A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor.

Synthetic peptides corresponding to the pseudosubstrate domains of protein kinase C (PKC) have been used as specific inhibitors of PKC in in vitro assays and permeabilized cell systems. However, their use in vivo was hampered by the impermeability of the plasma membrane for such peptides. Here, we show that N-myristoylation of the PKC pseudosubstrate nonapeptide Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln permits its use as an inhibitor of PKC in intact cells. The myristoylated peptide, myr-psi PKC, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, as induced by 12-O-tetradecanoyl-phorbol-13-acetate, and the activation of phospholipase D by bradykinin, which strictly depends on PKC. Half-maximal inhibition is obtained at concentrations of 8 and 20 microM, respectively. An N-myristoylated peptide derived from an inhibitor protein of the cAMP-dependent protein kinases, Myr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile, was ineffective. These results show that myr-psi PKC is a selective and cell-permeable inhibitor of PKC.

Synthetic oligopeptides based on pseudosubstrate sequences are potential candidates for specific inhibitors because they exploit the substrate specificity of the enzyme and presumably do not interfere with ATP binding. Peptides based on the pseudosubstrate sequence have previously been shown to inhibit PKC in vitro (3,7). These peptides have also succesfully been used in permeabilized cell systems, in which they inhibited phosphorylation of CD3 in T lymphocytes (8,9) and of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in fibroblasts (10). Finally, microinjection has been used as a means of delivering pseudosubstrate peptides to the intracellular medium in neuronal cells (11,12).
A recent approach utilizes modification of peptides by attachment of a fatty acid as a means to overcome the permeability barrier of the plasma membrane. Substrate peptides, whose sequences were based on the major phosphorylation site ( T h P 4 ) of PKC in the EGF receptor displayed, when Nmyristoylated, inhibitory activity against PKC in vitro (13).
In this study we have used a myristoylated nonapeptide, now based on the pseudosubstrate motif of PKC-a and -P subtypes, Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-$PKC), in an attempt to inhibit PKC in several cell systems. We find that in human fibroblasts this peptide inhibits ( a ) phosphorylation of the MARCKS protein and ( b ) activation of phospholipase D, both mediated by PKC. The myristoylated peptide Myr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile (myr-$PKA), also a pseudosubstrate, based on the inhibitory sequence of PKI, the inhibitor protein of the cyclic AMPdependent protein kinase (PKA) (16) was used as a control for the sequence specificity of the effect. We conclude that this myristoylated peptide is a specific PKC inhibitor that may have a wide potential range of applications, without the drawbacks of other, widely used PKC inhibitors that interfere with ATP binding, such as staurosporine and H7 (17, 18).

MATERIALS AND METHODS
Peptides-The PKC pseudosubstrate peptide Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (GPKC) and the myristoylated peptide Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-GPKC) were both synthesized by solid phase using the Fmoc methodology (19, 20) using a home-designed semiautomatic synthesizer. The first amino acid was attached to the 4-(hydroxymethyl)phenoxy acetic acid (Wang linker (21)) kieselguhr polydimethyl acrylamide resin (MilIigen@' ) as its benzotriazole ester analogous to the procedure of Van Nispen et al. (22). For the coupling steps, the appropriate amino acids (3 equivalents) dissolved in DMA were activated in situ with benzotriazole-1-yl-oxy-tris(dimethy1amino)phosphonium hexafluorophosphate (BOP, 3 equivalents) (23) in the presence of diisopropylethylamine (6 equivalents), transferred to the resin, and circulated for 1 h. The following side chain protective groups were used. The 4-methoxy-2,3,6-trimethylbenzene sulfonyl group (24) was used to protect the guanidinium functionality of arginine. The c-amino group of lysine was protected with a t-butyloxycarbonyl group, and the trityl group was used as a protection of the amide function of glutamine.
