Biochbnica et Bioph):vica Acta, ! 119 (1992) 19-26 ~:~ 1992 Elsevier Science Publishers B.V. All rights reserved 01t~7-4838/92/$05.00

19

BBAPRO 341)87

Circular dichroic studies of protein kinase C and its interactions with calcium and lipid vesicles J y o t s n a S h a h a n d G. G r a h a m S h i p l e y Departments of Biophysics and Biochemi.~trL Boston IJnirersity School of Medicine, Housman Medical Research Centel; Boston. MA (U.S.A.) (Received 18 July 1991)

Key words: Circular dichroism; Conformational change: Secondary structure: Protein kinase C

Circular dichroism was used to study the secondary structure of protein kinase C (PKC) in aqueous solution and the conformational changes resulting due to the presence of its regulatory cofactors (e.g. Ca z ÷, phosphatidylserine (PS) and phorbol 12-myristate 13-acetate (PMA)). Computer analysis of the CD data for the estimates of secondary structure showed that PKC maintains a highly ordered structure containing 36% a-helix, 57% fi-sheet and 7% fl-turu. PKC displays a minor conformational change upon addition of Ca z +. However, a larger change is observed on adding phosphatidyiserine vesicles in the presence of Ca z÷. In this case, the a-helix content is decreased by approx. 35% and fl-sheet increased by approx. 16%. The protein does not experience further significant changes in conformation on adding PMA.

Introduction

Protein kinase C (PKC), the Ca 2÷- and phospholipid-dependent protein kinase, has a crucial role in signal transduction and in tumorigenesis. It is especially interesting that the enzyme serves as the receptor for phorbol esters, a class of tumor-promoters. There is a correlation between the ability of individual phorbol esters to promote tumors and to activate the enzyme. It is probably the inappropriate signals given through PKC which result in the tumor promoting characteristics of these compounds. The enzyme was initially thought to be a single entity, but analysis of its complementary DNA (eDNA) clones indicate that PKC is a complex family of closely related structures [1-11]. The different forms of PKC exhibit subtle differences in enzymological characteristics [11,12]. Comparison of the predicted amino acid sequences (based on cDNAs), reveals that at least four subspecies (a, /3I, //2 and Y) of the enzyme may be present in mammalian tissue, particularly in brain.

Abbreviations: PKC, protein kinase C; CD, circular dichroism: PS, phosphatidylserine; PMA, phorbol myristate acetate. Correspondence: G.G. Shipley, Department of Biophysics, Boston University School of Medicine, Housman Medical Research Center, Boston, MA 02118, U.S.A.

Several groups have isolated the cDNA clones from libraries prepared from bovine, human, rat and rabbit brains and shown that the sequences (e.g., a-forms) are nearly identical. Seven subspecies of PKC have now been identified. Four subspecies emerged from the initial screening of a variety of complementary cDNA libraries. More recently, additional three cDNA clones having ~-, e- and ~'-sequences have been isolated from the rat brain cDNA library [8,13] but the eiution profile for these protein subspecies have not been identified on a hydroxTapatite column. Many studies have been focussed on the biochemical properties of PKC [14-17] which allowed the stoichiometry of PKC activation by phospholipid and diacylglycerol [14], the interdependence of phosphatidyiserine (PS), diacyiglycerol (DAG) and Ca 2÷ [15], the structural requirement for interaction of lipid cofactors with the enzyme [17], and phorbol ester binding and activation of PKC [16] to be determined. However, less attention has been paid towards biophysical and/or structural studies. Sequencing studies show that subspecies of protein kinase C (a, /3, y) are single polypeptide chains with four conserved regions and five variable regions [2-4]. The amino-terminal half of each polypeptide, containing regions C I and Ca is the regulatory domain which retains lipid- and Ca2÷-bind ing ability and thus, allows the enzyme to interact with the membrane; the carboxyl-terminal half containing

