Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes*

(Received for publication, September 8, 1996, and in revised form, October 16, 1996)

Kiyotaka Nishikawa Dagger §, Alex Toker Dagger , Franz-Josef Johannes , Zhou Songyang Dagger and Lewis C. Cantley Dagger

From the Dagger  Division of Signal Transduction, Beth Israel Hospital and Department of Cell Biology, Harvard Medical School, Boston Massachusetts 02115 and the  Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha , beta I, beta II, gamma , delta , epsilon , eta , µ, and zeta ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKCµ, selected peptides with basic amino acids at positions -6, -4, and -2. PKCalpha , -beta I, -beta II, -gamma , and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKCdelta , -epsilon , -zeta , and -µ preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKCalpha , -gamma , and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKCµ showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKCalpha , -beta I, -beta II, -gamma , -delta , and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKCalpha , -beta I, -delta , -zeta , and -µ were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.


INTRODUCTION

Protein kinase C (PKC)1 family members play crucial roles in the signal transduction of a variety of extracellular stimuli, such as hormones and growth factors (1). To date, twelve isozymes of PKC have been identified in mammalian tissues and subdivided into conventional PKC (cPKC) members comprising alpha , beta I, beta II, and gamma  isoforms (activated by calcium, acidic phospholipid, and diacylglycerol (DAG)), novel PKCs (nPKC) comprising delta , epsilon , eta , and theta  (activated by DAG and acidic phospholipid but insensitive to calcium), and atypical PKCs (aPKC) iota /lambda and zeta  (mechanism of regulation not clear) (1, 2, 3, 4, 5, 6). Another subgroup of PKCs may be defined by PKCµ, which has a potential signal peptide and transmembrane domain (7). Since these PKC isozymes differ in their expression in different tissues and in their mode of activation (1), each isozyme may play some specific role in signal transduction processes. Recent investigations using various approaches such as overexpression and down-regulation of specific isozymes support this idea (1, 5).

A large number of proteins have been shown to be phosphorylated by PKC in vivo and in vitro, such as growth factor receptors, ion channels, ion pumps, transcription factors, and translation factors (1, 8). Based on the sequences of the phosphorylated sites and the use of synthetic peptides based on these sites, a consensus phosphorylation site motif for PKC was determined to be RXXS/TXRX, where X indicates any amino acid (8). However, the optimal substrates have not been investigated by peptide library approaches, and relatively little information is available about differences in substrate selectivity between individual PKC isoforms. Histone IIIS, myelin basic protein, protamine, and protamine sulfate, which contain the above consensus phosphorylation site motif, are known to be efficient substrates for cPKCs, but poor substrates of the nPKC group (1). Recently, elongation factor eEF-1alpha was shown to be phosphorylated with much greater efficiency by nPKCdelta than by cPKCs, nPKCepsilon or -eta , or aPKCzeta (9). Heterogeneous ribonucleoprotein A1 is efficiently phosphorylated by PKCzeta but not by cPKCs or PKCepsilon (10). These findings suggest that the substrate specificity of each PKC isozyme is quite different.

We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates (11, 12, 13). In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. This technique predicts an optimal sequence and provides information about the relative importance of each position for selectivity. Here we have used this approach to determine optimal peptide substrates for human PKCalpha , -beta I, -beta II, -gamma , -delta , -epsilon , -eta , -zeta , and -µ and found that each PKC isozyme has a unique optimal substrate sequence. Furthermore, we prepared synthetic peptides based on the predicted optimal sequences for PKCalpha , -beta I, -delta , -zeta , and -µ and showed that these peptides are high affinity and selective substrates for the respective PKC isozymes.


EXPERIMENTAL PROCEDURES

Materials

DAG was purchased from Boehringer Mannheim. PS was purchased from Avanti Polar Lipids. Isozyme-selective antipeptide antibodies for PKCalpha , -beta I, -beta II, -gamma , -delta , -epsilon , -eta , and -zeta were purchased from Santa Cruz Biotechnology, Inc. PKCepsilon substrate peptide (PRKRQGSVRRRV) was purchased from Upstate Biotechnology Inc. Anti c-myc antibody was purchased from Oncogene Sciences. P81 phosphocellulose paper was purchased from Whatman. [gamma -32P]ATP (3000Ci/mmol) was obtained from DuPont NEN. Liquiscint was purchased from National Diagnostics. A ferric iminodiacetic acid (IDA) beads was purchased from Pierce. All other chemicals were obtained from Sigma. Synthesis of the degenerate peptide library was accomplished according to the standard 1-benzotriazolyloxy-tris-dimethylamino-phosphonium hexafluorophosphate (BOP/HOBt) N-hydroxybenzotriazole coupling protocols using Peptide BioSynthesizer (Minipore Model) as described previously (12).

Expression and Preparation of PKC Family Members

The full-length cDNAs for human PKCalpha , -beta I, -beta II, -gamma , -delta , -epsilon , -eta , and -zeta were subcloned into baculovirus transfer vectors as described previously (6). Recombinant baculoviruses were produced by co-infecting Spondoptera frugiperda cells (Sf9 cells) with the purified baculovirus transfer vectors and purified genomic AcNPV viral DNA using established protocols (14). Recombinant baculovirus encoding c-myc tagged PKCµ and anti-PKCµ monoclonal antibodies was prepared as described previously (7, 15). Baculoviruses expressing each of the nine different PKC isozymes were confirmed using PKC activity screens and immunoblotting with isozyme-selective antipeptide antibodies. Purification of PKCalpha , -beta I, -beta II, -gamma , -epsilon , and -delta were performed by several steps of column chromatography as described previously (16, 17). PKCeta , -zeta , and -µ were isolated by immunoprecipitation using isozyme-selective antipeptide antibodies (for PKCeta and -zeta ) and anti-c-myc antibody (for PKCµ), respectively. 1 unit of PKCalpha , -beta I, -beta II, -gamma , -delta , -epsilon , -eta , or -zeta is defined as the amount of kinase required to incorporate 1 nmol of phosphate into the epsilon -pseudosubstrate peptide per min. 1 unit of PKCµ is defined as the amount of kinase required to incorporate 1 nmol of phosphate into the µ-peptide (see Fig. 2) per min.


