(Received for publication, December 13, 1994; and in revised form, January 27, 1995)
From the
Protein kinase C (PKC) is a phospholipid-dependent isozyme family that plays a pivotal role in mammalian signal-transduction pathways that mediate cell growth and differentiation and pathological developments, such as the acquisition of drug resistance by cancer cells. Several peptide-substrate analogs have been shown to reversibly inhibit PKC with high potency and selectivity, but peptide-substrate analogs that antagonize PKC by forming a covalent complex with the enzyme have not been reported. The development of active site-directed irreversible inactivators of PKC could provide new insights into the catalytic mechanism and might ultimately lead to the design of novel therapeutics targeted at PKC.
In this report, we show that the peptide-substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates PKC in a dithiothreitol-sensitive manner. The inactivation mechanism most consistent with our results is the formation of a covalent linkage between the inhibitor-peptide and the enzyme at its active-site. Limited proteolysis of PKC produces a catalytic-domain fragment that is independent of the phospholipid cofactor. RKRCLRRL antagonized the histone kinase activity of PKC and its catalytic-domain fragment with similar efficacies, achieving >50% inactivation at an RKRCLRRL concentration of 10 µM. In contrast, RKRCLRRL analogs with single amino acid substitutions at Cys were non-inhibitory. The inactivated complex of the catalytic-domain fragment and RKRCLRRL was stable upon dilution, and the inactivation of PKC and the catalytic-domain fragment by RKRCLRRL was quenched by dithiothreitol, providing evidence that the enzyme and the synthetic peptide may be covalently linked in an inactivated complex by a disulfide bond. Substrates and substrate analogs protected the catalytic-domain fragment against inactivation by RKRCLRRL, providing evidence that inactivation entailed binding of RKRCLRRL at the active-site of the enzyme.
S-Thiolation is the formation of mixed disulfides between proteins and low molecular weight thiols. PKC is thought to have a highly reactive Cys residue in its active-site, and Cys residues that are flanked by basic residues, as is the case in RKRCLRRL, display enhanced reactivity. Our results support an inactivation mechanism that entails S-thiolation of the active-site of PKC by RKRCLRRL. This is the first report of irreversible inactivation of PKC by an active site-directed peptide.
Protein kinase C (PKC) ()is a phospholipid-dependent
isozyme family that plays key roles in signaling mechanisms involved in
normal physiological processes such as neurotransmission, muscle
contraction, and cell growth and differentiation, and also pathological
developments such as tumor promotion and the acquisition of drug
resistance by cancer cells in response to
chemotherapy(1, 2, 3, 4) . A number
of peptide-substrate analogs have been reported to inhibit PKC
reversibly, and in some cases with remarkable potency and
selectivity(5, 6, 7, 8, 9) .
Irreversible inactivation of the cAMP-dependent protein kinase has been
achieved with peptide-substrate analogs, some of which have been shown
to specifically label a highly reactive active-site Cys residue that is
conserved in PKC(10, 11, 12, 13) .
However, peptide-substrate analogs that inhibit PKC by forming a
covalent complex with the enzyme are still lacking. The development of
active site-directed irreversible inactivators of PKC could aid in the
identification of active-site residues and in the elucidation of the
catalytic mechanism of PKC (12) and could eventually lead to
the design of novel therapeutics, e.g. cancer therapeutics for
the reversal of drug resistance (14) .
We previously
reported that N-myristoylation of the PKC peptide-substrate
Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu (RKRTLRRL) and several
nonphosphorylatable analogs of the peptide transformed peptides that
lacked inhibitory activity against PKC-catalyzed histone
phosphorylation into potent inhibitors of the
reaction(9, 15) . The N-myristoylated
peptides inhibited PKC and a phospholipid-independent catalytic-domain
fragment of the enzyme by a reversible mechanism that involved binding
of the inhibitor-peptide at the active-site, according to
structure-activity studies and kinetic analysis(9) . Inhibition
of PKC also entailed interactions between the N-myristoylated
peptides and the phospholipid cofactor of the enzyme(9) . The
Cys-containing analog N-myristoyl-RKRCLRRL (NmRKRCLRRL) was by
far the most potent of the peptides examined in the inhibition of the
catalytic-domain fragment, but its inhibitory potency was reduced by an
order of magnitude in the presence of the sulfhydryl-reducing agent
dithiothreitol (DTT) (0.5 mM), providing evidence that its
inhibitory mechanism may include an irreversible component(9) .
