Regulation of Protein Kinase Cµ by Basic Peptides and Heparin
PUTATIVE ROLE OF AN ACIDIC DOMAIN IN THE ACTIVATION OF THE KINASE*

(Received for publication, March 20, 1997, and in revised form, April 23, 1997)

Michael Gschwendt Dagger §, Franz-Josef Johannes , Walter Kittstein Dagger and Friedrich Marks Dagger

From the Dagger  Division of Tumor Cell Regulation, German Cancer Research Center, D-69009 Heidelberg, Germany and the  Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Protein kinase Cµ is a novel member of the protein kinase C (PKC) family that differs from the other isoenzymes in structural and enzymatic properties. No substrate proteins of PKCµ have been identified as yet. Moreover, the regulation of PKCµ activity remains obscure, since a structural region corresponding to the pseudosubstrate domains of other PKC isoenzymes has not been found for PKCµ.

Here we show that aldolase is phosphorylated by PKCµ in vitro. Phosphorylation of aldolase and of two substrate peptides by PKCµ is inhibited by various proteins and peptides, including typical PKC substrates such as histone H1, myelin basic protein, and p53. This inhibitory activity seems to depend on clusters of basic amino acids in the protein/peptide structures. Moreover, in contrast to other PKC isoenzymes PKCµ is activated by heparin and dextran sulfate. Maximal activation by heparin is about twice and that by dextran sulfate four times as effective as maximal activation by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate, the conventional activators of c- and nPKC isoforms.

We postulate that PKCµ contains an acidic domain, which is involved in the formation and stabilization of an active state and which, in the inactive enzyme, is blocked by an intramolecular interaction with a basic domain. This intramolecular block is thought to be released by heparin and possibly also by 12-O-tetradecanoylphorbol-13-acetate/phosphatidylserine, whereas basic peptides and proteins inhibit PKCµ activity by binding to the acidic domain of the active enzyme.


INTRODUCTION

Protein kinase Cµ (PKCµ)1 is a serine/threonine protein kinase that is phospholipid-dependent and activated by diacylglycerol and the phorbol ester TPA (1-3). In this respect, PKCµ behaves similarly as most PKC isoenzymes (cPKCs and nPKCs, for reviews see Refs. 4 and 5) of the PKC family. However, PKCµ differs in some structural and enzymatic features from the other PKC isotypes known so far (1-3, 6), indicating that it may represent a novel subfamily of PKC. Thus, the two cysteine-rich domains, which serve as binding sites for cofactors and activators, are much further apart in PKCµ than in all the other PKCs and in contrast to the other PKC isoenzymes, PKCµ contains a pleckstrin homology domain and lacks a region corresponding to pseudosubstrate regions of the PKC family members. Moreover, PKCµ is not inhibited by a PKC-specific inhibitor (7) and is not down-regulated upon prolonged TPA treatment of murine keratinocytes and epidermis (8). As yet, no substrate proteins of PKCµ have been found (3, 9), neither in vivo nor in vitro, even though numerous proteins are known that are phosphorylated by the other PKC isoenzymes. Recently, Sidorenko et al. (10) claimed that the tyrosine kinase Syk and the phospholipase Cgamma 1 were substrates of PKCµ. However, incorporation of phosphate into these proteins was lower than into myelin basic protein, which was shown by us and others to be an extremely poor substrate of PKCµ (3, 9). As PKCµ substrates are not known so far, the function of PKCµ in cellular signaling is for the most part obscure. Very recently, data were presented suggesting that PKCµ is located at the Golgi apparatus and is involved in basal transport processes (11). Moreover, it was suggested that PKCµ regulates lymphocyte signaling (10).

Here, we report on a possibly differential regulation of the kinase activities of PKCµ and other PKC isoenzymes. In contrast to other PKCs, PKCµ is inhibited by various proteins and peptides, most likely due to clusters of basic residues in their structure, and is activated by heparin and dextran sulfate. Based on these results, the putative role of an acidic domain in the activation of PKCµ is discussed.