After the last coupling the resin was divided in two portions. One portion was deprotected as follows. The Fmoc protecting group was removed with a 20% solution of piperidine in DMA. Subsequently, the resin was washed with DMA, t-amyl alcohol, acetic acid, t-amyl alcohol, ether, each three times, respectively, and dried over PzO,in uucuo (19,20). Deprotection and cleavage from the resin were carried out as described earlier (25, 26) using a 0.5 M solution of trifluoromethanesulfonic acid in trifluoroacetic acid in the presence of metacresol and thioanisole (20 equivalents/protecting group). The deprotected PKC pseudosubstrate peptide was purified by Sephadex LH-20 gel filtration (eluent MeOH/HzO, 85:15 (v/v)). Sequencing showed that the pure (according to HPLC, Fig. la) nonmyristoylated PKC pseudosubstrate peptide (GPKC) had the expected amino acid sequence and further characterization by FAB-mass spectrometry confirmed the molecular mass (m/z = 1,032 (M+H+)). The second portion of the peptide-containing resin was subjected to myristoylation. After deprotection of the amino terminus of the immobilized peptide, myristic acid (3 equivalents) was activated in situ with BOP in the presence of diisopropylethylamine, transferred to the resin, and circulated for 1 h. This procedure was carried out twice to ensure completion of the reaction. Subsequently, the peptide attached to the resin was washed and deprotected as described above. The myristoylated PKC pseudosubstrate peptide was purified by Sephadex LH-20 (eluent CHzCl,/MeOH, 1:l (v/v)), characterized by FAB-mass spectrometry (m/z = 1,256 (M+H+)) ( Fig. 2) and pure according to HPLC (Fig. lb).

Asp-Ile and the myristoylated peptide Myr-Gly-Arg-Arg-Asn-Ala-Ile-
His-Asp-Ile were both synthesized on an Abimed (Langerfeld, Germany) multiple peptide synthesizer. The first amino acid was coupled to the 4-(hydroxymethyl)phenoxymethyl-copoly(styrene, 1% divinylbenzeneresin) using the symmetric anhydride of isoleucine in the presence of a catalytic amount of dimethylaminopyridine. Furthermore, for the coupling steps the appropriate amino acids (3 equivalents) dissolved in DMA were activated in situ with benzotriazole-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (27) (PyBOP) and N-methylmorpholine (6 equivalents). All amino acids were coupled twice to ensure completion of the reaction. The following side chain protective groups were used. The 2,2,5,7&pentamethylchromane-6-sulfonyl (28) group was used to protect the guanidinium group of arginine, the carboxylic acid function of aspartic acid was protected with a t-butyl group, and the trityl group was used as a protection of the amide of asparagine and the imidazole ring of histidine. Again, the resin was divided in two portions. One portion Arg-Asn-Ala-Ile-His-Asp-Ile (myr-$PKA) (panel d ) . LKB/ Bromma HPLC equipment with an analytical reverse phase (C'*) column (Lichrospher", 250 X 4 mm, 5 pM), at a flow rate of 1 ml/min and a temperature of 40 "C was used with a linear gradient from 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in acetonitrile. For detection, an UV detector operating at 206 nM was used. Absorption was measured from 0 to 0.5 and is displayed in arbitrary units. All peptides appear as a single peak. was myristoylated as described above except that myristic acid was activated with PyBOP instead of BOP, and the other portion was deprotected as follows. The Fmoc protecting group was removed as described above. Subsequently, the resin was washed with DMA, tamyl alcohol, acetic acidlt-amyl alcohol (1:9 (v/v)), t-amyl alcohol, and ether each three times, respectively. The peptide was cleaved from the resin and fully deprotected using trifluoroacetic acid/H20, 95:5 (v/v) as was described earlier (19,20). The nonmyristoylated PKA pseudosubstrate peptide ($PKA) was purified by preparative FPLC using a Pep HR 16/10 column using a gradient from water to acetonitrile both containing 0.1% trifluoroacetic acid. Pure (Fig. IC) $PKA had the expected amino acid sequence and was further characterized by FAB-mass spectrometry (m/z = 1,053 (M+H+)). The Nmyristoylated PKA pseudosubstrate peptide was cleaved from the solid support, deprotected and purified (see above). Myr-$PKA was characterized by FAB-mass spectrometry ( m / z = 1,262 (M+H+)) ( Fig. 3) and was pure according to reverse phase HPLC (Fig. Id).