NmRKRCLRRL appeared to be an active site-directed inhibitor, because of
its structural relatedness to the peptide substrate NmRKRTLRRL, and
because its K against the
catalytic-domain fragment in the presence of DTT, i.e. under
conditions of reversible inhibition (K
= 50 ± 2 µM) agreed within a
factor of two with the K
of NmRKRTLRRL (K
= 29 ± 2 µM)(9) .
The Cys residue present in the active-site of cAMP-dependent protein kinase and conserved in PKC isozymes (13) is a likely target for active site-directed irreversible inactivators of PKC. Cys residues surrounded by multiple basic residues have been shown to be highly reactive in studies of disulfide exchange reactions(16) . We therefore hypothesized that the DTT-sensitive inhibitory activity of NmRKRCLRRL against the catalytic-domain fragment of PKC could be due to the reactivity of the Cys residue in the sequence RKRCLRRL at the active site of PKC. In this report, we test the hypothesis by analyzing the effects of the peptide RKRCLRRL on PKC catalysis. We show that RKRCLRRL is a DTT-sensitive, active site-directed irreversible inactivator of PKC and its catalytic-domain fragment. This is the first report of irreversible inactivation of a protein kinase by a peptide-substrate analog composed entirely of naturally occurring, unmodified amino acids (12) , and RKRCLRRL is the first reported active site-directed irreversible inactivator of PKC.
[-
P]ATP was purchased from Amersham
Corp., frozen Sprague-Dawley rat brains were from Pel-Freez (Rogers,
AR), phosphocellulose paper was from Fisher Scientific (Houston, TX),
and hydroxylapatite resin was from Bio-Rad. Casein kinase II was from
Upstate Biotech, Inc. (Lake Placid, NY). DEAE-Sepharose, G-25 Sephadex,
ATP, PS, DTT, phenylmethylsulfonyl fluoride, leupeptin, histone III-S,
bovine pancreas L-1-tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin, Ellman's reagent, and all other reagents
were purchased from Sigma.
The histone kinase
reaction mixture (120 µl) contained 20 mM Tris-HCl, pH
7.5, 10 mM MgCl, 0.2 mM CaCl
,
30 µg/ml PS, 6 µM [
-
P]ATP
(5,000-8,000 cpm/pmol), 0.67 mg/ml histone III-S, purified PKC (5
ng), and where indicated, the peptide-substrate analog under
investigation. In assays of the catalytic-domain fragment, PS was
omitted, and CaCl
was replaced by 1 mM EGTA(9, 17) . Reactions were initiated by the
addition of [
-
P]ATP, proceeded for 5 to 10
min at 30 °C with linear kinetics, and terminated on
phosphocellulose paper(9, 17) .
P-Labeled
histone was quantitated as described previously (9, 17) . All assays were performed in triplicate and
expressed as the mean value ± S.D.
The peptide-substrate analog RKRCLRRL potently inhibited
Ca- and PS-stimulated PKC-catalyzed histone
phosphorylation (Fig. 1, closed circles), under
conditions where the closely related peptides RKRALRRL (Fig. 1, triangles) and RKRTLRRL (15) lacked inhibitory
activity. The inhibitory activity of RKRCLRRL was quenched by the
sulfhydryl-reducing agent DTT (0.5 mM) (Fig. 1, open circles). Limited trypsinolysis of PKC produces a
regulatory-domain fragment and an active catalytic-domain fragment,
which is independent of the cofactors Ca
and
PS(24) . When the catalytic-domain fragment was purified from
the regulatory-domain fragment and intact PKC by DEAE ion-exchange
chromatography, potent inhibition of its Ca
- and
PS-independent histone kinase activity was achieved by RKRCLRRL (Fig. 2, closed circles), and the inhibitory activity
of RKRCLRRL was abolished by the inclusion of 0.5 mM DTT in
the reaction mixtures (Fig. 2, open circles). RKRCLRRL
was equally effective against a catalytic-domain fragment preparation
that contained the regulatory-domain fragment and residual intact PKC
(data not shown). Neither RKRTLRRL (15) nor RKRALRRL
(1-40 µM) antagonized the histone kinase activity of
the catalytic-domain fragment. The DTT-sensitive nature of the
inhibitory activity of RKRCLRRL against PKC and its catalytic-domain
fragment and the lack of inhibitory activity observed with RKRCLRRL
analogs containing single amino acid substitutions at Cys provide
evidence that the inhibitory mechanism may involve the formation of a
DTT-sensitive covalent enzyme-inhibitor complex. Comparison of the
inhibitory curves generated by RKRCLRRL in Fig. 1and Fig. 2reveals the similarity of the inhibitory potencies of
RKRCLRRL against PKC and its catalytic-domain fragment. This supports
an inhibitory mechanism that involves interactions between RKRCLRRL and
the catalytic domain of PKC.