EXPERIMENTAL PROCEDURES

Materials

TPA was supplied by Prof. Dr. E. Hecker, German Cancer Research Center, Heidelberg, Germany. Gö6976 and Gö6983 were kindly provided by Gödecke, A.G., Freiburg, Germany. The tumor suppressor protein p53 was given by Dr. M. Frey, German Cancer Research Center, Heidelberg, Germany. Syntide 2, µ-peptide 1, µ-peptide 2, p53-peptide, and the Ser-pseudosubstrate peptides of PKCdelta , zeta , and eta  (delta -peptide, [zeta ]-peptide, [zeta ]-peptide-1, [zeta ]-peptide-2, [eta ]-peptide) were synthesized by Dr. R. Pipkorn, German Cancer Research Center, Heidelberg, Germany).

Other materials were bought from companies as indicated: [gamma -32P]ATP (specific activity, 5000 Ci/mmol), Hartman Analytic (Braunschweig, Germany); aldolase, Boehringer (Mannheim, Germany); heparin, phosphatidylserine (PS), protamine sulfate, dextran sulfate, histone H1 (III-S), myelin basic protein, poly-L-lysine (Mr 15,000-60,000), poly-L-lysine (Mr 1,000-4,000) poly-L-arginine (Mr 15,000-60,000), L-lysine, histamine, quinine, Sigma (Munich, Germany).

Recombinant PKCµ

Sf 158 cells were infected with recombinant PKCµ baculovirus, and cell extracts were prepared and used as source for PKCµ as described previously (3, 7).

Protein Kinase Cµ Assay

Phosphorylation reactions were carried out in a total volume of 100 µl containing buffer I (50 mM Tris-HCl, pH 7.5, 10 mM beta -mercaptoethanol), 4 mM MgCl2, 5 µl of a Sf 158 cell extract containing recombinant PKCµ, 35 µM ATP containing 1 µCi of [gamma -32P]ATP and 5 µg of syntide 2 or µ-peptide 1 as substrates. PS, TPA, heparin, Gö6976, Gö6983, or various other compounds (see Table I) were added at concentrations indicated in the legends of the figures and Table I. After incubation for 7 min at 30 °C, the reaction was terminated by transferring 50 µl of the assay mixture onto a 20-mm square piece of phosphocellulose paper (Whatman p81), which was washed three times in deionized water and twice in acetone. The radioactivity on each paper was determined by liquid scintillation counting. Phosphate incorporated into the substrate peptide was obtained by subtracting values determined in the absence of kinase.

Table I. Inhibition of PKCµ-catalyzed syntide 2 phosphorylation by various proteins, basic peptides, and other basic compounds

Incorporation of phosphate into syntide 2 by PKCµ was determined by applying the kinase assay as described under "Experimental Procedures." Mutated amino acids in the Ser-pseudosubstrates zeta 1 and zeta 2 are in bold. The elongation factor EF-1alpha was purified from porcine spleen as described previously (12).

Compound (5 µg) Inhibition of syntide 2 phosphorylation

%
Protamine sulfate 95
Histone H1 94
Myelin basic protein 78
p53 68
EF-1alpha 6
p53-peptide, SHLKSKKGQSTSRHKK 58
MARCKS-peptide, KKKKKRFSFKKSFKLSGFSFKKSK 92
Ser-pseudosubstrate zeta  (zeta -peptide), IYRRGSRRWRKL 62
Ser-pseudosubstrate zeta -1 (zeta -peptide-1), IYRRGSIRWRKL 48
Ser-pseudosubstrate zeta -2 (zeta -peptide-2), IYRRGSIRWAKL 29
Ser-pseudosubstrate eta  (eta -peptide), RKRQRSMRRRVH 51
Ser-pseudosubstrate delta  (delta -peptide), MNRRGSIKQAKI 15
µ-Peptide 2, GVRRRRL 5
Poly-L-arginine, Mr 15,000-60,000 90
Poly-L-lysine, Mr 15,000-60,000 76
Poly-L-lysine, Mr 1,000-4,000 64
L-Lysine, histamine, quinine (free base) 0

Autophosphorylation and Phosphorylation of Aldolase or Histone H1

These phosphorylations were carried out essentially as described for the protein kinase Cµ assay. However, no substrate was added for the autophosphorylation, and for the phosphorylation of aldolase or histone H1, these proteins instead of the substrate peptides were added, at the concentrations indicated in the text. The assay contained 7 µCi of [gamma -32P]ATP. Proteins of the reaction mixture were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.