Analysis of MARCKS Protein Phosphorylation-HF human foreskin fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum. Prior to the experiments cells were grown to confluence in 35-mm dishes and serum starved for 24 h. Cells were labeled with 0.5 mCi of 32Pi/ml/dish. Cells were preincubated during the last hour of the labeling in the presence of the indicated concentrations of peptide for the indicated times and stimulated with 100 nM TPA for 15 min. After stimulation, total cell lysates were prepared and analyzed on two-dimensional gel electrophoresis as described by O'Farrell (29). Isoelectric focusing in the first dimension was performed using 1% Ampholines 3.5-10 and 4% Ampholines 3.5-5 for acidic pH gradients. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (second dimension in two-dimensional analysis) was performed according to Laemmli (30). 32P-Labeled proteins were detected by autoradiography using Kodak XAR-5 films and Du Pont intensifying screens. Gel slices were cut out, and radioactivity in the slices was determined by liquid scintillation counting.
After preincubation of H F cells in the presence of either peptide, 96% of the cells still exclude the dye trypan blue. Upon removal of the peptide, cells resume growth. These observations argue against short term toxicity or lytic effcts of the peptides used.
Inhibition of Phospholipase D Actiuity-Analysis of phospholipase D activity was assayed as described (31). Shortly, serum-starved monolayers of H F cells were labeled for 3 h with 3 pCi of [3H]myristic acid. Prior to stimulation cells were incubated for 1 h in medium containing the indicated concentrations of peptide or for 2 min in the presence of 200 nM staurosporine. Cells were stimulated for 2 min with 1 p M bradykinin in the presence of 0.05% 1-butanol (32). Cell stimulation was stopped by removal of the medium and the immediate addition of 2 ml of chloroform/methanol (1:l). Carrier lipids were added, and extraction was performed according to Folch et al. (33). Lipids present in the lower phase were separated on Silica Gel 60 TLC plates (Merck) using ethyl acetate/isooctane/acetic acid/water (13:2:3:10, v/v; two runs with intermittent drying). Lipid spots were visualized by spraying with primulin and identified by comparison with reference compounds. The spot containing phosphatidylbutanol was scraped of the plates and prepared for liquid scintillation counting.

Effect of Pseudosubstrate Peptides on
MARCKS Phosphorylation-The state of phosphorylation of the so-called MARCKS protein (34, 35) has been widely used as a read-out system for the activation of PKC (e.g. 10, 36). The MARCKS protein is a 32-kDa protein and has an extended structure, causing it to migrate with an apparent molecular mass of about 80 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its degree of phosphorylation is easily monitored on two-dimensional gel electrophoresis. Because of its rather low isoelectric point, it separates well from other polypeptides. Serum-starved H F cells were labeled with orthophosphate for 4 h and treated with peptides as indicated during the last hour of labeling. As is shown in Fig. 4a (quantitation in Fig. 4b), stimulation of HF cells for 15 min with the phorbol ester TPA induces 2.5-fold stimulation of MARCKS protein phosphorylation. Control levels of phosphorylation are greatly reduced after preincubation with 100 p~ of myr-+PKC. Phosphorylation as induced by TPA is completely blocked by 100 p~ myr-+PKC, with an IC6o of approximately 7-8 p~. The total pattern of phosphorylation shows only few changes after incubation with myr-$PKC, as was determined by two-dimensional gel electrophoresis with a more neutral pH gradient (not shown), which is in line with the notion that MARCKS is the most prominent substrate of PKC in HF cells. Maximal inhibition of MARCKS phosphorylation stimulated by TPA was obtained after 1 h of preincubation with myr-$PKC (data not shown). The nonmyristoylated form of the PKC pseudosubstrate peptide (+-PKC) is a t least 2 orders of magnitude less effective in inhibiting MARCKS protein phosphorylation. This is consistent with a failure to reach the site of action of PKC, the inner surface of the plasma membrane, and thereby supports the necessity of the myristoyl modification for inhibitory action in intact cells. A similar peptide derived from PKI (see also below) could gain access to the cytoplasm in small amounts when present in concentrations as high as 1 mM (37). A similar phenomenon could explain the small decrease in MARCKS phosphorylation observed in the presence of 100 p~ nonmyristoylated +-PKC. However, concentrations of +-PKC higher than 100 p~ did not cause further inhibition (not shown).
