Vol. 180, No. 2, 1991


October 31, 1991


]'3Veterans Admlnistratlon Medical Center, Lexington, Ky 40511 Departments of

2Blochemistry, 3Neurology and 3pharmacology,

University of Kentucky Medical Center, Lexington, Ky 40536 Received




Summary: Protein kinase C (PKO is r o u t i n e l y assayed, after i t is p a r t i a l l y p u r i f i e d over DEAE-cellulose chromatography to eilmlnate any I n t e r f e r l n g protei~2kinases and phosphatases, by measuring the transfer of y-phosphate of [y- P]ATP to HI histone. Recently, i t has been shown that a synthetic peptlde, comprising residues 4-14 of myelin basic protein (MBP4 14) , is a very selective PKC substrate which Is not phosphorylated e~fectively-by c y c l i c AMP-dependent protein klnase, casein kinase I and I I , Ca~+/calmodulin dependent protein kinase I I or phosphory]ase klnase [Yasuda, I . , Klshlmoto, A., Tanaka, S - I . , Tominaga, M., Sakurai, A. and Nishlzuka, Y. (]990) BBRC 166, 1220-1227]. We report here that once MBP4_I4 is phosphorylated, i t is not dephosphorylated by okadaic a c i d - s e n s i t i v e phosphatases (proteln phosphatases I, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hlppocampa] homogenates. Therefore, MBP4_I4 can be used for PKC assay in crude extracts of neural tissue. ® 1991 Academic Press, Inc,

Protein klnase C (PKC), discovered in 1977 by Inoue et a l . (1), transfers the y-phosphate of ATP to the sery] or threony] residues of various protein substrates.

Some of the proteins phosphorylated in v i t r o by PKC are: the 40

kDa protein In p|atelets (2), plasma membrane Ca2+ ATPase (3), v l n c u l l n (4), ribosomal protein S6 (5), myelin basic proteln (MBP) (6, 7) and lysine rich histone (8). While searching for a speclfic substrate for PKC, Yasuda et a]. (9) compared various synthetic peptides and proteins, and reported that a synthetic peptide corresponding to the sequence surrounding the serine at position 8 in MBP, QKRPSQRSKYL (MBP4_I4), was the most selective substrate for To whom correspondence should be addressed at Department of Neurology, VAMC-Cooper Drive Division (127), Lexington, Ky 40511. Abbreviations used are: PKC, protein klnase C; PP I , PP 2A, PP 2B, PP 2C and PP 3, protein phosphatases I , 2A, 2B, 2C and 3 respectively; EDTA, ethylenedlamlne tetraacetic acid; EGTA, [ e t h y l e n e ( o x y e t h y l e n e n i t r l l o ) ] tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazlneethanesulfonlc acld; Tris, t r i s (hydroxy-methyl) am|nomethane; DEAE, dlethylamlnoethyl; MBP, mye]In basic protein; EGF, eplderma] growth factor.

0006-291X/91 $1.50 Copyright © 1991 by Academic Press, ~c. All rights of reproduction in any form reserved.


Vol. 180, No. 2, 1991


the ~-, B- and v-isoforms of PKC. Among the substrates compared wlth MBP4_I4 were the intact proteins, MBP and histone HI, as well as peptldes representing phosphorylatable sequences from MBP (GKGRGLSLSRFSHGA), EGF-receptor (VRKRTLRRL), histone HI (RRKASGPPV, AAASFKAKK, ASGSFKL), glycogen synthase (PLSRTLSVSS), ribosomal protein $6 (AKRRLSSLRA), and a generic peptide, GSRRRRRRY. The Km for the MBP4_14 peptlde was 7 oM with PKC =-, B- and e-lsoforms.

This peptide was not phosphorylated significantly by cyclic AMP

dependent protein klnase, casein kinase I and I I , Ca2+/calmodulin dependent protein klnase, phosphorylase kinase or by PKC a-, ~- or {- isoforms (9). We have studled further the usefulness of MBP4_14 as a selective substrate for PKC assay by investigating the possible enzymatic dephosphorylation of the phosphorylated product (phospho-MBP4_14). For MBP4_14 to be an Ideal selective substrate for PKC assay in a crude tissue extract, Its phosphorylated form should be resistant to any phosphatases present in the tissue preparation.