Fig. 2. Comparison of the phosphorylation efficiency of synthetic peptides by PKCalpha and PKCµ. A, synthetic peptides derived from the predicted optimal sequence of PKCalpha , -beta I, -delta , -zeta , and -µ were defined as alpha -, beta I-, delta -, zeta , and µ-peptides, respectively. An additional Lys was added to the carboxyl terminus of the predicted optimal peptide for PKCµ to ensure binding to phosphocellulose during the kinase assay. PKCalpha (B) or µ (C) was added to 30 µl of solution containing 100 µM of the indicated synthetic peptide, 100 µM ATP with [gamma -32P]ATP (1.1 × 107 cpm), 1 mM DTT, 5 mM MgCl2, 25 mM Tris-HCl (pH 7.5), 20 µg/ml PS, 10 µM DAG, 200 µM CaCl2 (for PKCalpha ), and 0.5 mM EGTA (for PKCµ). After incubation for the indicated periods at 30 °C, an aliquot (5 µl) of the reaction mixture was spotted onto P81 phosphocellulose paper and washed. Incorporation of 32P was determined by liquid scintillation counting.
[View Larger Version of this Image (29K GIF file)]


Kinase Reaction and Phosphopeptide Separation

Each PKC isozyme (0.2-0.5 units) was added to 300 µl of solution containing 1 mg of degenerate peptide mixture, 100 µM ATP with a trace of [gamma -32P]ATP (roughly 6 × 105 cpm), 1 mM DTT, 10 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 20 µg/ml phosphatidylserine (PS), 10 µM DAG, 200 µM CaCl2 (for PKCalpha , -beta I, -beta II, and -gamma ), and 0.5 mM EGTA (for PKCdelta , -epsilon , -eta , -zeta , and -µ). Reactions were started by addition of PKC and incubated at 30 °C. Reaction conditions were adjusted to allow phosphorylation of about 1% of the total peptide mixture. After reaction, peptide separation was performed as described previously (12). Briefly, the peptide supernatant was removed and diluted with 300 µl of 30% acetic acid. This mixture was then added to a 1-ml DEAE column previously equilibrated with 30% acetic acid, and the column was eluted with 30% acetic acid. After the 600-µl void volume, the next 1 ml contained both phosphorylated and non-phosphorylated peptides but was free of [gamma -32P]ATP. A 0.5-ml column of ferric iminodiacetic acid beads was charged with 2.5 ml of 20 mM ferric chloride, washed with 4 ml of water, then washed with 3 ml of 500 mM NH4HCO3 (pH 8.0), washed again with 3 ml of water, and then equilibrated with 3 ml of buffer A (50 mM MES, 1 M NaCl (pH 5.5)). The dried sample of peptide/phosphopeptide mixture was dissolved in 200 µl of buffer A and loaded onto the ferric column. The column was then eluted with 2.5 ml of buffer A followed by 2.5 ml of buffer B (2 mM MES (pH 6.0)). The phosphopeptides were then eluted with 2 ml of 500 mM NH4HCO3 (pH 8.0). Control experiments, in which the peptides were subjected to a mock phosphorylation, were conducted. The same column protocol was used and the fractions in which phosphopeptides usually elute were collected.

Sequencing and Data Analysis

Typically, 1-2 nmol of phosphopeptide mixture was added to the sequencer. The data analysis was performed as described previously (12). Briefly, the abundance of each amino acid at a given cycle in the sequence of the phosphopeptide mixture from the mock phosphorylation experiments was subtracted from the kinase experiments to correct for the background. To calculate the relative preference for amino acids at each degenerate position, the corrected data were then compared with the starting mixture to calculate the ratios of abundance of amino acids. The sum of the abundance of each amino acid at a given cycle was normalized to 14, 15, or 16 (the number of amino acids present at the degenerate positions) so that each amino acid would have a value of 1 in the absence of selectivity at a particular position.

PKC Assay

PKC activity was assayed in vitro essentially as described previously using the standard PKC vesicle assay (16, 17). The reaction mixture (30 µl) contained 100 µM ATP with [gamma -32P]ATP (5 µCi), 1 mM DTT, 5 mM MgCl2, 25 mM Tris-HCl (pH 7.5), 20 µg/ml PS, 10 µM DAG, 200 µM CaCl2 (for PKCalpha , -beta I, -beta II, and -gamma ), 0.5 mM EGTA (for PKCdelta , -epsilon , -eta , -zeta , and -µ) and indicated amount of synthetic substrate peptide. Reactions were started by addition of PKC (0.002-0.005 units) and incubated at 30 °C for 10 min. Reaction mixtures were spotted onto P81 phosphocellulose paper and washed 4 times in 500 ml of 1% phosphoric acid. Incorporation of 32P was determined by liquid scintillation counting. For each experimental condition, values for control reactions lacking substrate peptide were subtracted as blanks. In all assays to determine Km and Vmax, reaction rates were linear with respect to time for all conditions of peptide, and less than 10% of the peptide substrate was phosphorylated.


RESULTS

Identification of Optimal Substrate Sequence for Nine PKC Isozymes

In order to determine optimal substrate sequences for each of nine human PKC isozymes (alpha , beta I, beta II, gamma , delta , epsilon , eta , µ, and zeta ), we used a degenerate peptide library, comprising peptides of sequence: MAXXXXRXXSXXXXXAKKK (RS-peptide library), where X indicates all amino acids except Trp, Cys, Ser, or Thr. Trp and Cys were omitted to avoid problems with sequencing and oxidation, whereas Ser and Thr were omitted to ensure that the only potential site of phosphorylation was the Ser at position 10. The Met-Ala sequence at the amino terminus was included to verify that peptides from this mixture are being sequenced and to quantify the peptides present. Ala at position 16 provides an estimate of how much peptide loss has occurred during sequencing. The poly(Lys) tail prevents wash-out during sequencing and improves the solubility of the mixture. Arg was "locked-in" at position 7 since previous studies had shown the importance of Arg at the p-3 position for PKC substrates (8). The library was sequenced, and all 16 amino acids were present at similar amounts at all 11 degenerate positions (data not shown). Another Ser-kinase substrate library (12), comprising peptides of sequence MAXXXXSXXXXAKKK, was also used to investigate the 9 PKC isozymes, and was poorly phosphorylated compared with the RS-peptide library confirming the importance of Arg at the p-3 position.