Figure 1:
DTT-sensitive antagonism of the histone
kinase activity of PKC by RKRCLRRL. The Ca- and
PS-stimulated histone kinase activity of PKC was assayed as described
under ``Experimental Procedures'' in the presence of RKRCLRRL
(
), RKRALRRL (
), and RKRCLRRL plus 0.5 mM DTT
(
). 100% activity was 858 ± 34 nmol of
P
transferred per min/mg.
Figure 2:
DTT-sensitive antagonism of the histone
kinase activity of the catalytic-domain fragment of PKC by RKRCLRRL.
The Ca- and PS-independent histone kinase activity of
the catalytic-domain fragment of PKC was assayed as described under
``Experimental Procedures'' in the presence of RKRCLRRL
(
) and RKRCLRRL plus 0.5 mM DTT (
). The 100%
activity value was 678 ± 34 nmol of
P transferred
per min/mg.
To directly test whether RKRCLRRL forms an irreversible complex with PKC, we determined the effect of dilution of preincubation mixtures containing the catalytic-domain fragment of PKC and RKRCLRRL on the inhibitory potency of RKRCLRRL in histone kinase assays. In these experiments, we preincubated the catalytic-domain fragment of PKC with inhibitory concentrations of RKRCLRRL, and then the preincubation mixture was diluted into histone kinase assay mixtures so that the final RKRCLRRL concentration would be weakly inhibitory or non-inhibitory. The ability of RKRCLRRL to inhibit the catalytic-domain fragment in the histone kinase assay following preincubation with the catalytic-domain fragment was compared with its inhibitory potency in the absence of preincubation. Table 1shows that following preincubation with the catalytic-domain fragment, RKRCLRRL fully retained its inhibitory activity in the histone kinase assay, even when the final concentration of RKRCLRRL in the assay mixture was non-inhibitory. For example, when the catalytic-domain fragment was preincubated for 10 min at 30 °C with 6 µM RKRCLRRL and then added to histone kinase assay mixtures at a final RKRCLRRL concentration of 0.2 µM, RKRCLRRL inhibited the histone kinase reaction by 83 ± 7%, but 0.2 µM RKRCLRRL was non-inhibitory when it was added directly to the histone kinase assay mixtures (Table 1). Following preincubation of the catalytic-domain fragment with RKRCLRRL, a similar degree of inhibition was observed in the histone kinase assay across a broad range of final RKRCLRRL concentrations (0.2-2.0 µM) (Table 1). These data show that RKRCLRRL inactivates PKC by forming an irreversible complex with the catalytic domain of the enzyme.
The structural similarity between RKRCLRRL and the PKC
substrate RKRTLRRL (K = 20
µM) (15) strongly suggests that the PKC
inactivator RKRCLRRL is active site-directed. To test this, we examined
the ability of PKC substrates to protect the catalytic-domain fragment
of PKC against inactivation by RKRCLRRL(12) . The
catalytic-domain fragment was preincubated with 6 µM RKRCLRRL as described in the legend to Table 1, except that
various PKC substrates and substrate analogs were included in the
preincubation mixture. These experiments were designed so that
following dilution of the preincubation mixture into histone kinase
assay mixtures, the substrate Mg-[
-
P]ATP
was present in the reaction mixtures at its standard concentration (6
µM [
-
P]ATP), and the
peptide-substrate analogs, FARKGALRQ and RKRALRRL, were present at a
concentration (10 µM) where they had no inhibitory
activity against the histone kinase reaction(9) . Table 2shows that Mg-[
-
P]ATP,
Mg-[
-
P]ATP plus FARKGALRQ, and
Mg-[
-
P]ATP plus RKRALRRL each afforded
protection against inactivation of the catalytic-domain fragment of PKC
by RKRCLRRL. The protection of the catalytic-domain fragment of PKC
against RKRCLRRL that was afforded by
Mg-[
-
P]ATP alone and in the presence of
defined peptide-substrate analogs provides evidence that RKRCLRRL is an
active site-directed inactivator of PKC. As another test of whether the
peptide operates as an active site-directed PKC inactivator, we
examined the ability of the peptide to inactivate casein kinase II.