We report here that the phospho form of

MBP4_I4, phosphorylated by PKC, is not dephosphorylated by any phosphatases present in a cytosolic extract of rat hippocampus.

EXPERIMENTAL PROCEDURES Materials: Phospho-cellulose paper (P-81) and DEAE-cellulose (DE-52) ion exchange resin were purchased from H~tman. Pig brain phosphatldyl serlne was obtained from Serdary Research. [y-~P]ATP was from ICN. ATP, diolein, phenylmethylsulfonyl fluoride, aprotlnin, pepstatin A, soybean trypsin inhibitor, histone I I I s and catalytic subunit of cyclic AMP dependent protein klnase (bovine heart) were purchased from Sigma. Okadaic acid was purchased from Calbiochem. The peptide (MBP4 14) acetylated at the N terminal residue (AcMBP4_I4) was purchased from GIBCO-BRL. Peptides, RRRRAASVAand RRATVAwere synthesized and their identities were verifled by amino acid composition and sequence analysis by the Macromolecular Structure Analysis Facility of the University of Kentucky. Phosphorylated peptides, RRRRAAS(p)VA and RRAT(p)VA were prepared as described by Deanna et al. (lO) Tissue extraction and partial purification of PKC: Male Sprague-Dawley rats (HarIan Industries) were sacrificed by decapitation. Hippocampi were rapidly removed and immediately homogenized in 5 volumes of buffer A [20 mM Hepes (pH 7.5), 2 mM EDTA, 2 mM EGTA, I mM dlthiothreltol, I0% glycerol, 50 ~g/ml soybean trypsin inhibitor, 5 pg/ml aprotinln, 7 ~M pepstatln A, lO pM leupeptin, | mM phenylmethylsulfonyl fluoride], with a hand-held micro-centrlfuge tube pestle. The homogenate was centrifuged at 710 x g for lO min. The supernatant was saved and the pellet rehomogenlzed in an additional 5 volumes of buffer A and centrifuged at 710 x g for lO min to remove cell debris and unbroken whole cells. The two supernatants were combined and centrifuged at lO0,O00 x g (Beckman TL-IO0 ultracentrifuge) for 20 minutes, yielding a clear supernatant fraction which was diluted wlth buffer A to reach the desired protein concentrations. These preparations are termed crude cytosol extract in thls paper. For partial purification of PKC, | ml of crude cytosol extract was applied to a l ml DEAE-cellulose column equilibrated with buffer A. After washing the column wlth 4 ml of buffer A, PKC was eluted with buffer A containing 0.1M NaCI to yield a partially purified PKC fraction. All the above procedures were performed at 4° . 695