The RS-peptide library was incubated with each PKC isozyme under conditions in which approximately 1% of the total peptide mixture was phosphorylated. The phosphopeptide products were separated from non-phosphorylated peptides using the ferric-iminodiacetic acid column, and the mixture was sequenced. In Fig. 1, the relative abundance of amino acids at each of the 11 positions of degeneracy are presented from experiments using PKCalpha and -µ. These two enzymes clearly selected for distinct peptide substrates. PKCalpha preferred peptides with Arg at p-5 and p-4, while PKCµ selected peptides with Leu and Val, respectively, at p-5 and p-4. In fact, PKCµ had an extremely strong selectivity for peptides with Leu at position p-5. More than 40% of the phosphopeptide products of PKCµ had Leu at this position. At the p-2 position, both PKCs selected peptides with hydrophilic residues, with Gln and Lys preferred. PKCalpha selected for peptides with Gly at p-1, while PKCµ selected against peptides with Gly at p-1. Both PKCs preferred peptides with hydrophobic amino acids at p+1, though PKCalpha selected Phe while PKCµ selected Val. At p+2, p+3, and p+4 positions, PKCalpha strongly selected for peptides with the basic amino acids Arg or Lys. In contrast, PKCµ preferred peptides with hydrophobic amino acids in both positions.


Fig. 1.

Comparison of the substrate specificities of PKCalpha and PKCµ. Human PKCalpha and -µ were expressed in Sf9 cells using baculovirus. A degenerated substrate library with the sequence Met-Ala-X-X-X-X-Arg-X-X-Ser-X-X-X-X-X-Ala-Lys-Lys-Lys (where X indicates any amino acid apart from Trp, Cys, Ser, or Thr) was presented to PKCalpha and -µ. Each PKC isozyme was added to 300 µl of solution containing 1 mg of degenerate peptide mixture, 100 µM ATP with a trace of [gamma -32P]ATP (roughly 6 × 105 cpm), 1 mM DTT, 10 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 20 µg/ml PS, 10 µM DAG, 200 µM CaCl2 (for PKCalpha ), and 0.5 mM EGTA (for PKCµ). After 2 h at 30 °C, the phosphopeptides were separated on DEAE-Sephacel and a ferric chelation column. Reaction conditions were adjusted to allow phosphorylation of about 1% of the total peptide mixture. The phosphopeptides mixture was sequenced. Each panel indicates the relative abundance of the 15 (position -5 to -2 N-terminal to the phosphorylated Ser) or 16 (position -1 to +3 C-terminal to the Ser) amino acids at a given cycle of sequencing. The signal of Gly at position -7 to -2 and the signal of Ile for PKCµ at position +2 to +5 were not available because of a high background. The columns represent average values from two independent experiments. The one-letter amino acid code is used: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; V, Val; Y, Tyr.


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The results obtained for other PKC isozymes are summarized in Table I. All PKC isozymes selected for peptides with hydrophobic amino acid at p+1 position, and all isozymes except PKCµ selected for peptides with basic amino acid at the p-6, p-4 and p-2 positions. Interestingly, all PKC isozymes selected for peptide substrates with Gln or Glu at the p-2 position, although in most cases Lys at this position was optimal. PKCalpha , -beta I, -beta II, -gamma , and -eta selected for substrates with basic amino acid at positions p+2, p+3, and p+4, but PKCdelta , -epsilon , -zeta , and -µ preferred substrates with hydrophobic amino acid in these regions. At p-5, the selectivity was quite different among these isozymes. PKCalpha , -gamma , and -delta selected peptides with Arg; in contrast PKCbeta II, -zeta , and -µ selected for peptides with hydrophobic amino acids such as Phe, Leu, or Val. PKCbeta I and -eta preferred substrates with either Leu or Arg.

Table I.

Substrate specificities of protein kinase C isozymes

Values in parentheses indicate the relative selectivities for the amino acids; amino acids with values less than 1.5 are omitted. Bold letters indicate amino acids that are strongly selected; X indicates no selectivity. The one-letter amino acid code is used. All human PKC isozymes were expressed in Sf9 cells using baculovirus. A kinase substrate library with the sequence Met-Ala-X-X-X-X-Arg-X-X-Ser-X-X-X-X-X-Ala-Lys-Lys-Lys (where X indicates any amino acid apart from Trp, Cys, Ser or Thr) was presented to each PKC isozyme. The kinase reaction was performed as described in the legend for Fig. 1. Each PKC isozyme was evaluated at least twice; average values are shown.
PKC isozymes Position
 -7  -6  -5  -4  -3  -2  -1 0 +1 +2 +3 +4 +5

PKCalpha R(2.1) R/F(1.5) R(2.1) R(2.1) R K(2.2) G(2.0) S F(2.6) R/K(3.4) R/K(3.0) K(2.3) A(1.8)
L/F(1.7) F(1.6) Q(2.0) A/M(1.7) M/I/L(1.8) R(1.9) K(1.7)
R(1.8) V (1.5)
PKCbeta I R/K/F(1.5) K(1.8) L/R(2.1) K/R(2.3) R K(2.7) G(2.0) S F(3.1) K(2.5) K(2.0) F(1.8) A(2.3)
R(1.6) F(2.0) F(1.5) Q(2.2) A(1.9) M(1.8) R(1.9) F/R(1.7) V(1.6) F(1.8)
R/A(1.7) K(1.8) I/V(1.6) F(1.8)
PKCbeta II Y(1.8) K(1.6) L(1.9) K(1.9) R K(2.7) G(2.1) S F(3.0) K(3.0) K(2.6) K(1.9) A(1.7)
F(1.8) R(1.6) Q(2.0) K(1.8) M(2.1) R(2.0) Y(1.7) F(1.5) F/K(1.6)
E(1.5) M(1.7) L(1.8) F/M(1.5) F/R(1.5) M(1.5)
PKCgamma R(1.9) R(1.9) R(2.7) R(3.5) R K(3.5) G(2.7) S F(2.1) K(2.8) R(3.4) K(2.0) A(2.1)
K(1.6) F(1.5) K(1.6) R(1.9) K(2.4) K(1.9) R(2.3) K(2.3) R(1.9) K(1.6)
Q(1.8) A(1.6) M(1.5) R(1.5)
PKCdelta A(1.6) A/R(1.7) R(2.1) K/A(1.7) R K(2.1) G(2.6) S F(2.4) F(1.8) Y/F(1.5) G(1.9) G(2.2)
K(1.6) E(1.9) A(1.7) M/V(1.8) K/M(1.6) F(1.6) F(1.8)
Q(1.5) K(1.5) V(1.5)
PKCepsilon Y(1.9) Y/K(1.5) X K(1.7) R K(3.1) M(1.7) S F(1.9) F/A(1.7) E(1.7) F/E(1.5) D/E/F(1.5)
E(1.6) A/Q(1.5) E(2.7) K(1.6) I/M(1.7) Y/D(1.6)
R(2.1) V/L(1.6) F(1.5)
D(1.9)
PKCeta A(2.1) R(2.0) L/R(1.9) R(1.9) R R(2.2) R(2.3) S F(2.4) R(2.0) R(2.1) X R(1.6)
R(1.8) A(1.6) K(1.5) K(2.0) G(1.8) R(1.9) Y(1.9) Y(1.6)
Q(1.9) M(1.5) F(1.8)
PKCzeta R(1.8) R/K(1.6) F(2.5) K(1.8) R Q/K(1.8) G(1.7) S F(2.6) F(2.1) Y/F(1.8) F(1.9) F(1.7)
L/R(1.6) F(1.6) Y(1.6) K/M/Y(1.5) M(2.0) M(2.0) K(1.7) Y/K(1.6) K/Y(1.5)
A(1.5)
PKCµ A(1.9) A/K(1.7) L(6.2) V(2.2) R Q(2.4) M(2.2) S V(2.0) A/M(1.7) F(1.8) F(2.2) F(1.9)
Y(1.5) P(1.5) V(3.3) L(2.1) K(2.1) L/K(1.7) M(1.7) V(1.5) Y(1.5) V(2.0) P(1.6)
A(2.0) E(2.0) L(1.6) A(1.6) M(1.5)
M(1.8)