As a control for the sequence specificity of myr-+PKC inhibition, we synthesized a myristoylated PKA-pseudosubstrate peptide, myr-+PKA, based on a sequence found in the PKA inhibitor protein PKI (16). The CAMP-dependent protein kinase and PKC have distinct but obviously related consensus phosphorylation sites (38). Both sequences have basic amino acid determinants but at different positions relative to the phosphate acceptor. For PKC the relative positions of basic residues are more variable than in the case of PKA. Synthetic substrate and pseudosubstrate peptides can discriminate between the two kinases in vitro (39). When employed in the MARCKS phosphorylation assay, myr-+PKA at 100 PM only slightly effects the state of phosphorylation of MARCKS (Fig. 4, a and b), which clearly demonstrates the specificity of inhibition by myr-GPKC.
In the human carcinoma cell line KB, phosphorylation of the MARCKS protein is similarly inhibited by myr-GPKC (not shown). At the same time, myr-GPKC did not affect the tyrosine kinase activity of the EGF receptor, as was measured by the degree of autophosphorylation of the EGF receptor in the absence or presence of EGF (not shown).
Inhibition of Phospholipase D Activation-Stimulation of H F cells with the peptide hormone bradykinin results in a rapid activation of phospholipase D, which leads to an elevation of the levels of phosphatidic acid, or, in the presence of 1-butanol, to the formation of phosphatidylbutanol (32).
Phospholipase D activity strictly depends on activation of PKC in HF cells (31). As. is shown in Fig. 5, myr-GPKC fully inhibits the phospholipase D response induced by bradykinin. A half-maximal effect is obtained at about 20 PM. The myr-+PKA peptide has no effect on phospholipase D activity (Fig.   5), indicating that inhibition by myr-GPKC is specific. The nonmyristoylated GPKC peptide is also ineffective in inhibiting phospholipase D (Fig. 6). Staurosporine, at a concentration of 200 nM, inhibits activation of phospholipase D for only about 50%, thereby being less effective than myr-GPKC.

DISCUSSION
To probe the function of PKC, inhibitors competing with the substrate ATP, such as staurosporine (16) and H7 (17), have been used widely. None of these is really very specific in its action (40). With the discovery of the pseudosubstrate domains, new possibilities arose to devise more specific inhibitors: synthetic pseudosubstrate peptides. Their use was hampered thus far by the impermeability of the plasma membrane for such peptides. Earlier studies used a fatty acid modification of PKC substrate peptides to overcome this barrier (14,15). Pseudosubstrate peptides are preferred to substrate peptides because of their natural function in keeping kinases in the inactive state. Moreover, the absence of phosphate acceptor residues in most pseudosubstrate sequences presumably stabilizes the interaction with inhibitor peptides. We now show that myristoylation permits entry of a PKC pseudosubstrate peptide into the intracellular milieu, presumably by facilitation of membrane insertion, in such a way that it can inhibit PKC activity. MARCKS phosphorylation and activation of phospholipase D, well known cellular phenomena that have been attributed to the action of PKC, are inhibited efficiently, up to 98%, by 100 p~ myr-$PKC. Without the myristoylation modification, the peptide does not gain access to the site of action of PKC.
We designed a control peptide myr-$PKA on the basis of the sequence of PKI. The sequence specificity of the inhibitory effect of myr-#PKC is shown by the failure of myr-$PKA t o inhibit PKC. The potential use of myr-$PKA as an inhibitor of PKA may be restrained by the site of action of PKA, which is found in the inactive state in the cytosol and which might translocate into the nucleus upon activation (41). However, we did not test this hypothesis. The ability to activate the EGF receptor fully in the presence of myr-$PKC provides further evidence for the specificity of myr-$PKC. In vitro studies have indicated that the selectivity of different pseudosubstrate peptides for their respective kinases is sometimes relatively small (39), but the pseudosubstrate sequences of both PKC and PKI were found to be sufficiently specific to be used in inhibition studies. The presumed location of myr-+PKC at the site of action of PKC in the plasma membrane provides additional safeguard that PKC is the only kinaseaffected.
The pseudosubstrate sequence on which myr-$PKC was based is derived from the sequence of PKC-a and $3. PKC--y contains a pseudosubstrate with only a single substitution (Phe is replaced by Cys), which is probably not important for intramolecular control of the enzyme. We therefore presume that myr-$PKC also inhibits PKC-7. The other subtypes of PKC, sometimes referred to as nPKCs, show considerable sequence variability in their pseudosubstrate domains. It remains to be seen whether synthetic peptides can be used to discriminate between them. If so, myristoylated forms of these peptides perhaps could generate tools with which to address the intriguing question of the specific function of the different subtypes of PKC. 14.