(Received for publication, August 12, 1994; and in revised form, January 11, 1995)
From the
Using site-directed mutagenesis, we proposed that an
autoinhibitory domain(s) is located at the C-terminal region
(301-386) of the phosphorylase kinase -subunit (Huang, C.-Y.
F., Yuan, C.-J., Livanova, N. B., and Graves, D. J. (1993) Mol.
Cell. Biochem. 127/128, 7-18). Removal of the putative
inhibitory domain(s) by truncation results in the generation of a
constitutively active and calmodulin-independent form,
. To probe the structural basis of
autoinhibition of
-subunit activity, two synthetic peptides, PhK13
(
) and PhK5
(
), corresponding to the two
calmodulin-binding regions, were assayed for their ability to inhibit
. Competitive inhibition of
by PhK13 was found versus phosphorylase b (K
=
1.8 µM) and noncompetitive inhibition versus ATP.
PhK5 showed noncompetitive inhibition with respect to both
phosphorylase b and ATP. Calmodulin released the inhibition
caused by both peptides. These results indicate that there are two
distinct autoinhibitory domains within the C terminus of the
-subunit and that these two domains overlap with the
calmodulin-binding regions. Two mutant forms of
, E111K and E154R, were used to probe the
enzyme-substrate-binding region using peptide substrate analogs
corresponding to residues 9-18 of phosphorylase b (KRK
Q
ISVRGL). The data suggest that
Glu
interacts with the P-3 position of the substrate
(Lys
) and Glu
interacts with the P-2 site
(Gln
). Both E111K and E154R were competitively inhibited
with respect to phosphorylase b by PhK13, with 14- and 8-fold
higher K
values, respectively, than that
observed with the wild-type enzyme. These data are consistent with a
model for the regulation of the
-subunit of phosphorylase kinase
in which PhK13 acts as a competitive pseudosubstrate that directly
binds the substrate binding site of the
-subunit (Glu
and Glu
).
Phosphorylase kinase catalyzes the phosphorylation and
activation of glycogen phosphorylase b (for review see (1) and (2) ). Phosphorylase kinase has a subunit
composition in white skeletal muscle of
(,
,
,
)
. The
- and
-subunits
are regulatory subunits. The
-subunit is almost identical to
bovine brain calmodulin and confers Ca
sensitivity
upon the enzyme. The
-subunit is catalytically active, and its
N-terminal region (residues 1-276) shares sequence homology with
the catalytic domains of other protein kinases(3) . The
C-terminal region of
(residues 287-386) is thought to
encompass two distinct, non-contiguous high affinity calmodulin-binding
subdomains(4) . This speculation is supported by the results
that exogenous calmodulin can stimulate the
-subunit isolated from
rabbit skeletal muscle(5, 6) as well as the
recombinant full-length
forms(7, 8, 9, 10, 11, 12) ,
but not the truncated
forms(11, 13, 14) .
Several
calmodulin-dependent protein kinases, e.g. myosin light chain
kinase and Ca/calmodulin-dependent protein kinase II,
have been shown to contain an autoinhibitory domain or a
pseudosubstrate sequence in their primary structures, which partly
overlaps with their respective calmodulin-binding regions (reviewed in (15) and (16) ). In both instances, calmodulin plays an
important role in releasing the inhibition imposed by the inhibitory
domain. Because the autoinhibitory domain in calmodulin-dependent
protein kinases usually overlaps with their calmodulin-binding domain,
two calmodulin-binding peptides, PhK13 (
)
and PhK5 (
), from the C terminus of the
-subunit are potential models for studying autoinhibitory
mechanisms in the
-subunit. We have employed a simple system, the
truncated constitutively active
,
, and
the two synthetic peptides, PhK13 and PhK5, to investigate the
possibility that those two regions of the
-subunit regulatory
domain might act as autoinhibitory domains. Two mutants of
, E111K and E154R, which have been reported
previously to have altered substrate binding affinity(18) ,
were also used in this study to define substrate/pseudosubstrate
binding sites in the catalytic domain of the
-subunit.
Figure 1:
Comparison of the inhibition of
by PhK13 and PhK5. The final concentrations
in the assay were 50 mM Tris, pH 8.2, 50 mM HEPES, 10
mM MgCl
, 4 µM phosphorylase b, 1 mM dithiothreitol, 1 mM [
-
P]-ATP, 1 µg/ml
, and various concentrations of PhK13 (panel A) or PhK5 (panelB). The reaction
was carried out at 30 °C for 5 min.
Figure 2:
Inhibition of
activity by PhK13 or PhK5. The inhibition assay was performed as
described under ``Experimental Procedures.'' The final
concentrations of PhK13 or PhK5 were 10 and 250 µM,
respectively, and those of the other components were the same as
described in Fig. 1. In the calmodulin (or BSA) activation
assay, equimolar amounts (10 or 250 µM) of calmodulin (or
BSA) relative to PhK13 or PhK5 were used in the reaction mixture. The
assay conditions were described under ``Experimental
Procedures.''
