(Received for publication, June 22, 1995; and in revised form, September 4, 1995)
From the
AMP-activated protein kinase (AMPK) and
Ca/calmodulin (CaM)-dependent protein kinase I
(CaMKI) are protein kinases that are regulated both by allosteric
activation (AMP and Ca
/CaM, respectively) and by
phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and
CaMKI kinase (CaMKIK), respectively). We now report that AMPKK can
activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK
is 68-fold more effective at activating CaMKI than AMPK, while AMPKK is
17-fold more effective at activating AMPK than CaMKI. Our results
suggest that CaMKIK and AMPKK are distinct enzymes dedicated to their
respective kinase targets but with some overlap in their substrate
specificities. The availability of alternative substrates for AMPKK and
CaMKIK allowed the unequivocal demonstration that AMP and
Ca
/calmodulin promote the activation of AMPK and
CaMKI, respectively, via three independent mechanisms: 1) direct
activation of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3)
by binding to AMPK and CaMKI, inducing exposure of their
phosphorylation sites. Since AMP and Ca
/calmodulin
each has a triple effect in its respective system, in
vivo, the two systems would be expected to be exquisitely
sensitive to changes in concentration of their respective activating
ligands.
The AMP-activated protein kinase (AMPK), ()is
believed to be involved in protecting cells against ATP depletion due
to environmental stress by inactivating several key biosynthetic
enzymes(1, 2) . 5`-AMP allosterically activates AMPK
(up to 5-fold)(3, 4) , but also promotes the
phosphorylation and activation (up to 50-fold) of AMPK by an upstream
protein kinase, AMP-activated protein kinase kinase (AMPKK) (5, 6) . Since the two effects multiply, this
mechanism ensures a sensitive activation of the system when AMP rises.
This happens when ATP is depleted due to displacement of the adenylate
kinase reaction (7) . We have proposed that the effect of AMP
on AMPK phosphorylation is, at least in part, substrate-mediated, i.e. that binding of the nucleotide to AMPK makes it a better
substrate for AMPKK. The evidence was that AMP analogues, which either
mimic or antagonize the allosteric effect of AMP on AMPK activity, have
the same effects on the phosphorylation of AMPK(6) . However,
as no alternative substrates for AMPKK were available, it was difficult
to exclude the possibility that AMPKK was directly stimulated by AMP.
Calmodulin-dependent protein kinase-I (CaMKI) is one of a family of
Ca/calmodulin-dependent protein kinases (CaM
kinases), which also includes CaM kinases II and IV, elongation
factor-2 kinase, myosin light chain kinases, and phosphorylase kinase
(for review, see (8) and (9) ). Two isoforms of CaMKI,
CaMKIa and CaMKIb, have been separately purified from rat brain and
characterized(10, 11) . They are similar to purified
bovine brain CaMKI (12) and expressed rat and human CaMKI (13, 14) in size, subunit structure, and substrate
specificity. CaMKIa was isolated from rat brain in a relatively
inactive, apparently nonphosphorylated state, but its activity was
enhanced >10-fold by a protein activator. By contrast, CaMKIb was
isolated from rat brain in an active, phosphorylated state but could be
inactivated by treatment with purified protein
phosphatase-2A(10, 11) . The a and b forms of CaMKI
may therefore represent dephospho- and phospho- forms respectively, of
the same gene product. The activator of CaMKIa has been purified and
characterized from pig brain(15) . It is a protein kinase that
phosphorylates and activates (up to 50-fold) purified rat brain CaMKIa
or expressed human CaMKI(14, 15, 16) . We
will refer here to purified rat brain CaMKIa as CaMKI, and CaMKIa
activator as CaMKI kinase (CaMKIK). Intriguingly, the phosphorylation
and activation of CaMKI by CaMKIK requires
Ca
/calmodulin(10, 14, 15) .
For reasons analogous to those described above for AMPK, it has been
difficult to determine whether the effect of
Ca
/calmodulin is on CaMKI itself or on CaMKIK,
especially since both bind to calmodulin-Sepharose in a
Ca
-dependent manner(11, 15) .