20 C 3 and C4 constitutes the catalytic domain that has the ability to phosphorylate protein substrates in the absence of calcium and phosphatidylserine. The catalytic domain is highly homologous in all of the PKC subspecies [10]. The C ! region has a tandem repeat of cysteine-rich sequences (the sr subspecies has only one set of cysteine-rich sequence), that resembles the cysteine-rich zinc finger motif that is found in many metallo-proteins and DNA-binding proteins [18]. Recently, it has been demonstrated that at least one cysteine-rich zinc finger is necessary for phorbol ester binding whereas the conserved region C 2 is needed for Ca2+-dependent phorbol ester binding [19,20]. The conserved region C 3 contains the characteristic structure of an ATP binding sequence similar to other kinases [3,5,8,21]. The conserved region C 4 also contains a similar ATP-binding sequence Gly-X-Gly-X-XGly. . . . . . (X) and since ATP does not bind with this segment, its significance is unknown. The regulatory and protein kinase domains are cleaved by limited proteolysis catalyzed by a C a 2 +-dependent neutral proteinase. In terms of biophysical studies, Bazzi and Nelsestuen [22-24] suggested that binding of PKC to phospholipid vesicles and monolayers consisted of a calcium-dependent interaction followed by an insertion of the protein into the membrane to form an irreversible phospholipid-bound state. Also, monolayerbound PKC was unable to phosphorylate in vitro substrates despite simultaneous insertion of the enzyme and substrate with the phospholipid in the presence of Ca 2+ and phorbol ester. In contrast, a recent study by Brumfeld and Lester [25] showed that the association and subsequent penetration of protein kinase C into defined lipid bilayers was independent of divalent cations and had no significant effect on activator-independent substrate phosphorylation. The predictive algorithm for determining the secondary structure of proteins from the primary sequence, developed by Chou and Fasman in 1974 and later extended [26,27], was based on the X-ray diffraction studies of 29 proteins. Garnier et al. [28], examined four structure-type predictions based on sequence alone and found that only 49% of all residues were assigned correctly. However, when the secondary structure content was known from other physical measurements (e.g., circular dichroism, NMR), then 63% of all residues could be correctly assigned. While biochemical studies are beginning to be performed on the individual isoforms of PKC, due to the unavailability of the larger quantities of the individual isoforms, all previous biophysical studies have been conducted with 'pure' preparations of PKC but containing different isoforms. However, since the primary sequences of the isoforms are highly homologous [10], they are likely to adopt similar secondary and tertiary

structures. The secondary structures for the PKC a, fl and y forms, predicted by the Chou-Fasman algorithm, are very similar (see 'Results'), suggesting that the isoforms may have same structure. Therefore, at this point it is reasonable to perform the preliminary structural studies on the PKC preparations purified to homogeneity, although eventually similar studies will have to be done for the individual isozymes. Here we report circular dichroic (CD) spectroscopic studies of PKC purified to homogeneity from bovine brain, to quantitate its secondary structure and to examine the effect of various regulatory cofactors (e.g., Ca 2+, PS and PMA) on its secondary structure. Materials and Methods

Phosphatidylserine, histone (Ill S), EGTA, EDTA, ATP, dithiothreitol (D'IT), phenylmethylsulfonyl fluoride (PMSF) and trichloroacetic acid (TCA) were obtained from Sigma (St. Louis, MO). Leupeptin was obtained from Boehringer-Mannheim Chemicals (Indianapolis, IN) and [5,.32p] was obtained from New England Nuclear (Wilminton, DE).

Isolation and purification of protein kinase C Protein kinase C was purified by a modification of a previously described method [29,30]. All procedures were performed at 4°C. Bovine brains were obtained from a local slaughterhouse and fresh brains were homogenized for 90 s in a commercial blender in 2 vols. of homogenizing buffer containing 20 mM TrisHCI (pH 7.5), 2 mM DTT, 1 mM CaCl 2, 2 mM PMSF and 0.01% leupeptin. The homogenate was centrifuged at 1000 × g for 20 min. The supernatant was recentrifuged at 100000 x g for 1 h. This pellet was resuspended and sonicated for 2 min in the extraction buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, 2 mM EDTA, 2 mM PMSF and 0.01% leupeptin. The suspension was gently stirred for 1 h and recentrifuged at 300000 × g for 1 h. ( N H 4 ) 2 S O 4 was added to the supernatant to 21% (w/v) and the resulting suspension was centrifuged at 37000 x g for 15 min. ( N H 4 ) 2 S O 4 was again added to the supernatant to achieve a final concentration of 45% (w/v) and the mixture was centrifuged at 37000 × g for 15 min. The resulting pellet was suspended in 20 mM Tris-HCl (pH 7.5), 2 mM EGTA, 2 mM EDTA and 2 mM DTI' and then dialysed extensively against the dialysis buffer containing 20 mM Tris-HCl (pH 7.5) and 2 mM DTT. This soluble enzyme preparation was incubated with inside-out vesicles (20 mg of protein) prepared from red blood cells [31,32], in the presence of 3 mM MgCl 2, 1 mM CaCl 2 and 1 mg/ml poly(ethylene glycol) (PEG) 20000 for 15 min at 24 ° C. The vesicles were retrieved by centrifugation at 40000 × g for 15 min. The pellet containing the membrane vesicles is resuspended in

