(Received for publication, September 8, 1996, and in revised form, October 16, 1996)
From the 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
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 (,
I,
II,
,
,
,
, µ, and
) 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. PKC
, -
I, -
II,
-
, and -
selected peptides with basic amino acid at positions +2,
+3, and +4, but PKC
, -
, -
, and -µ preferred peptides with
hydrophobic amino acid at these positions. At position
5, the
selectivity was quite different among the various isozymes; PKC
,
-
, and -
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 PKC
, -
I, -
II, -
, -
, and -
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 PKC
, -
I, -
, -
, 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.
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 ,
I,
II, and
isoforms (activated by calcium, acidic
phospholipid, and diacylglycerol (DAG)), novel PKCs (nPKC) comprising
,
,
, and
(activated by DAG and acidic phospholipid but
insensitive to calcium), and atypical PKCs (aPKC)
/
and
(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-1 was shown to be phosphorylated with much greater
efficiency by nPKC
than by cPKCs, nPKC
or -
, or aPKC
(9).
Heterogeneous ribonucleoprotein A1 is efficiently phosphorylated by
PKC
but not by cPKCs or PKC
(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 PKC, -
I, -
II, -
, -
, -
,
-
, -
, 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 PKC
, -
I, -
, -
,
and -µ and showed that these peptides are high affinity and selective
substrates for the respective PKC isozymes.
DAG was purchased from Boehringer Mannheim.
PS was purchased from Avanti Polar Lipids. Isozyme-selective
antipeptide antibodies for PKC, -
I, -
II, -
, -
, -
,
-
, and -
were purchased from Santa Cruz Biotechnology, Inc.
PKC
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. [
-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).
The
full-length cDNAs for human PKC, -
I, -
II, -
, -
,
-
, -
, and -
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 PKC
, -
I, -
II, -
,
-
, and -
were performed by several steps of column chromatography
as described previously (16, 17). PKC
, -
, and -µ were isolated
by immunoprecipitation using isozyme-selective antipeptide antibodies
(for PKC
and -
) and anti-c-myc antibody (for PKCµ),
respectively. 1 unit of PKC
, -
I, -
II, -
, -
, -
, -
,
or -
is defined as the amount of kinase required to incorporate 1 nmol of phosphate into the
-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.
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 [-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
PKC
, -
I, -
II, and -
), and 0.5 mM EGTA (for
PKC
, -
, -
, -
, 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 [
-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.
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 AssayPKC 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 [-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 PKC
, -
I, -
II,
and -
), 0.5 mM EGTA (for PKC
, -
, -
, -
, 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.
In order to determine optimal substrate sequences for
each of nine human PKC isozymes (,
I,
II,
,
,
,
,
µ, and
), 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 PKC and -µ. These two enzymes clearly selected for distinct peptide substrates. PKC
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. PKC
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 PKC
selected Phe while PKCµ selected Val. At p+2,
p+3, and p+4 positions, PKC
strongly selected for peptides with the
basic amino acids Arg or Lys. In contrast, PKCµ preferred peptides
with hydrophobic amino acids in both positions.
Comparison of the substrate specificities of
PKC and PKCµ. Human PKC
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 PKC
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 [
-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 PKC
), 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.
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 p6, 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. PKC
, -
I, -
II, -
, and -
selected for substrates with basic amino acid at positions p+2, p+3,
and p+4, but PKC
, -
, -
, and -µ preferred substrates with
hydrophobic amino acid in these regions. At p
5, the selectivity was
quite different among these isozymes. PKC
, -
, and -
selected
peptides with Arg; in contrast PKC
II, -
, and -µ selected for
peptides with hydrophobic amino acids such as Phe, Leu, or Val. PKC
I
and -
preferred substrates with either Leu or Arg.
|
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 p3 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 PKC
. The predicted optimal sequences from p
3 to p+2
for PKC
, -
I, -
II, -
, -
, and -
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.
|
The optimal substrates predicted for the various PKC isozymes are in
good agreement with known substrates. In Table III, the optimal substrate sequence of PKC 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
PKC
, -
, and -
). Most of these proteins have the motif
R/K-X-R/K-R/K-X-S/T-
-R/K-R/K, where
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 PKC
, -
I, and -
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-1
is
reported to be a specific substrate for PKC
(9). The sequence of
426-436 from murine eEF-1
containing Thr-431 was compared with the
predicted optimal substrate of PKC
(Table III). PKC
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-1
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.
|
The predicted optimal peptide substrates for PKC,
PKC
I, PKC
, PKC
, 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 PKC
was
used to assay the various PKC isozymes. A set of experiments using 100 µM of the various peptides as substrates of PKC
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 PKC
, PKC
I,
PKC
, and PKCµ (as judged by
Vmax/Km ratios) were the
-peptide,
I-peptide,
-peptide, and µ-peptide respectively, in agreement with the library predictions. Curiously, the
-peptide was not the optimal peptide for PKC
as judged by
Vmax/Km ratio, although it
was the lowest Km substrate for PKC
. In fact, the
predicted optimal peptide substrate for each PKC was the lowest
Km substrate for that PKC, except in the case of
PKC
where the
I and
-peptides had slightly lower
Km values (2.7 µM and 2.8 µM, respectively) than the
-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).
|
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 -peptide is a good substrate for all the
PKC isozymes investigated except PKCµ. The
I-peptide is a
relatively good substrate for all the PKC isozymes, including PKCµ.
In contrast, the
-peptide is relatively specific for PKC
, 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
I-peptide allows it to be
phosphorylated by PKCµ (only the
I-peptide and µ-peptide have
Leu at p
5). Although Leu is not the optimal residue at the p
5
position for PKC
, PKC
, and PKC
, 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).
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 PKC, PKC
I, PKC
, 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 (
,
I,
II, and
)
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 (
,
, and
) and the aPKC
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,
and
(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 PKC,
I, -
, and -
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 PKC
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
-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 PKC 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 PKC
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
p5 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
I-peptide is optimal for PKC
I but is a useful general substrate
for all PKC isoforms (including PKCµ). The
-peptide (like the
-pseudosubstrate peptide) is a general substrate for all PKCs except
PKCµ. The µ-peptide is extremely specific for PKCµ, and the
-peptide is relatively specific for PKC
. The
-peptide is
phosphorylated by PKC
, PKC
, 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 p5 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 p5 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.
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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 PKC
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 PKC
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 , -
, and -
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.
We thank Michael Berne (Tufts University) for protein sequencing. We thank Caryn Ivanof for the preparation of Sf9 insect cells.