Vol. 180, No. 2, 1991


Protein concentration was measured using the Coomassie Blue dye bindlng assay (11) wlth bovine serum albumin as the standard. Preparation of Phospho-AcMBP4 14:AcMBP4 14 was phosphorylated in a rea3c2t~on mixture of 20 mM Tris-CI (pH 7.5), 20-mM MgCI2, I mM CaC12, 20 .M [y-J'P]ATP (5,000-10,000 cpm/pmole), 50 .M AcMBP4 14, 0.1 mM dithio~hreitol, 80.g/m1 phosphatidyl serine, 8 .g/ml diolein and-100 .I (30.g protein) of partially purified PKC fraction in the total volume of 500 . I . After one hour of incubation at 36° , the reaction mixture was applied to AGIX8 anion exchange resin (Bio-Rex sample preparation disk) to remove ATP. Phospho-peptide collected in the eluate was used for assaying phosphatases without furthur purification. Routinely, about 10% of AcMBP4_14 w~ phosphorylated under these conditions. Specific radioactivity of the [° P]p~sphate incorporated Into the peptide was assumed to be equal to that of [y-JLP]ATP, and the concentration of phospho-peptide was based on this assumption. Enzymatic Activity Assays: PKC activity was measured at 36° in a reaction m~ture of 20 mM Tris-CI (pH 7.5), 20 mM MgC12, l mM CaC12, 20 .M or 120 .M [y- P]ATP (5,000-10,000 cpm/pmole), 50 .M - 140 .M AcMBP4 14 or 0.4 mg/ml histone I I I s , 0.8 mM dithiothreitol, 80 .g/ml phosphatidylserine and 8 .g/ml diolein in a 50 .l volume. The reaction was initiated by an appropriate amount of crude cytosol extract. The phosphorylated product from 10 pl of reaction mixture was quantified using ph@~pho-cellulose paper and the washing me~hod of Cook et al. (12). Background J~P incorporation in the absence of Cat+ and phosphati~l serine/diolein and in the presence of I mM EGTAwas subtracted from total incorporation in the complete PKC assay reaction mixture to determine the extent of PKC-dependent phosphorylation. Any departure from this standard assay is noted in the figure legends. Protein phosphatase activity was measured by organic extraction of [32p]pi released (13) from phospho-AcMBP4 14" The 50 .l reaction mixture at 3~ contained 58 mM Hepes/6 mM Tris-Cl (pH 7.5), l mM dithiothreltol, 1.5 .M [ P]phospho-AcMBP4_14, 15 .M AcMBP4_14, 6 mM MgC12, 1 mM CaC]2 and the carried-over components of buffer A, diluted by 5-fold. Following 6 minute incubation with lO .l of hippocampal crude cytosol extract, a 10 .I a11quot of reaction mixture was removed and added to a mixture of 400 .l of 5 mM silicotungstate/1, mM H2S04 and 500 .] of I:1 (v/v) isobutano1:toluene. After vortex]ng for 5 seconds, 100 .l of 5% (w/v) ammonium molybdate/2 M H2SO4 was added, the solution vortexed for 10 seconds and centrifuged for 4 minutes to separate the~rganic and aqueous phases. From the organic phase, 200 ~I was removed for J P measurement by liquid sclntillat|on spectrometry. Phospho-peptides, RRRRAAS(p)VA and RRAT(p)VA shown to be dephosphorylated by phosphatases 2A > 2C >> I (10, 14, 15) were employed as positive controls.

RESULTS AND DISCUSSION The i n i t l a l velocity curves of AcMBP4_14 phosphorylatlon (Figure I) by various amounts of PKC in hippocampal crude cytosol extract show that the rate of phosphorylatlon Is linear wlth time and amounts of protein.

The assay

reaction mixture contained 120 .M MgATP and 140 ~M AcMBP4_14, both of which are 20 times their respective Mlchaelis constants (9, 16). Yasuda et a_11. (9), using 10 pM MgATP and 25 .M MBP4_14 to assay PKC in rat brain crude extracts, obtained linear i n i t i a l velocity up to I0 minutes followed by a slightly decreased rate of phosphorylation, and based on these data, suggested that dephosphorylatlon of the phospho-peptide might not be occuring.

The decreased

rate of phosphorylation observed at later times by those authors was probably 696

Vol. 180, No. 2, 1991


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E 600

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Q. 0





./> /o






Time (minutes) Flqure I. Phosphorylatlon of AcMBP4_| by hlppocampal crude cytoso| extract. Progress of phosphoryiatlon measured IR 50 ~1 reaction mixtures wlth varying amounts of protein ( • , 32 pg/ml; o , 16 pglml; • , 8 ~glmI; A , 4 ~g/ml). The concentrations of ATP and AcMBP4 14 were 120 ,M and 140 ~M,respectlvely. The carrled-over components of buffer-A, dlluted by 5-fold, were also present In the reaction mixture. A11 other reaction conditions were as described under Experlmental Procedures. Each point Is an average of dupllcates. due to the decrease of substrate concentration as the reaction proceeded since initial

concentrations of ATP and peptide were well below saturating levels.

The i n i t i a l

velocity curves presented here in Figure I do not show any

deviation from l i n e a r l t y , and the substrate concentrations were always saturating (more than 15 times t h e i r respective KmS) under the conditlons of reaction.

However, this does not eliminate the p o s s i b i l i t y that the

phosphorylated product mlght have been dephosphoryted by protein phosphatases at a linear rate, producing the progression curves observed by Yasuda et al. (9) and those shown in Figure I. There are numerous reports that PP I and PP 2A are inhibited by free ATP in a time-dependent manner (17, 18, 19) and that this inhibition is reversed by d|valent cations such as Co2+, Mn2+ and Mg2+ (20, 21, 22).