21 the extraction buffer l0 mM Tris-HC! (pH 7.5), 1 mM EDTA and 1 m g / m l PEG and incubated for 15 min at 24°C. The vesicles were recentrifuged at 140000 × g for 30 min and the supernatant enriched in enzyme was saved. The enzyme was further purified by chromatography on a phenyl-Sepharose column. The above preparation was rendered in 1.5 M NaCl and applied to a phenylSepharose CL-4B column (0.6 × 1.0 cm) which had been equilibrated with 20 mM Tris-HCl (pH 7.5), 1 mM D'IT, 1 mM E D T A and 1.5 M NaC! and the column was washed with 5 ml of the same solution. The enzyme was eluted from the column by a step-wise reduction in NaC! concentration to 1.0 M, 0.5 M and 0.0 M, respectively, in 2 ml fractions. About one-third of the enzyme was released in the presence of 0.5 M NaCI, and the rest of the enzyme appeared at the 0.0 M NaCI fraction. Sufficient quantity of PKC was purified to perform biochemical and biophysical analyses. Proteins were examined by SDS-PAGE using 8% acrylamide (0.7 mm thick) according to Laemmli [33]. Gels were stained by Coomassie brilliant blue and silver stain. SDS-polyacrylamide gel electrophoresis of the purified enzyme shows a major band corresponding to a molecular weight of 78 000-80000 daltons (Fig. 1). Protein concentration was measured by the method of Lowry et al. [34].

PKC activity assay Protein kinase C was assayed by measuring the incorporation of 32p into histone (III S) from [v-

9 7 K -->

V

6 6 K -->

4 3 K ---> 31K

1

2

Fig. 1. SDS-polyacrylamide (8%) gel electrophoresis (with Coomassie blue stain) of PKC; molecular weight standards (lane 1); and purified enzyme after phenyl-Sepharose column (lane 2).

32p]ATP [7]. The standard reaction mixture contained in a final vol. of 0.2 ml, 50 mM Tris-HC! (pH 7.5), 10 mM MgCI 2, 20 ~g phosphatidylserine, 10 nM PMA, with or without 3 mM CaCI 2, 2 mM EGTA, 50 #g of lysine-rich histone, 50 p.M ATP, 1 # M [y-32p]ATP and 100 ng of enzyme. The reaction was started with the addition of radioactive ATP and was carried out for 10 min at room temperature. The assay was terminated by the addition of 20% trichloroacetic acid and fitration over 0.45 /~m Millipore filters. Filters were washed with the same buffer at least three times and radioactivity was counted in a liquid scintillation counter. The specific activity of the protein was calculated to be 200 n m / m i n per mg. To make PS vesicles, PS (in chloroform) from bovine brain was dried under N 2. The lipid residue was resuspended in 20 mM Tris-HCl, (pH 7.5) and sonicated under N 2 for 5 min. Vesicle size was measured by electron microscopy. The lipid vesicles were layered on the carbon coated grids using a flotation method [35] and negatively stained with 1% uranyl acetate. The grids were examined in a Hitachi HU-11C electron microscope.