Comparison of the Predicted Optimal Substrate Sequences with Corresponding Pseudosubstrates and Other Known PKC Substrates

In Table II, the predicted optimal sequence for each PKC isozyme was compared with the pseudosubstrate region of the respective isozyme by lining up the pseudosubstrate Ala with the Ser of the substrate. The amino acid at p-3 in all the pseudosubstrate sequences was Arg, consistent with our observation that peptides with Arg at p-3 are preferentially phosphorylated. Furthermore, hydrophobic amino acids are present at the p+1 position in all the pseudosubstrate sequences except that of PKCzeta . The predicted optimal sequences from p-3 to p+2 for PKCalpha , -beta I, -beta II, -gamma , -delta , and -eta were in good agreement with pseudosubstrate sequences of the corresponding PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides.

Table II.

Comparison of the optimal sequence of each PKC isozyme determined by the peptide library with the pseudosubstrate region of each isozyme

The optimal sequence of each PKC isozyme determined by the peptide library (optimal.) and the pseudosubstrate region of each isozyme (pseudo.) are presented by the one-letter codes, respectively. Bold letters indicate the phosphorylated Ser in the optimal sequence and the corresponding Ala in the pseudosubstrate region. Boxed amino acids emphasize positions that are similar between optimal sequence and pseudosubstrate region.
PKC isozymes Position
 -7  -6  -5  -4  -3  -2  -1 0 1 2 3 4 5

PKCalpha
  optimal. R R R R R K G S F R R K A
  pseudo. N R F A R K G A L R Q K N
PKCbeta I F K L K R K G S F K K F A
V R F A R K G A L R Q K N
PKCbeta II Y K L K R K G S F K K K A
V R F A R K G A L R Q K N
PKCgamma R R R R R K G S F K R K A
P L F C R K G A L R Q K V
PKCdelta A R R K R K G S F F Y G G
P T M N R R G A I K Q A K
PKCepsilon Y Y X K R K M S F F E F F
R P R K R Q G A V R R R V
PKCeta A R R R R R R S F R R X R
F T R K R Q R A M R R R V
PKCzeta R R F K R Q G S F F Y F F
K S I Y R R G A R R W R K

The optimal substrates predicted for the various PKC isozymes are in good agreement with known substrates. In Table III, the optimal substrate sequence of PKCalpha is compared with known PKC substrates, most of which were determined to be PKC substrates using cPKCs or partially purified PKC isozyme mixtures (probably mixtures of PKCalpha , -beta , and -gamma ). Most of these proteins have the motif R/K-X-R/K-R/K-X-S/T-Phi -R/K-R/K, where Phi  indicates hydrophobic amino acids (F, L, V). This motif is in agreement with the predicted optimal peptide from the peptide library experiment. The strongest selectivities from the library were for hydrophobic amino acids at p+1 and R/K at p+2, and almost every protein substrate has these characteristics. As mentioned above, the presence of Arg at the p-3 position is important for PKC substrates. However, some known substrates do not have Arg at this position (Table III). Using another library, comprising MAXXXXSXXXXAKKK, we found that PKCalpha , -beta I, and -delta strongly selected for peptides with Arg at p-3 (selectivity values 6.0, 4.5, and 6.5, respectively), followed by His (1.9, 1.9, and 1.7) and Lys (1.3, 2.8, and 1.4) (data not shown). Thus, although Arg is preferred, substrates with His or Lys at p-3 are also selected. It is also expected that peptides lacking a basic amino acid at p-3 but with optimal amino acids at the other critical positions could still be reasonable substrates. Recently, a few proteins has been shown to be isozyme-specific substrates. eEF-1alpha is reported to be a specific substrate for PKCdelta (9). The sequence of 426-436 from murine eEF-1alpha containing Thr-431 was compared with the predicted optimal substrate of PKCdelta (Table III). PKCdelta strongly selects for substrates with Arg at p-5, basic at p-2, hydrophobic at p+1, and Gly at p+4. The site in eEF-1alpha meets these criteria. The failure of cPKCs to phosphorylate this site could be explained by the lack of basic residues at p+2 and p+3. PKCµ is shown to phosphorylate glycogen synthase-derived peptide (15) much better than MARCKS, which is known to be a good substrate for other PKC isozymes (18, 19). The glycogen synthase-derived peptide sequence is in good agreement with the optimal peptide sequence that we predicted for PKCµ. Hydrophobic amino acids are found at positions -1 and +1 to +5, Arg is at -3, and most importantly, Leu is at position -5, the position at which PKC shows greatest selectivity (Table III). None of the PKC phosphorylation sites in the MARCKS protein have Leu at the p-5 position (Table III), explaining why MARCKS is not a good substrate for PKCµ.

Table III.