In the kinetic analyses, the
concentration ranges of substrate (ATP and phosphorylase b)
were varied from 1/2 K to 5 times K
. With wild-type
,
studies were done using (a) varying concentrations of
phosphorylase b (10-90 µM) and inhibitor
peptides (0-7.2 µM for PhK13 and 0-51
µM for PhK5) at a fixed concentration of ATP (400
µM), and (b) varying concentrations of ATP
(40-400 µM) and inhibitor peptides (0-54
µM for PhK13 and 0-51 µM for PhK5) at a
fixed concentration of phosphorylase b (90 µM).
The K
values for phosphorylase b using
the E111K and E154R mutants of
were both
around 100 µM, but due to insolubility it was impossible
to obtain a final concentration of 500 µM phosphorylase b (5 times K
). Therefore, a concentration
of 225 µM phosphorylase b was used as the
greatest concentration in the E111K and E154R mutant assay. In the case
of the kinetic analyses using Phos(9-18) peptide and
Phos(9-18) peptide analogs (Table 2) as substrates, the
concentration ranges of these peptides were varied from 0.5 to 15
mM.
Kinetic data were analyzed with the program, Enzfitter
(Elsevier Science Publishers). The initial velocity data (Table 2) were fitted to the equation for a two-substrate random
Bi Bi mechanism. The inhibition data (Table 1) with PhK13 and
PhK5 were fitted to the equations for competitive or noncompetitive
inhibition with the program Enzfitter. The inhibition constants, K for competitive inhibition and K
and K
for noncompetitive inhibition, were
calculated from the linear secondary plots as illustrated in Fig. 3(A and B). All the kinetic analyses were
repeated at least three times.
Figure 3:
Initial velocity patterns for inhibition
of by PhK13 (A) and PhK5 (B). Activity was assayed as described under
``Experimental Procedures.'' The final concentrations of
peptides in the assay were PhK13 (0, 2.7, 4.5, and 7.2 µM)
and PhK5 (0, 17, 34, and 51 µM). K
and K
were estimated from the
linear secondary plots (insets in A and B).
To determine
if either Glu or Glu
in
participates in the binding of Lys
at the P-3 site in Phos(9-18), wild-type
and both E111K and E154R mutants were
subjected to kinetic analyses by using Phos(9-18) and E11
peptides as substrates. If either residue Glu
or
Glu
in
interacts with the
residue at the P-3 site of 9-18 peptide, it would be expected
that charge-reversal mutations at these positions would negatively
affect substrate kinetics and that charge reversal at the P-3 site in
the peptide E11 (Lys
Glu) would reverse or
attenuate these negative effects. The kinetic parameters and relative
catalytic efficiencies calculated from these experiments are presented
in Table 2. Using wild-type
, a
10-fold greater K
value was observed with E11 as
substrate than Phos(9-18). There were no significant differences
in the K
or V
values of the
E154R mutant using either E11 or Phos(9-18) peptide. These data
suggest that Glu
in
does not
interact with the P-3 residue in the substrate. Our results agree with
the results of the co-crystal structure of cyclic AMP-dependent protein
kinase and PKI, which shows that Glu
(or Glu
in
) does not contact the P-3 site of PKI.
A dramatic
increase in the relative efficiency of the E111K mutant toward E11
(33-fold) was found. The results indicate that the charge reversal in
(Glu
Lys) was
compensated by the peptide substrate charge reversal (Lys
Glu). We interpret these data to mean that Glu
in
interacts with Lys
at
the P-3 site of the Phos(9-18) peptide substrate.
To learn
whether Glu interacts with the P-2 site in the substrate
as it does with cyclic AMP-dependent protein kinase, the kinetics of
phosphorylation of Phos(9-18) and E12 peptides were analyzed.
Charge reversal at residue 154 in
(Glu
Arg) and substitution at the P-2
residue in the substrate peptide (Gln
Glu) might be
predicted to improve substrate kinetics if there are interactions
between residue 154 and the P-2 site in the substrate. The kinetic data
confirmed this prediction (Table 2). As shown previously, in
wild-type
, a 3-fold decrease in relative
enzymatic efficiency was seen using E12 as a substrate compared with
Phos(9-18). There was no significant difference in relative
enzymatic efficiency with E111K toward Phos(9-18) (0.0013) or E12
(0.0017). A greater relative enzymatic efficiency was seen with E154R
toward E12 peptide (0.32) than Phos(9-18) peptide (0.11). The
results support the view that Glu
interacts with the P-2
site in the Phos(9-18) peptide.