Circular dichroism CD spectra were recorded at room temperature on a Cary 61 CD Spectropolarimeter (Varian; Palo Alto, CA, U.S.A.) calibrated from 500 to 190 nm with d-10camphorsu!phonic acid (1 m g / m l of protein). Spectra were recorded from 250 nm to 200 nm in a 0.1 cm quartz cell. The spectra reported are the average of three scans for each sample and have been corrected for baseline contribution due to buffer or buffer plus cofactors. For each type of sample two independent preparations were examined. The CD data were expressed as the mean residue ellipticity [O] using the following equation [O ]A = Oobs"MRW/10- d" C

where [O]~ is the mean residue ellipticity, A is the wavelength of irradiation, Oobs represents the displacement from the baseline value, full range in degrees, M R W equals the mean residue weight of the amino acids, d is the path length of the cell in cm and C equals the concentration of the protein in g/ml. Data points were analysed at 1 nm intervals between 200 and 240 nm by using both a normalized unconstrained fitting procedure and a constrained procedure (which required the fraction of each type of secondary structure to be positive and the sum of the fractions to equal unity) using the LINEQ program of Cynthia Teeters as described by Map and Wallace [36,37] using reference data sets of Brahms and Brahms [38] and Chang et al. [39]. An average helix length of l 1 residues was used in the analysis. The normalized root mean

22 square deviation (NRMSD) parameters reported are a measure of the quality of the fit of the calculated secondary structure to the observed CD data [36,37]. The secondary structures from the primary sequences were calculated using the program "Predict" provided by Dr. G.D. Fasman. The secondary structure potentials of the PKC-a sequence (shown in Fig. 4) were analyzed by using the program SEQ [40]. SEQ is a protein primary sequence analysis program which is strictly based on the work of Chou and Fasman [26,27]. This requires some estimate of the fractions of each secondary type. In addition to the fraction of secondary types, SEQ requires the % of the fraction of the secondary type to be initially designated, i.e. SEQ first assigns the user specified percent of the secondary structure type (e.g., helix), then it goes to the next secondary structure type (e,g., sheet). After the initial assignment SEQ fills in the sequence one residue at a time starting with secondary structure type that is furthest from being assigned. Once the helices, sheets and turns have been assigned, the rest of the residues are assigned to random coil. Mean hydrophobicity ( H i) and the mean a-helical hydrophobic moment (/.tH) were analyzed with a program entitled 'Computational Analysis of Physicochemical Property Scales' or CAPPS t. This program was developed to provide the calculation of hydrophobicity with local windowed averages, an extension to the method originally described by Kyte and Doolittle [41], and average moment calculations, a generalization of the helical hydrophobic moment analysis of Eisenberg et al. [42,43]. The windowed averaging calculation can be carried out by the program in either a central or left/right edge justified manner with a variable window size. Moment calculations are reported as the averaged vector sum around a central position with the individual vector magnitudes derived from the selected scale, and the relative orientation determined by both position from the central residue and the selected angle of analysis as follows.

Results and Discussion

Circular dichroic studies The CD spectra of PKC (0.8-1.0 /~M), in 10 mM Tris buffer (pH 7.5), in the presence and absence of different cofactors are shown in Fig. 2. The spectrum in 10 mM Tris buffer shows a broad negative minimum with a peak at approx. 218 nm (Fig. 2, curve a). Mean residue ellipticity at the negative maximum was 20 728 deg cm-" dmol -I. Secondary structure calculations based on the CD data showed that PKC is a highly ordered protein with a very high content of ,0-sheet and a significant content of a-helix i.e. 57% g-sheet, 36% a-helix, 7% g-turn and 0% random coil (Table I) as determined by a constrained linear square fit [38,39]. Since these data gave normalized root mean square standard deviation (NRMSD) values < 0.04 (Table I), the calculated secondary structure may closely represent the actual structure. A comparison of the normalized and fitted CD-data set for PKC in 10 mM Tris-HC! buffer (pH 7.5) is shown in Fig. 3. It is obvious that the two curves show good agreement indicating the accuracy of the prediction. The effect of different cofactors (i.e. Ca 2+, PS and PMA) on the CD spectra of PKC was examined. First, the CD spectrum of PKC was measured in the same buffer (Tris-HCl, pH 7.5) in the presence of 100 # M Ca -,+ (Fig. 2, curve b). The curve shows a change in negative ellipticities (18844 deg cmz dmol-~) without any significant change in the spectral shape. The secI

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Fig. 2. C D spectra of P K C in I0 m M

; This program was developed in this department by Robert T. Noite and Dr. D. Atkinson.