CaMKI and AMPK therefore display one striking similarity in that the
activating ligand (Ca/calmodulin or AMP respectively)
has a dual effect, causing both direct activation of the
downstream protein kinase, and promotion of its phosphorylation and
activation by the upstream kinase kinase. A further similarity lies in
their substrate specificities, since both CaMKI and AMPK recognize the
consensus
XRXX(S/T)XXX
, where
is a hydrophobic residue(17, 18, 19) .
Furthermore, although AMPK is not regulated by
Ca
/calmodulin, the sequence of its kinase domain (and
that of its yeast homologue, Snf1p) is closely related to those of
members of the calmodulin-dependent protein kinase
subfamily(20, 21) . These observations raised the
possibility that AMPKK and CaMKIK could be similar or even identical,
enzymes. We report here that CaMKIK and AMPKK are distinct, with each
preferring its respective kinase target. However their abilities to
phosphorylate and activate the heterologous kinases allowed us to
dissect the mechanisms of action of the activating ligands, AMP and
Ca
/calmodulin, in these two protein kinase cascades.
Figure 1:
Activation of CaMKI by AMPKK. CaMKI
(0.33 µg/ml) was incubated with MgATP as described under
``Experimental Procedures'' in the presence (filled
symbols) or absence (open symbols) of AMPKK (8000
units/ml). Incubations also contained no further additions (circles), AMP (triangles), CaCl and
calmodulin (Ca
/CaM) (squares), or AMP,
CaCl
and calmodulin (inverted triangles) all at
concentrations stated under ``Experimental Procedures.''
Where CaCl
and calmodulin were not added, EGTA was added to
a final concentration of 2 mM. The small time-independent
increase in activity in the presence of AMPKK (compare open and filled circles) is due to activation occurring during
the subsequent CaMKI assay, which contains
Ca
/calmodulin. AMP and/or
Ca
/calmodulin had no effect in the absence of AMPKK
(not shown).
To reproducibly observe the effects of AMP, we included purified
5`-nucleotidase in the minus AMP controls. We previously showed that
inclusion of 5`-nucleotidase in minus AMP controls is necessary to
observe complete AMP-dependence of the reactivation of AMPK by AMPKK. ()The 5`-nucleotidase removes traces of AMP that might have
been present in the protein preparations or AMP formed due to trace
contamination of one of the preparations with adenylate kinase. The
latter enzyme would produce AMP from ADP generated in the kinase
reaction. The purified 5`-nucleotidase utilized does not hydrolyze ATP. (
)
Figure 2:
Reactivation of dephosphorylated AMPK by
CaMKIK. AMPK (6 units/ml) was dephosphorylated using the catalytic
subunit of PP2A as described under ``Experimental
Procedures.'' At the point shown by the arrow, okadaic
acid was added to inhibit PP2A, with MgATP either with (filled
symbols) or without (open symbols) CaMKIK (0.07
µg/ml). Where indicated, AMP and/or Ca/calmodulin
were also added (concentrations and symbols as for Fig. 1). AMP
and/or Ca
/calmodulin had no effect in the absence of
CaMKIK (not shown).
We also measured
the rate of CaMKIK-catalyzed AMPK reactivation as a function of
calmodulin concentration in the presence of 200 µM AMP and
1 mM CaCl. To measure initial rates, a lower
concentration of CaMKIK was used than in Fig. 2. Under these
conditions, the rate of activation of AMPK was stimulated 5.6 ±
1.8-fold by Ca
/calmodulin with a K
of 15 ± 4 nM (Fig. 3).
Figure 3:
Effect of calmodulin concentration on the
reactivation of AMPK by CaMKIK. Reactivation of AMPK (3 units/ml) was
carried out, after dephosphorylation using the catalytic subunit of
PP2A, as described under ``Experimental Procedures,'' for 20
min with 0.033 µg/ml CaMKIK in the presence of AMP (100
µM) and CaCl (1 mM) at the indicated
calmodulin concentrations.
Figure 4:
Activation of CaMKI by CaMKIK. CaMKI (0.33
µg/ml) was incubated with MgATP as described under
``Experimental Procedures,'' in the presence (filled
symbols) and absence (open symbols) of CaMKIK (0.055
µg/ml). In some incubations, AMP and/or
Ca/calmodulin were also added (concentrations and
symbols as for Fig. 1). The time-independent increase of
activity in the presence of CaMKIK (compare open and filled circles) is due to activation occurring during the
subsequent CaMKI assay, which contains Ca
/calmodulin.