There are also

conflicting reports that the presence of Mg2+ when phosphatases are preincubated with ATP relieves the ATP inhibition (23), or has no effect (24) on ATP inactivation.

Furthermore, these effects of ATP and/or divalent

cations on phosphatases are dependent on phospho-protein substrates (25, 26, 27).

Therefore, I t is also possible that under the reaction conditions of the


velocity curves shown in Figure l , PP I and PP 2A might have been

inhib|ted by ATP. From the above discussions, the possible presence of phosphatases in crude cytoso] extract that might dephosphorylate phospho-AcMBP4_14 could not be ruled out.

To investigate whether phospho-AcMBP4_14 is dephosphorylated by 697

Vol. 180, No. 2, 1991





2.0 ]



,o WO

o,1 °~, • k~°,~ ° ~ c o . - ~ , . -1






o.o I . . . . . . . . . . . . . -1 0 1 2


log Okadaic Acid (nM)


log Okadaic Acid (nM)

Flqure 2. Effect of okadalc acld on phosphorylatlon of AcMBP4_14 (panel A) and hlstone IIIS (Panel B) by hlppocampal crude cytosol extract. For phosphorylatlon of AcMBP4_14and hlstone I l l s , the respective amounts of protein were 13 ,g protein/ml and 32 pg proteln/ml In the assay reaction mixture. The concentrations of ATP and AcMBP4 14 were 20 ,M and 50 pM, respectively. The carrled-over components of buffer A, diluted by lO-fold, were also present In the reactlon mixture. Y-axls denotes nmole phosphate incorporated Into peptlde or histone IIIs per minute per mg protein based on the 5 mlnute reaction. The arrow heads point to phosphate Incorporatlon In th e absence of okadaic acid. • , phosphorylation in the presence of CaL+/phosphatldyl serlne~dloleln; o , phosphorylatlon In the presence of EGTA and In the absence of CaL+/phosphatldyl serine/dioleln. All other reaction conditions were as described under Experimental Procedures. Each point Is an average of duplicates.

PP 1 (28), PP 2A (28) and/or PP 3 (29), the phosphorylation of AcMBP4_I4 by PKC in hlppocampal crude cytosol extract was measured in the presence and absence of okadaic acid, a specific inhibitor of these three protein phosphatases (29, 30, 31).

As shown in Figure 2 (Panel A), okadalc acid did

not affect the level of PKC-dependent or background phosphorylatlon of AcMBP4_I4 at concentrations s u f f i c i e n t to i n h i b i t all three phosphatases, indicating that oKadaic acid-sensitive phosphatases do not dephosphorylate phospho-AcMBP4_14.

In contrast, the level of phosphorylation of hlstone I I I s

(Figure 2, Panel B) increased substantially at higher okadaic acid concentrations. The Ca2+/phosphollpid/diolein-dependent phosphorylation was increased more than the phosphorylation in the absence of Ca2+/phospholipid/diolein, indicating the presence of okadaic acid-sensitlve phosphatases that dephosphorylated the phospho-hlstone I I I s produced by PKC as well as by other kinases independent of Ca2+ or phospholipid/diolein such as cyclic AMP dependent protein kinase.

Thus, the inhibition of phosphatase can

affect the apparent incorporation of 32p into protein (or peptide substrate) i f its phosphorylated form is susceptible to these phosphatases. The results with okadalc acid do not address dephosphorylation that may occur by phosphatases not inhibited by okadaic acld [e.g., PP 2B (calcineurin) and PP 2C].

In order to estimate the amount of dephosphorylatlon of

phospho-AcMBP4_14 by okadalc acld-insensitive phosphatases in the hippocampal 698

Vol. 180, No. 2, 1991



A E Q.