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Tris-HCl (pH 7.5):(a) P K C only

(!.0 g.M); (b) PKC (1.0 # M ) + C a -~÷ (100 t~M); (c) PKC (I.0/~M)+ Ca 2+ (100 /~M)+PS (60 v,M); (d) PKC (1.0 # M ) + C a 2+ (i00 # M ) + P S (60/~M)+ PMA (0.1 p,M); and (e) PKC (!.0 # M ) + C a 2. (100 v.M)+PC (60 v,M). All the curves represent an average of three scans.

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Ca 2+ (Fig. 2, curve b), if PS vesicles are substituted by 60 It M phosphatidylcholine (PC) vesicles; this suggests relatively minor changes in the conformation of PKC in the presence of PC vesicles. The secondary structure calculations show a slight increase in a-helix (37%), no change in fl-sheet (55%), increase in fl-turn (6%) and a decrease in random coil (2%) and the NRMSD value is 0.024 (Table It. Previously several CD studies of different proteins have been analyzed successfully in vesicular systems [37,44]. The lipid-induced changes in the CD profiles cannot be attributed to the distortions due to the differential scattering by the lipid vesicles because: (i) the vesicle size used in this study is fairly small (average size ---25 nm, measured by electron microscopy), hence the scattering by these vesicles is minimal [37]; (ii) Ca 2+ concentrations used in these experiments are much lower than that required to induce fusion in PS vesicles [45] and no visual aggregation was observed; (iii) the rotational strengths of the electronic transitions of the ester and phosphate groups of the phospholipids are very weak compared to the peptide backbone, so that nearly all the signal will be due to the protein [37]; and (iv) PC vesicles (vesicle size ---25 nm) do not show same effect as that of PS vesicles (compare Fig. 2, curve c and e, and see Table I). The full activation of the enzyme requires Ca" , phospholipid and D A G / p h o r b o l esters as essential activators [2,20] and strong interdependence of these activators has been demonstrated [15,22]. In order to see the effect of phorbol ester on PKC conformation, PMA (0.1-1.0 ItM) was added in the presence of Ca 2+ (100 ItM) and PS (60 ItM). Low concentrations of PMA are used since for higher levels of PS in mixed micelles, even 0.1 moi% of PMA is sufficient lo activate the enzyme [14,15]. Addition of PMA at 0ol ItM (PMA to protein ratio of 0.1" 1.0 (mol'mol); Fig. 2, curve d) or even at 1.0 # M (PMA to protein ratio of 1" 1 (mol:mol); data not shown) does not induce any further significant changes in the CD spectrum, suggesting that the overall secondary structure of PKC is preserved. The secondary structure calculations predict

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Wavelength (nm) Fig. 3. Comparison of the normalized and fitted CD-data for PKC in 10 mM Tris-HCI (pH 7.5): The dashed line is the observed spectrum Isee Fig. 2a) and the solid line is the calculated spectrum using the LINEQ program as described in "Material and Methods'.

ondary structure calculations show that binding of Ca 2÷ introduces some disorder into the protein conformation of PKC i.e. 32% a-helix, 55% 3-sheet, 0%/J-turn and 13% random coil and the NRMSD value is 0.021 (Table I). In the presence of 60 It M PS vesicles (four molecules of PS are required to activate monomeric PKC, therefore, PS concentration used in this study i.e. protein : lipid ratio of 1 : 60 (mol : mol) should provide the maximal binding of PKC (see Refs. 14 and 15)), a larger change is observed in the negative ellipticities (16 369 deg cm-" dmol- ~) accompanied by a significant change in the CD profile (Fig. 2, curve c). The secondary structure calculations show that the helical content decreases significantly (a-helix = 22%) and both g-sheet (fl-sheet = 64%) and fl-turn (/3-turn = 14%) contents increase significantly. This increase in fl-sheet and /3-turn content is mostly at the expense of helix and random coil (random coil =0%). However, as shown in Fig. 2, curve e, the CD spectrum remains almost the same as that of PKC in the presence of

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TABLE I Predicted secondao" stnlcture " o f P K C based on CD-data

PKC PKC + Ca 2 * PKC + Ca'-* + PC PKC + Ca-" ÷ + PS PKC + Ca 2+ + PS+ PMA

a-helix

/3-sheet

fl-turn

Randomcoil

NRMSD

ActiviB, ~

36% 32% 37% 22~ 21%

57% 55% 55% 64% 61%

7% 0% 6% 14% 18%

0R 13% 2% 0% 0%

0.032 0.021 0.024 0.037 0.034

393 427 700 4467 4804

a The predicted secondary structures are an average of three experiments. h PKC was assayed as described in Material and Methods and activity is expressed as counts per minute. Each value is the average of triplicate determinations.