AMP and/or Ca
/calmodulin had no effect in the absence
of CaMKIK (not shown).
Figure 5:
Reactivation of AMPK by AMPKK. AMPK (6
units/ml) was dephosphorylated using the catalytic subunit of PP2A as
described under ``Experimental Procedures.'' At the point
shown by the arrow, okadaic acid was added to inhibit PP2A,
together with MgATP either with (filled symbols) or without (open symbols) AMPKK (1600 units/ml). In some incubations, AMP
and/or Ca/calmodulin were also added (concentrations
and symbols as for Fig. 1). AMP and/or
Ca
/calmodulin had no effect in the absence of AMPKK
(not shown).
Figure 6:
Phosphorylation of CaMKI by CaMKIK and
AMPKK. CaMKI (0.8 µg/ml) was incubated with
[-
P]ATP for 40 min at 30 °C, with or
without CaMKIK (0.055 µg/ml), AMPKK (4000 units/ml), and AMP and/or
CaCl
and calmodulin (concentrations as given under
``Experimental Procedures''), as indicated. The reaction was
stopped by adding SDS sample buffer, and the mixture was analyzed by
SDS-PAGE and autoradiography. The area containing CaMKI (Ia form, 43
kDa) is shown with its migration indicated by the arrow. The
less prominently labeled band below CaMKIa probably represents a minor
amount of the Ib form of CaMKI (39 kDa). Values at the bottom show the
activation of CaMKI obtained under each condition, expressed as a
percentage of the maximal activation obtained with
CaMKIK.
Figure 7:
Phosphorylation of p63, the catalytic
subunit of AMPK, by AMPKK and CaMKIK. AMPK (55 units/ml) was incubated
with [-
P]ATP for 15 min at 30 °C with
or without AMPKK (1600 units/ml), CaMKIK (0.05 µg/ml), and AMP
and/or CaCl
and calmodulin (concentrations given under
``Experimental Procedures''), as indicated. The reaction was
stopped by adding EDTA to a final concentration of 5 mM, and
AMPK was immunoprecipitated. The photograph shows an autoradiogram of
the SDS-PAGE gel of the immunoprecipitate. Values for p63
phosphorylation represent radioactivity in the p63 band estimated using
phosphorimaging. The migration of p63 is indicated by the arrow. Values at the bottom show the activation of
AMPK obtained under each condition, expressed as a percentage of the
maximal activation obtained with AMPKK.
Collectively, the results of Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7are
consistent with a model in which AMP and
Ca/calmodulin, promote the phosphorylation and
activation of AMPK and CaMKI by both substrate- and enzyme-directed
effects. In the activation of CaMKI by AMPKK,
Ca
/calmodulin binds to the former (exposing a
phosphorylation site), and AMP binds to the latter (directly enhancing
its activity). In the activation of AMPK by CaMKIK, AMP binds to the
former (exposing a phosphorylation site) and
Ca
/calmodulin binds to the latter (directly enhancing
its activity). In the homologous reactions, a single allosteric
activator (either Ca
/calmodulin or AMP depending on
the system) is sufficient for both substrate- and enzyme-directed
effects.
The first alternative explanation
is ruled out by the following observations. 1) CaMKIK and AMPKK are
highly selective toward their respective kinase targets. This was
quantified by determining the preference of each kinase kinase for the
two kinase substrates. We expressed the activity of the two
preparations as the percentage of maximal activation (i.e. of
that achieved with maximal amounts of the homologous kinase
kinase)/min/µl of preparation. The kinase kinase preparations were
diluted such that a linear increase in activity was obtained with time.
For CaMKIK, the activity was 24.4% of maximum/min/µl using CaMKI as
substrate and 0.36% of maximum/min/µl with AMPK as substrate. For
AMPKK, the activity was 13.4% of maximum/min/µl with AMPK as
substrate and 0.78% of maximum/min/µl with CaMKI as substrate.