Casein kinase II differs from PKC in that its substrate recognition
motif does not include basic residues but instead requires acidic
residues. In assays of the phosphorylation of casein by casein kinase
II performed under conditions described in (25) , we found that
RKRCLRRL did not inhibit casein kinase II activity at peptide
concentrations up to and including 100 µM.
To identify
structural features of RKRCLRRL that play important or essential roles
in its DTT-sensitive inactivation of PKC, we examined the abilities of
RKRCLRRL analogs to inactivate PKC and its catalytic-domain fragment in
a DTT-sensitive manner. Among the peptides studied, RKRCLRRL was the
most potent inactivator of PKC and its catalytic-domain fragment (Table 3). A truncated analog lacking the C-terminal Leu residue
(RKRCLRR) was also a potent inactivator of PKC and its catalytic-domain
fragment (Table 3). However, analogs lacking a single basic
residue (KRCLRRL and RKRCLR) either failed to inactivate PKC and its
catalytic-domain fragment or did so only very weakly, and KRCLR, which
lacked two of the basic residues of the parent peptide, was inactive (Table 3). These results indicate the critical importance of the
basic residues of RKRCLRRL in its DTT-sensitive inactivation of PKC.
The failure of KRCLR to inactivate PKC and its catalytic-domain
fragment could be due to weak reactivity of the Cys residue as a
consequence of the limited number of basic residues in the peptide
compared to RKRCLRRL (16) and/or to a weak affinity for the
active-site of the enzyme compared to RKRCLRRL, since the closely
related peptides RKRTLRRL and KRTLR serve as PKC substrates with K values of 20 and 300
µM, respectively(15, 26) . The affinity
of the N-myristoylated analog NmKRCLR at the active-site of
PKC can be expected to be superior to that of KRCLR, because the
related peptide NmKRTLR reversibly inhibits the histone kinase activity
of the catalytic-domain fragment of PKC (IC
= 200
µM), whereas KRTLR does not(27) . Therefore, we
analyzed the effects of NmKRCLR on the histone kinase activity of the
catalytic-domain fragment of PKC, in order to determine whether the
sequence KRCLR was sufficiently basic to provide the Cys residue with
DTT-sensitive reactivity at the active-site of the enzyme. NmKRCLR
effected 50% antagonism of the histone kinase activity of the
catalytic-domain fragment at a concentration of 8 ± 2 µM in the absence of DTT and at a concentration of 27 ± 3
µM in the presence of 0.5 mM DTT. At 20
µM NmKRCLR, the loss of catalytic-domain fragment activity
was 95 ± 9% in the absence of DTT and 33 ± 2% in its
presence. These results provide evidence that NmKRCLR antagonizes the
catalytic-domain fragment of PKC by a mixed mechanism that includes
reversible and irreversible components. Thus, three flanking basic
residues appear sufficient to endow the Cys residue of the
peptide-substrate analog with the ability to undergo a reaction with
the catalytic domain of PKC that irreversibly inactivates the enzyme in
a DTT-sensitive manner. Table 3shows that substitution of Cys
with its D-isomer in RKRCLRRL weakened the peptide in the
inactivation of PKC and its catalytic-domain fragment, indicating a
degree of stereoselectivity in the inactivation of the enzyme by the
peptide.
The studies described above were carried out using a
purified preparation of a PKC isozyme mixture that contained PKC-,
PKC-
, and PKC-
. These isozymes have closely related peptide
and protein substrate specificities(28, 29) . Using
preparations of the purified isozymes, we determined that RKRCLRRL
effected DTT-sensitive inactivation of each isozyme with comparable
efficacy. 50% inactivation of PKC-
, PKC-
, and PKC-
was
achieved at RKRCLRRL concentrations of 11 ± 2, 6 ± 2, and
4 ± 1 µM, respectively, and in each case,
inactivation was quenched by 0.5 mM DTT.