24 only very small changes in a-helix, fl-sheet and fl-turn contents (Table I). In order to correlate the activity and secondary structure changes,. PKC activity was measured under the above mentioned conditions. As shown in Table I, essentially no activity is observed in presence of C a 2+ o r C a 2 + + PC. However, in the presence of Ca 2+ and PS, a significant increase in activity is observed. Very little change in activity is observed with the further addition of PMA. This is consistent with the observation that PKC activation occurs with 100% PS vesicles in the absence of DAG or phorbol esters [12]. However, this may not be true for the mixed vesicles/micelles where maximal activity occurs in the presence of DAG/PMA [22,14,15]. These results do not, however, rule out the possibility of some conformational changes upon PMA binding. Compensatory changes in different portions of the molecule would not be observed by this method, as only net changes can be observed.

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Sequence analysis of PKC Secondary structures were also calculated for the different PKC isoforms from their primary sequences using the widely accepted method of Chou and Fasman [26,27]. The predicted secondary structure for PKC-a sequence gives 40% of a-helix, 26% fl-sheet, 26% fl-turn and 8% random coil, for PKC-fl sequence is 40% a-helix, 27% fl-sheet, 25% fl-turn and 8% random coil and for PKC-y is 38% a-helix, 31%/3-sheet, 26% fl-turn and 5% random coil. Thus, the predicted secondary structures for the three PKC isoforms (a,/3, y) are very similar which justifies conducting the CD studies with a preparation containing a mixture of isoforms. The secondary structure predicted by the Chou-Fasman algorithm differs considerably from the experimentally derived CD data, neverthless, the ahelix content predicted by both the algorithms is close. Since all the three isoforms have similar secondary structures, only the PKC-a sequence has been used for the further analysis. Fig. 4 shows the details of the computer-aided analysis of the secondary structure potentials for a-helix (Fig. 4A), /]-sheet (Fig. 4B) and /3-turn (Fig. 4C) of the PKC-a sequence using the program SEQ [40]. As illustrated in Fig. 4A and B the sequence contains many strongly predicted a-helical regions (e.g. amino-acid residues 1-45, 160-176, 238245, 264-269, 276-'283, 299-309, 351-357, 380-394, 482-491,542-552 and 589-610) and fl-sheet structure (e.g. amino acid residues 63-83, 120-128, 137-146, 334-345, 402-419, 454-466, 520-529 and 666-672). The mean hydrophobicity (H i) profile (Fig. 4D) and the mean (x-helical hydrophobic moment (/.LH) (Fig. 4E) were also determined using the program CAPPS. Analysis of the mean hydrophobicity and hyc;rophobic moment profiles does not predict any particular location of a structural domain which could definitely asso-

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ciate with the plasma membrane. Most of the helical domains show low hydrophobicity with low hydrophobic moments (cf Fig. 4A, D and E). However, the regulatory domain has some hydrophobic stretches (e.g. amino acid residues 52-58, 67-77, 82-87, 116-127, 142-150 and 258-268). Out of these six stretches, five stretches fall in the cysteine-rich region which extends from amino acid residue 50 to 150. Interestingly both the zinc-fingers (amino acid residue 50-86 and 115151) also fall in this region. Although it is premature to conclude any definite relationship at this point, it is attractive to suggest that zinc-fingers play some role in the lipid binding. Recently, Gschwendt et al. [46] proposed a model for the binding of DAG and PMA to PKC. In this model, it is suggested that each activator is hydrogen bonded to sulfhydryl groups of cysteine