CaMKIK was therefore 68-fold more active toward CaMKI than AMPK,
whereas AMPKK was 17-fold more active toward AMPK than CaMKI. 2) CaMKIK (15) but not AMPKK (data not shown) bound to
calmodulin-Sepharose in a Ca-dependent manner. 3) The
allosteric activators of AMPKK and CaMKIK are different. This is most
clearly seen when the two kinase kinases are compared using a single
kinase target and an allosteric activator that does not bind to the
kinase target itself. Activation of CaMKI by AMPKK (Fig. 1), but
not by CaMKIK (Fig. 4), was stimulated by AMP. And, activation
of AMPK by CaMKIK (Fig. 2), but not by AMPKK (Fig. 5),
was stimulated by Ca
/calmodulin. Thus, AMPKK is
stimulated by AMP but not Ca
/calmodulin, while CaMKIK
is stimulated by Ca
/calmodulin, but not AMP.
The
second alternative explanation, that the preparations of AMPKK and
CaMKIK were cross-contaminated, is ruled out for the following reasons.
1) CaMKIK was purified by Cadependent affinity
chromatography on calmodulin-Sepharose(15) , to which AMPKK
does not bind. 2) During the purification of AMPKK, CaMKI-activating
activity exactly comigrated with AMPK-activating activity on the last
two column steps, Q-Sepharose, and Mg
gradient
elution from Mono-Q, making it very unlikely that the minor
CaMKI-activating activity detected was due to residual unresolved
CaMKIK but indicating rather that this activity was an intrinsic
property of AMPKK itself. 3) The acceleration in the rate of CaMKI
activation by AMP in the presence of AMPKK (Fig. 1) but not
CaMKIK (Fig. 4) can only be explained by an authentic capacity
of AMPKK to phosphorylate and activate CaMKI. Similarly, the
accelerated activation of AMPK by Ca
/calmodulin in
the presence of CaMKIK (Fig. 2) but not AMPKK (Fig. 5)
can only be explained by an authentic capacity of CaMKIK to
phosphorylate and activate AMPK.
As shown by these results, AMPKK and CaMKIK are kinase kinases each with some capacity to phosphorylate and activate the respective heterologous kinase target. Given that the heterologous reactions are catalyzed at a small fraction of the rate of the homologous reactions, these cross-reactivities may not be physiologically significant, although this is difficult to exclude at present. The cross-reactivities are explainable by assuming that AMPKK and CaMKIK are representatives of a related family of kinase kinases, which recognizes similar phosphorylation site sequences. In the case of CaMKI, this site has been recently identified as Thr-177 located in the ``activation loop'' of the catalytic domain(14) . Efforts are currently underway to identify the activating phosphorylation site in AMPK.
The studies described here have also
identified for the first time an alternative substrate for AMPKK, and
for CaMKIK, the first instance of an alternative substrate regulated by
a distinct activating ligand, since although CaMKIK does phosphorylate
and activate CaMKIV(27) , the latter is also
Ca/calmodulin-regulated. The availability of
alternative substrates regulated by distinct activating ligands has
allowed us, by analysis of the heterologous systems, to demonstrate
that in these two protein kinase cascades, the regulatory molecules
(Ca
/calmodulin and AMP) both have three
independent effects (schematically illustrated in Fig. 8).
Figure 8:
Triple effects of AMP and
Ca/calmodulin (Ca
/CaM) on the AMPK
and CaMKI protein kinase cascades. The heavy arrows indicate
that the activating ligands have three effects: 1) direct allosteric
activation of the downstream protein kinase; 2) binding to the
downstream protein kinase and making it a better substrate for the
upstream protein kinase; 3) direct activation of the upstream protein
kinase. Existing evidence suggests that activation of AMPK is reversed
by protein phosphatase 2C (PP2C)(25, 26) . PP? indicates that the protein phosphatase responsible for
dephosphorylation of CaMKI is not known, although CaMKI can be
deactivated in vitro by the catalytic subunit of PP2A (10) .
The first role described for Ca/calmodulin in the
regulation of CaMKI activity was that of a direct activator (10, 11, 12) . The mechanism of this
activation involves relief of intrasteric autoinhibition via identified
calmodulin-binding and autoinhibitory domains(14) . In addition
to this classic role, Ca
/calmodulin is also required
for the phosphorylation and activation of CaMKI by CaMKIK (Refs. 10,
14, and 15; see also Fig. 4). We show here that this latter role
itself occurs through two distinct effects. Since phosphorylation and
activation of AMPK by CaMKIK was stimulated by
Ca
/calmodulin ( Fig. 2and Fig. 7),
CaMKIK is itself a Ca
/calmodulin-stimulated enzyme.