RESULTS AND DISCUSSION

No substrate protein of PKCµ, neither in vitro nor in vivo, has been found so far. In accordance with previous reports (3, 9), we observed that typical PKC substrates, such as histone H1, myelin basic protein, and protamine sulfate, were phosphorylated very weakly by PKCµ and, therefore, cannot be considered as PKCµ substrates. Recently, the tyrosine kinase Syk and the phospholipase Cgamma 1 were claimed to be substrates of PKCµ. However, phosphorylation by PKCµ in vitro of these proteins was even weaker than that of myelin basic protein (10). Here we show that aldolase can serve as a substrate for PKCµ in vitro. Aldolase was phosphorylated by PKCµ much more effectively than histone H1 (Fig. 1). To our surprise, when aldolase (5 µg) and histone (5 or 10 µg) were both present in the kinase assay, histone H1 suppressed the phosphorylation of aldolase almost completely and also autophosphorylation of PKCµ was inhibited (Fig. 1).


Fig. 1. Phosphorylation of aldolase and histone H1 by PKCµ and inhibition of aldolase phosphorylation by histone H1. Aldolase (A) and histone H1 (H) alone or together (the concentration is given as µg/100 µl in brackets) were phosphorylated by recombinant PKCµ as described under "Experimental Procedures." Proteins were separated by polyacrylamide gel electrophoresis (10% gel) and phosphorylated proteins visualized by autoradiography. Molecular masses (kilodaltons) were determined from standard proteins, as indicated.
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To determine the dose dependence of the inhibitory effect of histone H1 on PKCµ, we used syntide 2 as well as a novel synthetic peptide that we termed µ-peptide 1 as substrates for PKCµ. The µ-peptide 1 with the amino acid sequence RKRYSVDKTLSHPWL, corresponding to the sequence 825-839 of human PKCµ, proved to be a potent PKCµ substrate. It incorporated around 30% more phosphate than syntide 2 on phosphorylation with PKCµ.2 Phosphorylation of syntide 2 and µ-peptide 1 by PKCµ was inhibited by histone H1 depending on the concentration of the inhibitor (Fig. 2). Half-maximal inhibition was reached with 0.2-0.3 µg of histone H1/100 µl (IC50).