Comparison of the optimal sequence of each PKC isozyme determined by the peptide library with sequences at the same regions of known PKC substrates

Amino acids are represented by the one-letter code. Positions that showed great selectivity for each PKC are indicated by an asterisk.
Peptide/protein position
 -5  -4  -3  -2  -1 0 +1 +2 +3 +4 +5

* * * *
PKCalpha optimal seq. R R R K G S F R R K A
K K
  MARCKS proteina K K K R F S F K K S F
  MARCKS proteina F S F K K S F K L S G
  MARCKS proteina K L S G F S F K K N K
  Neuromodulina A K I Q A S F R G H I
  GABA type A receptor gamma 2Lb L L R M F S F K A P T
  EGF receptora I V R K A T L R R L L
  Ribosomal protein S6a R R R L S S L R A S T
  PTP1Bc R V V G G S L R G A Q
  Troponin Id K F K R P T L R R V R
  Insulin receptor tyrosine kinasee N G R I L T L P R S N
  P-glycoproteinf R S T R R S V R G S Q
  P-glycoproteinf L I R K R S T R R S V
  Kit/SCFRg A D K R R S V R I G S
  Annexin IIa P S A Y G S V K P Y T
* * * * * * *
PKCdelta optimal seq. R K R K G S F F Y G G
  Elongation factor-1alpha h R D M R Q T V A V G V
* * * * * * *
PKCµ optimal seq. L V R Q M S V A F F F
  GS-peptidei L S R T L S V A A L L

a  From Ref. 8.
b  From Ref. 26.
c  From Ref. 27.
d  From Ref. 28.
e  From Ref. 29.
f  From Ref. 30.
g  From Ref. 31.
h  From Ref. 9.
i  From Ref. 15.

Determination of Vmax and Km Values of Synthetic Peptides Derived from the Predicted Optimal Substrate Sequences

The predicted optimal peptide substrates for PKCalpha , PKCbeta I, PKCdelta , PKCzeta , and PKCµ were synthesized (Fig. 2A) and investigated as substrates of the various PKC isozymes. For comparison, a commercially-available PKC substrate peptide based on the pseudosubstrate region of PKCepsilon was used to assay the various PKC isozymes. A set of experiments using 100 µM of the various peptides as substrates of PKCalpha and PKCµ are presented in Fig. 2, B and C. The Vmax and Km values determined for each of the peptides with each of the PKC isozymes are summarized in Table IV. It is clear from Fig. 2 and Table IV that different PKC family members selectively phosphorylate different subsets of the peptides. The optimal substrates for PKCalpha , PKCbeta I, PKCdelta , and PKCµ (as judged by Vmax/Km ratios) were the alpha -peptide, beta I-peptide, delta -peptide, and µ-peptide respectively, in agreement with the library predictions. Curiously, the zeta -peptide was not the optimal peptide for PKCzeta as judged by Vmax/Km ratio, although it was the lowest Km substrate for PKCzeta . In fact, the predicted optimal peptide substrate for each PKC was the lowest Km substrate for that PKC, except in the case of PKCalpha where the beta I and delta -peptides had slightly lower Km values (2.7 µM and 2.8 µM, respectively) than the alpha -peptide (3.8 µM). These results are in agreement with previous results with the peptide library approach that have indicated that this technique selects substrates on the basis of low Km and/or high Vmax/Km ratios rather than on the basis of Vmax alone (11, 12, 13).

Table IV.

Determination of Vmax and Km of synthetic peptides derived from the optimal sequences of PKCalpha , -beta I, -delta , -zeta , and -µ using these PKCs

Phosphorylation reaction was performed in the same condition as described in the legend for Fig. 2 in the presence of various amounts of synthetic peptides. Values are the average of three independent experiments. * Values couldn't be determined because of the low phosphorylation efficiency. Vmax was expressed by the relative value; the highest value was calculated as 100 for each isozyme. The 100 of Vmax for PKCalpha , -beta I, -delta , -zeta , and -µ are corresponding to 1.6, 1.2, 2.5, 2.1, and 1.1 nmol/min/unit, respectively. Bold values indicate the best value (the smallest value for Km, the largest value for Vmax and Vmax/Km) among the five synthetic peptides (a-, beta I-, delta -, zeta -, and µ-peptides).
PKC isozymes Peptides
 alpha  beta 1  delta  zeta µ  epsilon

PKCalpha
  KmM) 3.8 2.7 2.8 * * 2.6
  Vmax 100 33 48 * * 62
  Vmax/Km 26.4 12.0 17.1 * * 24.1
PKCbeta I 8.2 2.8 4.8 * * 3.8
100 63 76 * * 83
12.2 22.9 16 * * 22.0
PKCdelta 3.9 1.6 0.98 1.6 * 2.1
100 24 39 9.6 * 36
25.9 14.6 39.9 6.1 * 17.5
PKCzeta 23 15 26 14 69.8 13
100 45 8.4 10 4.7 51
4.3 3.0 0.34 0.72 0.07 4.0
PKCµ * 27 106 10 9.1 *
* 100 6.4 15 60 *
* 3.8 0.06 1.5 6.6 *

An interesting inference from the results in Table IV is that some sequences in peptides/proteins can act as generic substrates of almost all PKC isozymes, whereas other sequences are highly specific substrates for only one or two PKC isozymes. For example, in agreement with previous studies, the epsilon -peptide is a good substrate for all the PKC isozymes investigated except PKCµ. The beta I-peptide is a relatively good substrate for all the PKC isozymes, including PKCµ. In contrast, the delta -peptide is relatively specific for PKCdelta , and the µ-peptide is very specific for PKCµ. In some cases, these results can be explained on the basis of a key residue at a specific position in the sequence. For example, as discussed above, PKCµ strongly selects for substrates with Leu at the p-5 position while other PKC isoforms are less sensitive to the amino acid at this location. Thus, the Leu at position p-5 in the beta I-peptide allows it to be phosphorylated by PKCµ (only the beta I-peptide and µ-peptide have Leu at p-5). Although Leu is not the optimal residue at the p-5 position for PKCalpha , PKCdelta , and PKCzeta , peptides with Leu at this position are clearly good substrates based on the peptide library results (Fig. 1 and Table I). Thus, including a Leu at this position broadens the number of kinases that could phosphorylate the substrate. The high specificity of the µ-peptide for PKCµ can probably be explained by the lack of basic residues at p-2, p+2, and p+3 that are critical for substrates of the other PKC isoforms (Fig. 1, Table I).


DISCUSSION

We have determined the optimal peptide substrates of nine human PKC isozymes using an oriented peptide library approach. The predicted optimal peptides are in good agreement with sequences at phosphorylation sites of known PKC substrates. Different PKC isozymes selected for different optimal peptide sequences based on residues both N-terminal and C-terminal of the site of phosphorylation. These differences can explain why distinct PKC isoforms phosphorylate distinct substrates in vivo and in vitro. The predicted optimal peptides for PKCalpha , PKCbeta I, PKCdelta , and PKCµ were synthesized and shown to be excellent substrates for the respective enzymes.

