml leupeptin, and 1 ug/ml pepstatin,final concentration)] . Homogenates were combined and cen-trifuged at 35,000 g for 25 min. The supernatant was loadedonto a 2.5 X 27-cm column of DEAE-Sephacel equilibratedin buffer B (20 mM Tris-Cl, pH 7.5, 2 mM EDTA, 5 mMEGTA, and 2 mM dithiothreitol) . After washing the columnwith 400 ml of buffer C (20 mM Tris-Cl, pH 7.5, containing1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol),proteins were eluted with a linear NaCl gradient (0-0.5 M)in buffer C at a flow rate of 2 ml/min . Five-milliliter fractionswere collected; two peaks of 14-3-3 immunoreactivity werepooled and combined .NaCl was added to immunoreactive fractions (2 .5 M final
concentration) and they were applied to a 2.5 X 8-cm phe-nyl-Sepharose 4B column (Pharmacia, Inc.) equilibrated in2.5 M NaCl in buffer C. After washing with 120 ml of 2.5MNaCl in buffer C, protein was eluted from the column bya linear gradient of 2.5-0 M NaCl in 100 ml of buffer C,followed by 80 ml of buffer C. The flow rate was 1 ml/minand 3-ml fractions were collected. Immunoreactive fractionswere pooled and dialyzed against buffer C overnight .
J. Neurochem ., Vol . 63, No . S, 1994
The desalted phenyl-Sepharose immunoreactive pooledfractions were divided into 11 fractions and each chromato-graphed separately on a MonoQHR5/5 column (Pharmacia,Inc.) equilibrated in buffer C. Protein was eluted with agradient of 0-0.6 M NaCl in 10 ml of buffer C, followedby 0.6-1.0 M NaCI in 5 ml of buffer C. The flow ratewas 0.5 ml/min and 0.5-ml fractions were collected. Eachfraction was analyzed by immunoreactive assay, westernblotting, and tyrosine hydroxylase activation assay (see be-low) . Although three immunoreactive peaks were eluted,labeled P1, P2, and P3, only the last to elute, P3, activatedtyrosine hydroxylase and migrated on sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE) withthe molecular weight subunits characteristic of 14-3-3 (mo-lecular weight of 29,000, 30,000, and 31,000 detected byeither Coomassie Blue staining or western blotting with theantibody to 14-3-3) . Peak P3 was pooled and dialyzed over-night against a saturated ammonium sulfate solution(pH 7.0).
Precipitated protein was collected by centrifugation andredissolved in 500 ul of buffer C followed by gel filtrationon a Superose-12 HR10/30 column (Pharmacia, Inc.) equili-brated with buffer C. The flow rate was 0.1 ml/min and 0.5-ml fractions were collected. A single major immunoreactiveand protein peak eluted immediately after the void volume .Two-thirds of the purified 14-3-3 was stored at -80C andthe remainder stored at 4C.
Determination of molecular massThe molecular mass of purified 14-3-3 was determined by
polyacrylamide-gel electrophoresis and gel-filtration chro-matography . Before polyacrylamide-gel electrophoresis, theprotein was dissolved in 5-10 /.cl of gel buffer (90 mM Tris-borate, pH 8.3, containing 3 mM EDTA) containing 12%glycerol and 0.8 mg/ml bromophenol blue and applied to4-30% polyacrylamide gel. Commercially available highmolecular mass markers (Pharmacia, Inc.) were used as stan-dards. Electrophoresis was performed at 8.8 V/cm for 16 hat room temperature . After electrophoresis, the gel wasstained for protein with Coomassie Brilliant Blue R-250.The molecular mass determination by gel-filtration col-
umn chromatography was performed using a Superose-6HR10/30 column (Pharmacia, Inc.) equilibrated with bufferC containing 0.1 M NaCl . Thyroglobulin, ferritin, catalase,and bovine serum albumin (1-3 Nag) were used as molecularweight standards. The flow rate was 0.05 ml/min .
Reverse-phase HPLCPurified 14-3-3 was analyzed by reverse-phase HPLC ac-
cording to the procedure of Toker et al . (1992) . Forty micro-grams of protein from the Superose-12 HR10/30 columnwas applied to a Brownlee RP 300, 100 X 2.1-mm column(Applied Biosystems, Inc.) equilibrated in 0.1% heptofluor-obutyric acid and eluted with a two-step gradient of H2O/acetonitrile containing 0.1% heptofluorobutyric acid as anion-suppressing agent. Acetonitrile was increased to 45%over 10 min, then further increased to 50% over 50 min ata flow rate of 0.2 ml/min . 14-3-3 isoforms eluted during the45-50% acetonitrile gradient . Protein was monitored at A215-The 14-3-3 isoform peaks were collected and analyzed bySDS-PAGE .
Assay of tyrosine hydroxylase and PKC activationActivation of tyrosine hydroxylase by 14-3-3 was exam-
ined by a modification of the method of Yamauchi et al .
J. Neurochem., Vol . 63, No . 5, 1994