Fig. 2. Inhibition by histone H1 of phosphorylation of syntide 2 and µ-peptide 1 by PKCµ. Phosphorylation of the peptides (5 µg each) in the presence of various concentrations of histone H1 and determination of incorporated phosphate were performed as described under "Experimental Procedures."
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To prove the hypothesis that the basic properties of histone H1 were responsible for the suppression of PKCµ activity, we tested various proteins and peptides containing basic regions and other basic compounds (5 µg/100 µl each) for their inhibitory capacity (Table I). All proteins tested suppressed the PKCµ-catalyzed phosphorylation, with the exception of the elongation factor EF-1alpha . Protamine sulfate and histone H1 were most effective in this respect, followed by myelin basic protein and the human tumor suppressor protein p53. The p53-peptide with the amino acid sequence SHLKSKKGQSTSRHKK, corresponding to sequence 367-382 of human p53, was similarly active as the p53 protein. Peptides derived from the pseudosubstrate domain of PKCzeta (IYRRGSRRWRKL) and eta  (RKRQRSMRRRVH), which contain serine instead of alanine and therefore serve as substrates for several PKC isoenzymes, were also found to effectively inhibit PKCµ. However, the respective peptide derived from the pseudosubstrate of PKCdelta (MNRRGSIKQAKI) as well as the µ-peptide 2 (GVRRRRL), corresponding to the amino acid sequence 198-204 of human PKCµ, did not show such an inhibitory effect. A major difference between the two peptides and the inhibitory peptides (zeta -peptide, eta -peptide, myristoylated alanine-rich protein kinase C substrate-peptide, and p53-peptide) exists in the total number and clustering of basic amino acids (Arg/Lys). The delta -peptide and the µ-peptide 2 contain four basic amino acids and one cluster of two or four basic amino acids, respectively, whereas the inhibitory peptides have at least six basic amino acids arranged in two or three clusters. The myristoylated alanine-rich protein kinase C substrate-peptide (KKKKKRFSFKKSFKLSGFSFKKSK) with 12 basic residues and three clusters was the most effective inhibitor peptide. Thus, a peptide might require a minimal positive net charge and/or specific clusters of basic amino acids to be able to inhibit PKCµ. In fact, an exchange of one or two basic amino acids in the zeta -peptide for neutral residues resulting in zeta -peptide-1 (IYRRGSIRWRKL) and zeta -peptide-2 (IYRRGSIRWAKL) caused a gradual loss of inhibitory activity (Table I). zeta -peptide-2 has a similar arrangement of basic amino acids as the delta -peptide. EF-1alpha protein, which does not contain any cluster of basic amino acids even though it is basic (pI = 9), did not inhibit PKCµ thus further supporting our notion. The strongly basic polypeptides poly-L-arginine and poly-L-lysine (molecular weights of 15-60 kDa), and even the smaller poly-L-lysine (molecular mass of 1-4 kDa) inhibited PKCµ effectively. L-Lysine and other basic low molecular weight compounds, such as histamine and quinine, were on the other hand unable to inhibit PKCµ activity. This indicates that structural features, such as the above mentioned clusters of basic amino acids, rather than a positive net charge, determine the suitability of a compound to act as PKCµ inhibitor, thus pointing to some specificity of the interaction with the kinase. Most of the proteins and peptides inhibiting PKCµ are substrates rather than inhibitors of the other PKC isoenzymes, and some of them, such as protamine and poly-L-arginine, have been found to activate other PKC isoenzymes (13). On the other hand, none of the inhibitory proteins and peptides was significantly phosphorylated by PKCµ. Thus, inhibition of PKCµ was not likely to be due to a competition of the inhibitory compound with the substrate syntide 2 for ATP. Inhibition was not reduced by increasing substrate concentrations, as demonstrated in Fig. 3 for the inhibition by protamine sulfate of syntide 2 phosphorylation by PKCµ. For comparative purposes, Fig. 3 shows also the inhibition of PKCdelta by the pseudosubstrate peptide. In this case, inhibition decreased upon increasing the concentration of the substrate syntide 2. This clearly demonstrates that the PKCµ-inhibiting peptides do not act like the well known pseudosubstrate peptides that inhibit other PKC isoenzymes by competing with the PKC substrate for its binding site (14). Therefore, we postulate that PKCµ contains an acidic domain, different from the acidic substrate-binding motif of other PKCs (see Ref. 15), which is involved in enzyme activation or stabilization of the active state of the kinase. In the active state PKCµ is inhibited by proteins and peptides containing clusters of basic residues probably due to an interaction with this "activating" domain. In the inactive state the acidic domain might not be accessible, due to an interaction with an autoregulatory basic domain of the enzyme. Indeed, PKCµ exhibits a highly acidic domain (amino acid sequence 336-391 of human PKCµ) in the regulatory part close to the C terminus of the cysteine-rich regions. This domain contains 40% acidic and just 2% basic residues and, in a smaller region (342-362), even 48% acidic residues. It is intriguing that the other PKC isoenzymes lack a comparable domain.