25 residues and to the carbonyl group of an asparagine within the cysteine-rich regions of PKC. In conclusion, the secondary structure of PKC has been examined under a variety of conditions by CD spectroscopy. The data presented here suggest (1) PKC maintains a highly ordered structure, (2) the protein is dominated by fl-structure but has a significant a-helical content, (3) PKC can interact with Ca `'+ in the absence of PS and this binding introduces a slight disorder, and (4) a large and specific conformational change (i.e. a large increase in /J-structure at the expense of a-helix) is observed upon binding to phosphatidylserine vesicles. Little is known about the mode of interaction of PKC with Ca 2+, lipid and phorbol esters. Several approaches have been employed to characterize the physical interaction between PKC and its cofactors. Biophysical techniques such as fluorescence energy transfer and light scattering have been utilized to demonstrate the association of PKC with lipid [22]. These studies, however, do not provided detailed information on the type of interaction a n d / o r the conformational changes of PKC. In a study by Brumfeld and Lester [25], it has been demonstrated that the interaction and association between PKC and lipid is controlled by both electrostatic and hydrophobic interactions. On the basis of their fluorescence quenching experiments, they suggested a rearrangement in some regions of PKC during the lipid-divalent cation-dependent activation process. In another publication, Lester and Brumfeld [47] reported some ligand-induced conformational changes in cytosolic protein kinase C and suggested that the enzyme can adopt different secondary structures in the active state. In a more recent study by Mosior and McLaughlin [48] reported that the peptides that mimic the pseudosubstrate region (amino acid residue 19-36) of PKC bind to acidic phospholipids in the membrane and support the hypothesis that the binding of lipids to the enzyme induces conformational changes in the enzyme to stabilize the active form. Our data clearly demonstrate that PKC actually undergoes a conformational change upon binding the specific phospholipid phosphatidylserine. These altered conformational properties, particularly those arising as a result of binding of these cofactors, may determine the mechanism by which PKC interacts at the cytoplasmic surface of the plasma membrane. Acknowledgements

This work was supported by research grant HL-26335 from the National Institutes of Health. We express our gratitude to Dr. Mary T. Walsh for guidance in performing the CD experiments. We also wish to thank Robert T. Nolte for his valuable advice on structure prediction and Dr. D. Atkinson for helpful suggestions.