By using various combinations of peptide substrate analogs
and mutants of the -subunit (
), we have
identified amino acids in the catalytic domain of the
-subunit of
phosphorylase kinase which recognize the substrate specificity
determinants P-2 and P-3 of the phosphate acceptor substrate. The
results indicate that the residues at P-3 and P-2 of the substrate
(Lys
and Gln
, respectively) may directly
interact with residues Glu
and Glu
in the
active site of
. This result is consistent with the results of
cyclic AMP-dependent protein kinase and myosin light chain kinase (30, 31, 32, 33) that two conserved
glutamyl residues in the active site region play an important role in
substrate recognition. However, Glu
in cyclic
AMP-dependent protein kinase, which also interacts with the P-2 Arg of
Kemptide, is replaced by Thr
in the
-subunit (Table 3, part B). If the same alignment exists in these two
protein kinases at the P-2 site, H-bonding might occur between residues
in the
-subunit, i.e. Glu
and
Thr
, and Gln
at the P-2 site. Unlike
Kemptide in the cyclic AMP-dependent protein kinase system, Arg
at the P+2 site of Phos(9-18) was shown early on to be
an important substrate specificity determinant (26, 27) for
. The basic residues on both sides
of Ser
in phosphorylase seem to be required for substrate
recognition for phosphorylase kinase, but the basic residue at P+2
is inhibitory for cyclic AMP-dependent protein kinase. This may partly
explain why cyclic AMP-dependent protein kinase and phosphorylase
kinase have different substrate specificity, although they share
similar substrate recognition residues, Glu
and
Glu
.
The objective of the present study was to gain
insight into the structure-function relationships of phosphorylase
kinase and its regulation. Previous studies suggested that two
autoinhibitory domains may exist at the C terminus of ,
(11) . One is located before
residue 331 and another after residue 331. Two
-subunit
calmodulin-binding peptides, PhK13 (
) and
PhK5(
), were tested and found to be
inhibitory to
. Based on the present
findings, we found that the region
(PhK13) functions as a pseudosubstrate autoinhibitory inhibitor
for
and the region
(PhK5) acts as an allosteric autoinhibitory inhibitor. Both PhK13
and PhK5 can bind calmodulin simultaneously(4) ; however, these
two peptides have different structures(4) , suggesting they can
interact with calmodulin by different ways and may have different
functions in regulating activity. It is not surprising to see these two
peptides have different inhibitory mechanisms observed here. Other
studies (
)(17) done using phosphorylase kinase
holoenzyme instead of a truncated form of the
-subunit,
, gave different results suggesting that
other subunits of the enzyme can influence PhK5 and PhK13 binding
events.
Like other calmodulin-dependent protein kinases, the
autoinhibitory effect of both PhK13 and PhK5 can be released
specifically by calmodulin (Fig. 2). It is likely that the
autoinhibitory action of this region is regulated by calmodulin or the
-subunit. However, in our experiments no calcium was needed for
good activation. This could be due to the fact that tight binding of
calmodulin to the
-subunit can occur in the absence of
calcium(34) . At this point, the physiological significance of
this activation mechanism seen in vitro is not established.
Four protein kinase structures have been solved, including cyclic
AMP-dependent protein kinase(28, 29, 35) ,
cyclindependent kinase 2 (36) , ERK2 (mitogen-activated protein
kinase)(37) , and twitchin kinase (38) . Overall, the
architecture of these kinases is similar. Co-crystallization of cyclic
AMP-dependent protein kinase and the tight binding competitive
inhibitory
PKI(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) indicates
that the amino acids of PKI participate in binding to the active site
and maintain cyclic AMP-dependent protein kinase in an inactive state
by steric inhibition. The pseudosubstrate inhibition mechanism has been
proposed for myosin light chain kinase,
Ca/calmodulin-dependent protein kinase II, and
protein kinase C (reviewed in (15) ). Putative structural
models for these protein kinases have been proposed based on homolog
modeling using the cyclic AMP-dependent protein kinase and PKI as a
template(33, 39, 40, 41, 42) .
Recently, crystallization of the twitchin kinase, which contained the
catalytic core and the autoinhibitory C-terminal tail, provides the
direct evidence for the intrasteric mechanism of protein kinase
regulation(38) . Multicontact sites occur between the
autoinhibitory tail and the active site of twitchin kinase. Based on
the twitchin kinase crystal structure, Glu
and
Glu
in the catalytic core of twitchin kinase (equivalent
to Glu
and Glu
in
) make an
electrostatic contact with its autoinhibitory C-terminal tail. The data
support our findings about the important role of these two glutamyl
residues in
regulation.
So far, there are at least three
peptides ((43) ,
(44) , and PhK13) derived from
phosphorylase kinase that show competitive inhibition toward
phosphorylase b using a catalytic subunit. However, there is
no evidence suggesting there is more than one active site in the
-subunit. This raises another question about which sequence is a
real pseudosubstrate sequence regulating
-subunit activity.
Although PhK13 is a more potent inhibitor than
and
, the exact interpretation of
the inhibitory mechanisms awaits future structural studies of the
catalytic subunit and the holoenzyme.