Although each PKC isozyme had a unique optimal peptide substrate, there were some features common to optimal substrates for all PKC family members and other features common to optimal substrates of subgroups of PKC family members. For example, all PKCs preferred substrates with a basic residue at position -3 and a hydrophobic residue (usually Phe) at position +1. The cPKC family members (alpha , beta I, beta II, and gamma ) could be distinguished from other subfamilies in that they selected for substrates with basic residues at positions -6, -4, -2, +2, and +3. The nPKC family members (delta , epsilon , and eta ) and the aPKCzeta also selected for substrates with basic residues at -6, -4, and -2, but these kinases were not as selective for basic residues at +2 and +3. Instead, peptides with hydrophobic residues at these positions were usually selected. PKCµ was unique in that it selected for substrates with hydrophobic residues at -4, as well as at positions +2, +3, +4, and +5. However, the most critical residue for selectivity of PKCµ is a Leu at the -5 position.

The results we obtained for the specificities of the cPKC family members are in good agreement with previous studies. For example, substitution of the +1 Phe with Ile or the +2 Arg with Ile in the neurogranin peptide substrate (AAKIQAS*FRGHMARKK, asterisk indicates phosphorylation site) reduced phosphorylation by cPKCs (20). This result is consistent with our finding that Phe and Arg are optimal at the +1 and +2 positions for substrates of cPKCs. In another study, amino acid substitutions in the glycogen synthase-derived peptide (GGPLARALS*VAAG, asterisk indicates phosphorylation site) have shown the importance of having basic residues at positions -4, -3, -2, +2, and +3 for phosphorylation by the cPKCs, alpha  and gamma  (21). These results are also in agreement with the predictions of the peptide library (Table I).

As discussed under "Results," the optimal peptides for the various PKC isozymes are similar but not identical to the pseudosubstrate regions of the respective enzymes (Table II). Peptide substrates based on the pseudosubstrate regions of PKCalpha , beta I, -gamma , and -epsilon were previously used to investigate the specificity of these enzymes (22). All four peptides had similar Vmax values with the four enzymes and had the lowest Km when used as substrates for PKCbeta I. This result was not surprising considering how similar these sequences are. In contrast, the optimal peptides predicted by the library are more divergent than the pseudosubstrate sequences and, with the exception of the zeta -peptide, these peptides are preferential substrates of the kinases for which they were designed (Table IV).

A few isozyme-specific PKC substrates have been previously reported. A synthetic peptide based on region 422-443 of eEF-1a (RFAVRDMRQT*VAVGVIKAVDKK) was reported to be phosphorylated at Thr-431 by PKCdelta but not by other PKC isoforms (9). Conversion of Met-428 (the p-3 position) to Arg somewhat increased the ability of this peptide to be phosphorylated by cPKCs, consistent with known selectivity of these enzymes. Conversion of Ala-433 and Val-434 (the p+2 and p+3 positions) to Lys made this peptide a good substrate for all PKC isozymes. These results are consistent with our finding that the cPKCs prefer substrates with basic residues at p+2 and p+3, whereas PKCdelta will utilize substrates with either hydrophobic or basic residues at these positions but with a slight preference for hydrophobic residues (Table I).

Prior to this study, very little was known about the substrate specificity of PKCµ. The peptide library results show that the two most critical residues for substrates of PKCµ are Leu at position p-5 and an aliphatic residue (preferentially Val) at position p-4 (Table I). PKCµ also differed from the other PKCs in that it selected for peptides with Val rather than Phe at the p+1 position. A recent study (15) showed that PKCµ was very poor at phosphorylating known PKC substrates but phosphorylated the glycogen synthase-derived peptide (LSRTLS*VAALL). This peptide has Leu at p-5, Arg at p-3, Val at p+1, and hydrophobic residues at p+2 through p+5 and thus is predicted to be a good PKCµ substrate based on the peptide library results (Table I). Syntide 2 (PLARTLS*VAGLPGKK), a synthetic peptide derived from glycogen synthase, is also an efficient substrate of human PKCµ (15) and of the mouse homologue called PKD (23). This peptide also has the critical Leu at p-5 along with Arg at p-3, Val at p+1, and hydrophobic residues C-terminal of the phosphorylation site. Since PKCµ (and PKD) have very different substrate specificities than the other PKCs and are reported to be activated by phorbol esters (15, 23), these enzymes are likely to mediate novel phorbol ester signaling pathways distinct from those mediated by other PKCs.

The synthetic peptides we designed based on the predicted optimal substrates could be quite useful for further studies. For example, the beta I-peptide is optimal for PKCbeta I but is a useful general substrate for all PKC isoforms (including PKCµ). The alpha -peptide (like the epsilon -pseudosubstrate peptide) is a general substrate for all PKCs except PKCµ. The µ-peptide is extremely specific for PKCµ, and the delta -peptide is relatively specific for PKCdelta . The zeta -peptide is phosphorylated by PKCzeta , PKCdelta , and PKCµ but not by the cPKCs, so it would be useful for assaying the nPKCs and aPKCs without interference from cPKCs.

Finally, the crystal structure of protein kinase A (PKA) bound to the Walsh inhibitor (PKI) (24, 25) has provided a basis for explaining how protein kinases select for specific substrates. Recently, we proposed a model to explain substrate specificity of protein-Ser/Thr kinases based on the PKA/PKI structure and the alignments of various protein kinase sequences with that of PKA (13). Crystal structures of several protein-Ser/Thr kinases and protein-Tyr kinases indicate that these structures are highly conserved in the catalytic core, supporting the idea that homologous regions of sequences predicted to be in the catalytic cleft of diverse enzymes will be at analogous locations in the folded structures. The model we proposed assumes that all peptide substrates fit into the catalytic cleft in an extended structure similar to that of PKI. Thus, the residues from the kinase that contact the side chains of substrate residues p-5 to p+3 can be predicted from the alignments with PKA.