Fig. 3. Inhibition of PKCµ by protamine sulfate and of PKCdelta by the pseudosubstrate peptide at various substrate concentrations. The kinase assay was performed as described for PKCµ under "Experimental Procedures." For inhibition of PKCdelta , 0.6 µl of a Sf9 cell extract containing recombinant PKCdelta (12) and 7.5 µg of the pseudosubstrate peptide MNRRGAIKQAKI, and for inhibition of PKCµ, 5 µl of a Sf 158 cell extract containing recombinant PKCµ and 1 µg of protamine sulfate were used. The kinase assays were performed both in the absence and presence of the inhibitors using various concentrations of the substrate syntide 2. Inhibition of phosphorylation in the presence of the inhibitor is given in percent and is based on the incorporation of phosphate in the absence of the inhibitor at each substrate concentration (0% inhibition). Values are the means of three determinations (bars, ± S.E.).
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Our hypothesis would imply that polyanions are able to break up the autoinhibitory interaction between the acidic and the basic domain. In fact, the highly sulfated polysaccharides heparin and dextran sulfate were found to function as potent activators of PKCµ. Maximal activation of PKCµ by heparin alone, i.e. in the absence of any other cofactor, was about twice and that by dextran sulfate four times as effective as maximal activation by PS/TPA (Fig. 4). Dextran sulfate contains more sulfate groups than heparin and is, therefore probably, more active than heparin in stimulating PKCµ. Application of PS/TPA together with heparin or dextran sulfate did not further increase the activity of PKCµ. Activation of PKCµ by heparin or dextran sulfate was saturable at low concentrations (Fig. 5). The Ka values for heparin and dextran sulfate, as determined by a Lineweaver-Burk plot, were approximately 0.36 and 0.05 µM (based on an average molecular weight of 20,000 and 500,000, respectively, as given by the supplier). Thus, heparin and dextran sulfate are very effective activators as compared for instance with diacylglycerol (e.g. the Ka value of dioctanoylglycerol for PKCdelta is 10 µM, see Ref. 16). As shown in Fig. 6, A and B, the maximal velocities (Vmax) of the heparin- and dextran sulfate-activated syntide 2 phosphorylations (23.3 pmol/min and 41.7 pmol/min, respectively) were much higher than that of the PS/TPA-activated phosphorylation (9.5 pmol/min). On the other hand, the affinity of the enzyme for the substrate was rather lower upon activation with the two polyanions (same Km for both: 9.5) than with PS/TPA (Km: 4.8 µM). Thus, the much more effective incorporation of phosphate into syntide 2 by the polyanion-activated PKCµ than by the PS/TPA-activated kinase is due to an increase in the maximal velocity of the phosphorylation reaction. As the mechanisms of action of heparin and dextran sulfate are likely to be identical, we will in the following just deal with heparin. Autophosphorylation of PKCµ was also more efficiently stimulated by heparin alone than by PS/TPA (Fig. 7). Both, heparin- and PS/TPA-activated (7) autophosphorylation could be strongly suppressed by 1 µM of the PKC inhibitor Gö6976, but not at all by 1 µM of Gö6983, an effective inhibitor of the other PKC isoenzymes. These inhibitors are known to interact with the ATP binding site of PKC.