In this report, we present data in support of a mechanism of
PKC inactivation by the peptide-substrate analog RKRCLRRL that most
likely involves the formation of a covalent complex between the active
site of the enzyme and the inhibitor peptide. Our evidence for this is
as follows. 1) RKRCLRRL antagonized the histone kinase activity of PKC
and its catalytic-domain fragment with similar efficacies, and in both
cases activity was fully restored by 0.5 mM DTT. In addition,
analogs of RKRCLRRL with single amino acid substitutions at Cys
(RKRALRRL and RKRTLRRL) were non-inhibitory against the histone kinase
activity of PKC and the catalytic-domain fragment. The unique ability
of RKRCLRRL compared with other RKRXLRRL peptides to inactivate PKC
provides evidence that the high reactivity of the Cys residue in the
basic peptide environment compensates for the weak binding affinity of
the peptide at the active site in the inactivation mechanism. 2) The
inactivated complex of catalytic-domain fragment and RKRCLRRL was
stable upon dilution. 3) Substrates and substrate analogs protected the
catalytic-domain fragment of PKC against inactivation by RKRCLRRL. 4)
The isozymes PKC-, PKC-
, and PKC-
each have a Cys
residue that is homologous to a highly reactive active-site Cys residue
in the cAMP-dependent protein kinase(13) . RKRCLRRL inactivated
PKC-
, PKC-
, and PKC-
in a DTT-sensitive manner with
similar efficacies. This is the first report of irreversible
inactivation of PKC by an active site-directed peptide.
S-Thiolation is the formation of mixed disulfides between
proteins and low molecular weight thiols; this reaction has been
observed between endogenous low molecular weight thiols and several
cellular proteins, including glyceraldehyde-3-phosphate dehydrogenase
and creatine kinase(30, 31) . PKC appears to be a good
candidate for S-thiolation, because a highly reactive
active-site Cys residue present in cAMP-dependent protein kinase is
conserved in PKC isozymes(13) . In addition, the
peptide-substrate analog RKRCLRRL appears to be a good candidate for an S-thiolation reaction at the active-site of PKC, because the
reactivity of Cys residues has been shown to be increased by as much as
10-fold by flanking basic residues, in studies of
disulfide-exchange reactions (16) . DTT reversal of the
inactivation of PKC by RKRCLRRL provides evidence that the enzyme and
synthetic peptide are linked in the inactivated complex by a disulfide
bond. This supports an inactivation mechanism that entails S-thiolation of the active-site of PKC by the peptide. In view
of these results, the possibility that cellular proteins subject to
regulation by S-thiolation in vivo(30, 31) may include PKC and perhaps other
protein kinases deserves attention.
Attempts to analyze the kinetics
of PKC inactivation by RKRCLRRL revealed an extremely rapid reaction
that was complete within several seconds, even at 4 °C. ()Thus, successful analysis of the kinetics of PKC
inactivation by RKRCLRRL will require the development of an applicable
continuous assay of PKC activity, e.g. an assay system based
on spectral or fluorescent changes associated with product formation.
However, even in the absence of kinetic analysis, it is clear that
RKRCLRRL, which achieves >50% inactivation of PKC and its
catalytic-domain fragment at a concentration of 10 µM ( Fig. 1and Fig. 2), is a highly potent active
site-directed peptidic inactivator of PKC. In addition, the K
of the closely related peptide
RKRTLRRL (K
= 20
µM) (15) provides an estimate of the affinity of
RKRCLRRL for the active-site of PKC. Recently, peptide-substrate
analogs of cAMP-dependent protein kinase that contain an intrachain
disulfide bond, e.g. Leu-Arg-Arg-Ala-Cys = Cys-Gly,
were shown to affinity label the enzyme by undergoing a
disulfide-exchange reaction at its active site(12) . This
report shows that the presence of a single cysteine residue at the
phospho-acceptor position of a synthetic peptide-substrate analog
containing multiple basic residues may result in novel active-site
affinity labels of PKC and perhaps other protein kinases that
phosphorylate highly basic sequences.