Table V presents in single letter codes the residues from PKA that make contact with the p-5 to p+3 positions of PKI along with the residues at the analogous positions of the PKC isozymes. The optimal amino acid at each position, as determined with the peptide library, is also presented. It is clear that, as with PKA, the p-3 pockets of all the PKC isozymes are very acidic, and the p+1 pocket is very hydrophobic. This explains why a basic residue is selected at the p-3 position and a hydrophobic residue is selected at P+1 for all these enzymes. The p+1 pocket of PKCµ is more similar to the p+1 pocket of PKA than it is to the p+1 pockets of the other PKC isozymes. This may explain why this enzyme selects for peptides with Val at the p+1 pocket (similar to the Ile selected at p+1 by PKA), while the other PKCs select for peptides with Phe at p+1.

Table V.

Alignment of residues of each PKC isozyme that are predicted to contact with side chains of optimal substrate sequences

Substrate position indicates the N- or C-terminal position of Ala present in Walsh inhibitor pseudosubstrate (PKI, TGRRNAIIHD) or of phosphorylated Ser present in the obtained optimal substrate sequence of each PKC isozyme (see Fig. 1). Residue number indicates the residue present in the indicated subdomain of PKA. Residues present in each PKC isozyme, corresponding to the indicated residue number of PKA, are shown in the same column. Amino acids are represented by the one-letter code for the residue of each kinase or three-letter code for the residue of PKI or each optimal substrate sequence.
Pocket
Substrate position -5 Pocket
Substrate position -4 Pocket
Substrate position -3
Subdomain
Subdomain
Subdomain
V
VI
VIII
V
V
VI
Residue no.
Residue no.
Residue no.
129 133 170 203 127 133 328 127 170 330

PKA F R E E THR E R D GLY E E Y ARG
PKCalpha M Q D D ARG D Q D ARG D D F ARG
PKCbeta I M Q D D LEU/ARG D Q D LYS/ARG D D E ARG
PKCbeta II M Q D D LEU D Q D LYS D D F ARG
PKCgamma M Q D D ARG D Q D ARG D D F ARG
PKCdelta M Q D D ARG D Q D LYS/ALA D D E ARG
PKCepsilon M Q D D X D Q D LYS D D D ARG
PKCeta M Q D D LEU/ARG D Q D ARG D D D ARG
PKCzeta M Q D N PHE D Q D LYS D D Q ARG
PKCµ M S E A LEU M S E VAL M E Y ARG
Pocket
Substrate position -2 Pocket
Substrate position -1
Subdomain
Subdomain
VI
VIII
IX
I
Residue no.
Residue no.
170 201 203 204 230 236 52 53

PKA E T E Y E P ARG G S ASN
PKCalpha D T D Y E P LYS G S GLY
PKCbeta I D T D Y E A LYS G S GLY
PKCbeta II D T D Y E A LYS G S GLY
PKCgamma D T D Y E P LYS G S GLY
PKCdelta D T D Y E S LYS G S GLY
PKCepsilon D T D Y E P LYS G S MET
PKCeta D T D Y E A ARG G S ARG
PKCzeta D T N Y E S GLN/LYS G S GLY
PKCµ E T A Y V F GLN G Q MET
Pocket
Substrate position +1 Pocket
Substrate position +2 Pocket
Substrate position +3
Subdomain
Subdomain
Subdomain
VII
VIII
X
I
II-III
II-III
VIII
Residue no.
Residue no.
Residue no.
187 198 202 205 247 53 54 82 84 81 82 83 198

PKA F L P L Y ILE S F L Q HIS K L K L ASP
PKCalpha M F P I F PHE S F D D ARG/LYS Q D D F ARG/LYS
PKCbeta I M T P I F PHE S F D D LYS Q D D T LYS
PKCbeta II M T P I F PHE S F D D LYS Q D D T LYS
PKCgamma M F P I F PHE S F D D LYS Q D D F ARG
PKCdelta M F P S F PHE S F D D PHE I D D F TYR/PHE
PKCepsilon M F P L F PHE S F D D PHE/ALA Q D D F GLU/TYR/ASP
PKCeta M F P L F PHE S F D D ARG L D D F ARG
PKCzeta M F P I F PHE S Y D D PHE/MET D D E F TYR/PHE
PKCµ F V P L Q VAL Q F K E ALA/MET T K Q V PHE

The -5, -4, and -2 pockets of most of the PKC isozymes are quite acidic, consistent with basic residues being selected at these positions. The major exception is PKCµ, which has fewer acidic residues in these pockets. For example, all the PKCs except PKCzeta and PKCµ have an Asp at position 203 of the p-5 pocket. The Ala rather than Asp at this position may explain why PKCµ is unique in its strong selection for peptides with Leu at p-5. Likewise, all the PKCs except PKCµ have an Asp at position 127 in the p-4 pocket and all except PKCµ select for peptides with a basic residue at p-4. PKCµ has a Met in the p-4 pocket and selects for substrates with Val at p-4. The p-2 pockets of PKCzeta and PKCµ are less acidic than those of the rest of the PKCs, and these two enzymes select for peptides with Gln rather than Lys at p-2. Several PKC isoforms selected substrates with either hydrophobic amino acids (Leu or Phe) or Arg at the p-5 position. The selection for hydrophobic amino acids may be explained by a hydrophobic residue in this pocket (e.g. a Met conserved in the PKC family members, Table V). The selection for Arg could be rationalized if the aliphatic part of the Arg side chain interacts with the hydrophobic Met in the pocket, while the guanidium group interacts with hydrophilic residues (Asp, Gln).

The differences in selectivity of the various PKCs at the p+2 and p+3 pockets can also be rationalized. All the PKCs except PKCµ have acidic residues in these pockets (Table V). This can explain why the cPKCs strongly select substrates with basic residues at p+2 and p+3 and why PKCµ fails to select for substrates with basic residues at these positions. PKCs delta , -epsilon , and -zeta weakly select for substrates with basic residues at p+2 and p+3 but prefer substrates with hydrophobic residues at these positions. This might be explained by subtle changes in the packing of residues in these regions such that the surfaces of the Phe residues in these pockets are more available for contact with substrate side chains (positions 54 and 198).

In summary, the oriented peptide library approach has provided information about substrate specificity of PKC isozymes that can explain selectivity for in vivo and in vitro substrates. In addition, the selectivity of individual PKC isozymes can be rationalized on the basis of analogies to the PKA/PKI crystal structure. Ultimately, these models will be testable by mutational studies and by co-crystals of PKC/peptide complexes.