We also thank Dr. G.D. Fasman for providing the 'Chou-Fasman program' for the calculation of secondary structure from the primary sequence of the protein. We thank Ms. Cheryl Oliva for technical assistance. References ! Nishizuka, Y. (19841 Nature 308, 693-697. 2 Nishizuka, Y. (19881 Nature 334, 661-665. 3 Parker, P.J., Coussens. L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D. and Ullrich, A., (1986) Science 233, 853-859. 4 Coussens, L., Parker, P.J., Rhee, L,, Yeng-Feng, T.L., Chert, L., Waterfield, M.D., Francke, U. and Uilrich, A. (1986)Science 233, 859-866. 5 Knopf, J.L., Lee, M.H., Sultzman, L.A., Kriz, R.W., Loomis, C.R., Hewick, R.M. and Bell, R.M. (19861 Cell 46, 491-502. 6 Kikkawa, U., Ogita, K., Asaoka, Y., Shearman, M.S. and Fujii, T. (1987) FEBS Lett. 223, 212-216. 7 Kikkawa, U., Ono, Y., Ogita, K., Fugii, T., Asaoka, Y., Sekiguchi, K., Kosaka, Y., Igarashi, K. and Nishizuka, Y. (19871 FEBS Lett. 217, 227-231. 8 0 n o , Y., Kurukawa, T., Fujii, T., Kawahara, K., Igarashi, K., Kikkawa, U., Ogita, K. and Nishizuka, Y. (19861 FEBS Lett. 206, 347-352. 9 0 n o , Y., Kikkawa, U., Ogita, K., Fujii, T., Kurokawa, T., Asaoka. Y., Sekiguchi, K., Ase, K., Igarashi, K. and Nishizuka, Y. (19871 Science 236, il 16-1120. 10 Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. and Nishizuka, Y. (19881 J. Biol. Chem. 263, 6927-6932. I I Huang, K.P., Nakabayashi, H. and Huang F.L. (1986) Proc. Natl. Acad. Sci. USA 83, 8535-8539. 12 Sekiguchi, K., Tsukuda, M., Ase, K., Kikkawa, U. and Nishizuka, Y. (19881 J. Biochem. 103, 759-765. 13 Ono, Y., Fujii, T., Ogita, K., Kikkawa, U. and Igarashi, K. (19881 FEBS Lett. 226, 125-128. 14 Hannun, Y.A., Loomis, C.R. and Bell, R.M. (19851J. Biol. Chem. 26{I, 10039-10043. 15 Hannun, Y.A., Loomis, C.R. and Bell, R.M. (19861 J. Biol. Chem., 261, 7184-7190. 16 Hannun, Y.A. and Bell, R.M. (19861 J. Biol. Chem. 261, 93419347. 17 Ganong, B.R., Loomis, C.R., Hannun, Y.A. and Bell, R. M. (19861 Proc. Natl. Acad. Sci. USA 83, 1184-1188. 18 Berg, J. (19861 Science 232, 485-487. 19 Ono, Y., Fujii, T., lgarashi, K., Kuno, T., Tanaka, C., Kikkawa, U. and Nishizuka, Y. (19891 Proc. Natl. Acad. Sci. USA 86, 4868-4871. 20 Kaibuchi, K., Fukumoto, Y., Oku, N., Takai, Y., Arai, K. and Muramatsu, M. (19891 J. Biol. Chem. 264, 13489-13496. 21 Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Yokokura, H., Sakoh, T. and Hidaka, H. (1987) Nature 325, 161-166. 22 Bazzi, M.D. and Nelsestuen, G.L. (19871 Biochemistry 26, 115122. 23 Bazzi, M.D. and Nelsestuen, G.L. (19881 Biochemistry 27, 75897593. 24 Bazzi, M.D. and Nelsestuen, G.L. (1988) Biochemistry 27, 67766783. 25 Brumfeld, V. and Lester, D.S. (19901 Archives of Biochem. Biophys. 277, 318-323. 26 Chou, P.Y. and Fasman, G.D. (19741 Biochem. 13, 222-244. 27 Chou, P.Y. and Fasman, G.D. (19781 Adv. Enzymoi., 45-148. 28 Gamier, J., Osguthorpe, D.J. and Robson, B. 11978) J. Mol. Biol. 120, 97-120.

26 29 Niedei, J.E., Kuhn, L.J. and Vandenbark. G.R. (1983) Proc. Nat. Acad. Sci. USA 80, 36-40. 30 WolL M., Cuatrecasas, P. and Sahyoun, N.J. (1985) J. Biol. Chem. 260, 15718-15722. 31 Steck, T.L. and Kant, J.A. (1Q74~ Methods Enzymol. 31,172-180. 32 Steck, T.L. (1978) Methods Memb. Biol. 2, 311-324. 33 Laemmli, U.K. (1970) Nature 227, 680-685. 34 Lowry, O.H., Rosebrough, N.J., Parr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 35 Valentine, R.C, Shapiro, B.M. and Stadtman, E.R. (1968) Biochemistry 7, 2143-2152. 36 Mao, D., Wachter, E. and Wallace. B.A. (1982) Biochemistry 21, 4960-4968. 37 Mao, D. and Wallace, B.A. (1984) Biochemistry 23, 2667-2673. 38 Brahms, S. and Brahms, J. (1980) J. Mol. Biol. 138, 149-178.

39 Chang, C.T., Wu, C.C. and Yang, J.T. (1978) Anal. Biochem. 91, 13-21. 40 Teeter, M.M. and Whitlow, M.C. (1988) Proteins 4, 262-272. 41 Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. 42 Eisenberg, D., Weiss, R.M. and Terwilliger, T.C. (1982) Nature 299, 371-374. 43 Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623. 44 Walsh, M.T. and Atkinson, D. (1983) Biochemistry 22, 3170-3178. 45 Ohki, S. and Arnold, K. (1990)J. Memb. Biol. !14, 195-203. 46 Gschwendt, M., Kittstein, W. and Marks, F. (1991) TIBS 16, 167-169. 47 Lester, D.S. and Brumfeld, V. (1990) Int. J. Biol. Macromol. 12, 251-256. 48 Mosior. M. and McLaughlin. S. (1991) Biophys. J. 60, 149-159.