Fig. 4. Activation of PKCµ by PS, TPA, heparin, and dextran sulfate. Phosphorylation of syntide 2 (5 µg) by PKCµ in the absence or presence of PS (10 µg/100 µl), and/or TPA (100 nM), and/or heparin (3 µg/100 µl), and/or dextran sulfate (3 µg/100 µl) was performed and incorporated phosphate measured as described under "Experimental Procedures." Data are expressed as the means of two independent experiments.
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Fig. 5. Activation of PKCµ by heparin and by dextran sulfate. PKCµ activity was determined in the presence of various concentrations of heparin or dextran sulfate as described in the legend to Fig. 4.
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Fig. 6. Dependence of PKCµ activation by PS/TPA, heparin, and dextran sulfate on substrate kinetics. A, phosphorylation of syntide 2 (concentrations as indicated) was performed as described in the legend to Fig. 4 and under "Experimental Procedures." B, Lineweaver-Burk plots of the data shown in A. The intercepts of the double-reciprocal plots with the x axis give the Km values and those with the y axis the Vmax values.
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Fig. 7. Stimulation of PKCµ autophosphorylation by heparin. Autophosphorylation of PKCµ was performed in the absence or presence of heparin (various concentrations as indicated), 3 µg/100 µl of heparin (Hep), 10 µg/100 µl of PS, 100 nM TPA, and 1 µM each of the PKC inhibitors Gö6976 and Gö6983, as described under "Experimental Procedures." Upon polyacrylamide gel electrophoresis, phosphorylated PKCµ was visualized by autoradiography.
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TPA is generally thought to activate PKC by a conformational change that results from its binding to the zinc finger regions of the enzymes (4). As a consequence, an inhibitory pseudosubstrate domain is removed from the substrate binding site. Whether this mechanism can explain the activation by TPA/PS of PKCµ remains an open question, since a domain corresponding to the pseudosubstrate regions of other PKC isoenzymes has not been found in the PKCµ structure (7). This does not exclude, however, that upon identification of bona fide in vivo substrates of PKCµ, a specific pseudosubstrate sequence will be identified in the future. Within the PKC family the activation by heparin may, on the other hand, turn out to be a specific feature of PKCµ, since PKCdelta activity was not affected by heparin (data not shown) and a PKC preparation from rat brain (containing mainly PKC alpha , beta , gamma ) was even inhibited by heparin (17). The latter result is in agreement with the finding that heparin might block smooth muscle cell proliferation by inhibition of PKCalpha (18). Several other protein kinases, such as casein kinase 1 and 2, nuclear kinases, the tyrosine kinase Syk, and G-protein-coupled receptor kinases are inhibited by heparin (17, 19, 20, and references in Ref. 17). On the other hand, activation by heparin was reported for instance for a RNA-activated protein kinase (21) and a Lyn-related tyrosine protein kinase (22). Activation of each of the two kinases by heparin was shown to occur through mechanisms different from those of other known activators of these kinases, thus resembling the activation of PKCµ by heparin. Moreover, many growth factors are known to bear specific heparin-binding sites that contain a cluster of basic amino acid residues (23, 24).

The activation by heparin or dextran sulfate of PKCµ appears to be rather specific, as other acidic compounds, such as chondroitin sulfate, cholesterol sulfate, double-stranded polyinosinic-polycytidylic acid, DNA (calf thymus), poly-L-aspartic acid, and poly-L-glutamic acid, did not or just very weakly activate PKCµ (data not shown). This supports the notion that heparin and dextran sulfate specifically break up the intramolecular interaction of basic residues with an acidic domain of PKCµ. The apparent specificity of the stimulatory effect may be taken as an indication for a physiological function of heparin or heparin-like compounds in the control of PKCµ activity. Heparin has been shown to affect various intracellular signaling pathways, including PKC-dependent pathways, and to be a potent proliferation inhibitor of several cell types (Refs. 25 and 26 and references therein). However, these effects are thought to be mediated by binding of heparin to cell surface binding sites or growth factors (23, 24, 27). Little is known about possibly direct actions of heparin on signaling pathways inside the cell. As heparin is synthesized in the Golgi complex and PKCµ has recently been shown to be localized there (11), a direct action of heparin on PKCµ in this cellular compartment is conceivable. Alternatively, heparin might mimic the effects of heparin-like factors in vivo. Such factors are produced, for instance, by endothelial and smooth muscle cells and are growth-inhibitory for these cells (28, 29).


FOOTNOTES

*   This work was supported by Deutsche Forschungsgemeinschaft Grants Ma 381/12-2 and Jo 227/4-2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 49-6221-424505; Fax: 49-6221-424554; E-mail: m.gschwendt{at}dkfz-heidelberg.de.
1   The abbreviations used are: PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; PS, phosphatidylserine.
2   M. Gschwendt, F.-J. Johannes, W. Kittstein, and F. Marks, unpublished results.

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