FOOTNOTES

*   This work was supported by the NAITO Foundation (42-6, Hongo 3, Bunkyo, Tokyo 113, Japan), the American Cancer Society, the Lucille P. Markey Charitable Trust, and the Medical Foundation, Inc., Boston, MA. 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: Beth Israel Hospital, Division of Signal Transduction, Harvard Institute of Medicine, 10th Floor, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-0935; Fax: 617-667-0957; E-mail: knishika{at}mercury.bih.harvard.edu.
1    The abbreviations used are: PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; DAG, diacylglycerol; eEF-1alpha , elongation factor-1alpha ; Sf9 cells, Spondoptera frugiperda cells; PS, phosphatidylserine; PKA, protein kinase A; PKI, Walsh inhibitor pseudosubstrate; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid.

Acknowledgments

We thank Michael Berne (Tufts University) for protein sequencing. We thank Caryn Ivanof for the preparation of Sf9 insect cells.


REFERENCES

  1. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291, 329-343 [Medline] [Order article via Infotrieve]
  2. Nishizuka, Y. (1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  3. Selbie, L. A., Schmitz-Peiffer, C., Sheng, Y., and Biden, T. J. (1993) J. Biol. Chem. 268, 24296-24302 [Abstract/Free Full Text]
  4. Akimoto, K., Mizuno, K., Osada, S., Hirai, S., Tanuma, S., Suzuki, K., and Ohno, S. (1994) J. Biol. Chem. 269, 12677-12683 [Abstract/Free Full Text]
  5. Dekker, L. V., and Parker, P. J. (1994) Trends Biochem. Sci. 19, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  6. Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M., and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367 [Abstract/Free Full Text]
  7. Johannes, F.-J., Prestle, J., Eis, S., Oberhagemann, P., and Pfizenmaier, K. (1994) J. Biol. Chem. 269, 6140-6148 [Abstract/Free Full Text]
  8. Pearson, R. B., and Kemp, B. E. (1991) Methods Enzymol. 200, 62-81 [Medline] [Order article via Infotrieve]
  9. Kielbassa, K., Muller, H.-J., Meyer, H. E., Marks, F., and Gschwendt, M. (1995) J. Biol. Chem. 270, 6156-6162 [Abstract/Free Full Text]
  10. Municio, M. M., Lozano, J., Sanchez, P., Moscat, J., and Diaz-Meco, M. T. (1995) J. Biol. Chem. 270, 15884-15891 [Abstract/Free Full Text]
  11. Songyang, Z., Carraway III, K. L., Eck, M. J., Harrison, S. C., Feldman, R. A., Mohammadi, M., Schlessinger, J., Hubbard, S. R., Smith, D. P., Eng, C., Lorenzo, M. J., Ponder, B. A. J., Mayer, B. J., and Cantley, L. C. (1995) Nature 373, 536-539 [CrossRef][Medline] [Order article via Infotrieve]
  12. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., and Cantley, L. C. (1994) Curr. Biol. 4, 973-982 [Medline] [Order article via Infotrieve]
  13. Songyang, Z., Lu, K. P., Kwan, Y., Tsai, L., Filhol, O., Cochet, C., Soderling, T. R., Bartleson, C., Graves, D. J., DeMaggio, A. J., Hoekstra, M. F., Blenis, J., Hunter, T., and Cantley, L. C. (1996) Mol. Cell. Biol. 16, 6486-6493 [Abstract]
  14. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman, New York
  15. Dieterich, S., Herget, T., Link, G., Bottinger, H., Pfizenmaier, K., and Johannes, F. J. (1996) FEBS Lett. 381, 183-187 [CrossRef][Medline] [Order article via Infotrieve]
  16. Burns, D. J., Bloomenthal, J., Lee, M.-H., and Bell, R. M. (1990) J. Biol. Chem. 265, 12044-12051 [Abstract/Free Full Text]
  17. Ogita, K., Ono, Y., Kikkawa, U., and Nishizuka, Y. (1991) Methods Enzymol. 200, 228-234 [Medline] [Order article via Infotrieve]
  18. Fujise, A., Mizuno, K., Ueda, Y., Osada, S., Hirai, S., Takayanagi, A., Shimizu, N., Owada, M. K., Nakajima, H., and Ohno, S. (1994) J. Biol. Chem. 269, 31642-31648 [Abstract/Free Full Text]
  19. Herget, T., Oehrlein, S. A., Pappin, D. J., Rozengurt, E., and Parker, P. J. (1995) Eur. J. Biochem. 233, 448-457 [Abstract]
  20. Chen, S. J., Klann, E., Grower, M. C., Powell, C. M., Sessoms, J. S., and Sweatt, J. D. (1993) Biochemistry 32, 1032-1039 [Medline] [Order article via Infotrieve]
  21. Marais, R. M., Nguyen, O., Woodgett, J. R., and Parker, P. J. (1990) FEBS Lett. 277, 151-155 [CrossRef][Medline] [Order article via Infotrieve]
  22. Marais, R. M., and Parker, P. J. (1989) Eur. J. Biochem. 182, 129-137 [Abstract]
  23. Van Lint, J., Sinnett-Smith, J., and Rozengurt, E. (1995) J. Biol. Chem. 270, 1455-1461 [Abstract/Free Full Text]
  24. Bossemeyer, D., Engh, R. A., Kinzel, V., Ponstingl, H., and Huber, R. (1993) EMBO J. 12, 849-859 [Abstract]
  25. Zheng, J., Knighton, D. R., Ten, E. L. F., Karlsson, R., Xuong, N., Taylor, S. S., and Sowadski, J. M. (1993) Biochemistry 32, 2154-2161 [Medline] [Order article via Infotrieve]
  26. Moss, S. J., Doherty, C. A., and Huganir, R. L. (1992) J. Biol. Chem. 267, 14470-14476 [Abstract/Free Full Text]
  27. Flint, A. J., Gebbink, M. F., Franza, B. R., Jr., Hill, D. E., and Tonks, N. K. (1993) EMBO J. 12, 1937-1946 [Abstract]
  28. Noland, T. A., Jr., Raynor, R. L., and Kuo, J. F. (1989) J. Biol. Chem. 264, 20778-20785 [Abstract/Free Full Text]
  29. Ahn, J., Donner, D. B., and Rosen, O. M. (1993) J. Biol. Chem. 268, 7571-7576 [Abstract/Free Full Text]
  30. Chambers, T. C., Pohl, J., Glass, D. B., and Kuo, J. F. (1994) Biochem. J. 299, 309-315 [Medline] [Order article via Infotrieve]
  31. Blume-Jensen, P., Wernstedt, C., Heldin, C.-H., and Ronnstrand, L. (1995) J. Biol. Chem. 270, 14192-14200 [Abstract/Free Full Text]

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