"o rr















Time (minutes)

Figure 3. Effect of 500-fold molar excess addition of non-radloactlve MgATPon the phosphorylatlon of AcMBP4_I4 by hlppocampal crude cytosol extract (chase experiment). The rea~2t|on mixture at 36° consisted of 20 mMTrls-Cl (pH 7.5), 2 mMMgCl2, lO .M [v-a' P]ATP, 20 pM AcMBP4_I4, l.O mMCaCl2, 80 pg/ml phosphatlayl serlne, 8 pg/ml dloleln, I mMdlthlothrelto], the carrled-over components of buffer A (diluted by 6-fold) and If.5 pg/ml protein In 120 pl reaction volume. At 24 minutes (arrow head), 25 ~I of glacial acetic acid ( o ), 21 mMMgCl2/21 mMATP ( a ), or water ( A ) was added to the respective reaction mixtures. Ten .l allquots up to the 20 minute point and 13.1 pl allquots after 24 minutes were removed to measure radioactive phospho-AcMBP4_14. Total radloactlvity In each aliquot was 2.2 million cpm. Each point Is an average of duplicates.

crude extract, we performed a 60 minute chase experiment as shown in Figure 3.

When a 500-fold molar excess of MgATP was added at the 24 minute time

point (arrow head), the amount of radioactive phospho-AcMBP4_14 did not decrease either immediately after or during the additional 36 minute incubation, demonstrating that the phospho-AcMBP4_14 is not rapidly dephosphorylated by enzymes In the hippocampal crude cytosol extract. However, a low level of dephosphorylation might not be apparent in this type of experiment since a large amount of the non-radloactive phospho-peptide would be produced in the presence of large excess of non-radioactive MgATP. To address this question d i r e c t l y , phospho-AcMBP4_14 was prepared and Its dephosphorylation by hlppocampal crude cytosol extracts was measured. Concentrations of Mg2+ and Ca2+ in the assay reaction mixture for dephosphorylation of phospho-AcMBP4_14 were 6 mM and l mM respectively, s u f f i c i e n t to support PP 2C and PP 2B a c t l v l t i e s (28).


not dephosphorylated at protein concentrations up to 30 ~g/ml, which is the highest amount of protein used to obtain the data for Figure I.

When lO .M

concentrations of two other phospho-peptides known to be substrates for PP i , PP 2A and PP 2C (I0, 14, 15) were tested under similar assay conditions, 5% of phospho-RRRRAASVA and 25% of phospho-RRATVA were dephosphorylated wlth 34 .g 699

Vol. 180, No. 2, 1 9 9 1


protein/ml and 9 ,g protein/ml, respectively.

I t should be noted that in

routine 5-10 minute phosphorylation assays of PKC. 2-8 ~g of crude cytosollc extract protein/ml is sufflcient using AcMBP4_14. The above results demonstrate that phospho-AcMBP4_14 is not dephosphorylated by protein phosphatases present in the hlppocampal crude cytosol extract. We therefore conclude that AcMBP4_14 can be successfully employed for PKC assay in these extracts without p r i o r p u r i f i c a t i o n to eliminate i n t e r f e r i n g protein klnases or phosphatases.

ACKNOWLEDGMENTS This research was supported by the VA Medical Research Funds and the Bertha LeBus Educational and Charitable Trust.

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Hsiao, K . - J . , Sandberg, A.R. and Li, H.-C. (1978) J. Bioi. Chem., 253, 6901-6907. Khatra B.S. and Soderling, T.R. (1978) B1ochem. Biophys. Res. Commun., 85 647-654. Merlevede, W., Goris, 3. and DeBrandt, C. (1969) Eur. J. B1ochem., 11, 499-502. Khandelwal, R.L. (1978) Arch. Blochem. Biophys., 191, 764-773. LI, H.-C. and Hsiao, K.-J. (1977) Eur. J. Biochem., 77, 383-391. Li, H.-C. and Hsiao, K.-J. (1977) Arch. B1ochem. Biophys., 179, 147-156. Khandelwal, R.L. (1977) Biochim. Biophys. Acta, 485, 379-390. Shenolikar, S. and Nairn, A.C. (1991) Advances in Second Messenger and Phosphoproteln Research, 23, 1-121. Honkanen, R.E., Z w i l l e r , 3., Daily, S.L., Khatra, B.S., Dukelow, M. and Boynton, A.L. (1991) 3. BioI. Chem., 266, 6614-6619. Bialojan, C. and Takai, A. (1988) Biochem. 3., 256, 283-290. Takal, A. (1988) 3. Muscle Res. Cell Mot1]., 9, 563-565.


A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein ki...
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