And since phosphorylation and activation of CaMKI by AMPKK was
stimulated by Ca
/calmodulin ( Fig. 1and Fig. 6), the latter is also exerting an effect at the level of
the substrate (CaMKI), to expose the site of phosphorylation. These
results are supported by experiments with expressed native and mutated
forms of human CaMKI in that mimicking calmodulin-binding by C-terminal
truncation of the calmodulin-binding/autoinhibitory domain of CaMKI
promoted its phosphorylation by CaMKIK, and phosphorylation of the
calmodulin-independent CaMKI mutant by CaMKIK was still accelerated by
Ca
/calmodulin(14) .
Similarly, AMP both directly activates AMPK and promotes its phosphorylation by AMPKK ( (5) and (6) ; see also Fig. 5). We show here that this latter role itself occurs through two distinct effects. Since phosphorylation and activation of CaMKI by AMPKK was stimulated by AMP ( Fig. 1and Fig. 6), AMPKK is itself an AMP-activated protein kinase. AMP also stimulated the phosphorylation of several polypeptides in the AMPKK preparation (data not shown), further supporting the idea that AMPKK is an AMP-activated protein kinase. And since phosphorylation and activation of AMPK by CaMKIK was also stimulated by AMP (Fig. 2, 7), the latter is also exerting an effect at the level of the substrate (AMPK), to expose the site of phosphorylation.
Since CaMKIK is
Ca/calmodulin-activated, we determined the
concentration of calmodulin that gave half-maximal CaMKIK activation.
The value obtained, 15 ± 4 nM, is within the range
found for other calmodulin-activated enzymes(8, 9) .
Interestingly, CaMKIK appears to have a significant basal activity in
the absence of Ca
/calmodulin ( Fig. 2and Fig. 3) and thus differs from most other calmodulin-activated
enzymes, which are essentially completely dependent on calmodulin. We
also consistently find that
25% of CaMKIK does not bind to
calmodulin-Sepharose (data not shown; (15) ). One explanation
is that CaMKIK exists in an equilibrium between active and inactive
conformations, and calmodulin binds with high affinity only to the
latter, converting it to the active conformation. Our results also
demonstrate that AMPKK is itself an AMP-activated protein kinase.
However, because it was necessary to add 5`-nucleotidase to the minus
AMP controls to observe the effects of AMP, it was not possible to
determine the concentration dependence of AMPKK on AMP.
These
findings that in the AMPK and CaMKI systems both the upstream and
downstream protein kinases are regulated by the same activating ligand
is not without precedent, since cyclin-dependent kinase-activating
kinase, the protein kinase that phosphorylates the activating threonine
of cyclin-dependent protein kinases, is itself a cyclin-dependent
protein kinase(28, 29, 30) . As in our
systems, binding of the activating ligand (cyclin) to the downstream
protein kinase stimulates both the activity of the downstream kinase
and its phosphorylation by the upstream protein kinase(31) .
The cyclin-dependent kinase-activating kinase/cyclin-dependent protein
kinase system differs in that the cyclin proteins that activate the
upstream and downstream protein kinases are
distinct(32, 33, 34) . We are not aware of a
precedent where the identical activating ligand (AMP or
Ca/calmodulin) has three effects on a protein kinase
cascade. We propose that the triple actions of AMP and
Ca
/calmodulin on their respective systems would make
these systems respond in an exquisitely sensitive manner to small
changes in the concentrations of the activating ligand. Although the
allosteric regulators differ between the two systems, our study
demonstrates a remarkable analogy between CaMKI and AMPK. We have
already shown another striking similarity, in that the substrate
sequence motifs recognized by these two protein kinases are very
similar(17, 18, 19) . These similarities in
specificity, and in recognition by the upstream protein kinases
presumably reflect the fact that the kinase domains of rat CaMKI and
rat AMPK (13, 20) are 40% identical in sequence and
phylogenetic analysis of kinase domain sequences places both within the
same protein kinase subfamily(21) . On the other hand, the two
kinases are unrelated outside of the kinase domain, which is presumably
where the binding sites for the distinct activating ligands are
located.
Okuno et al.(35) and Tokumitsu et
al.(36, 37) have recently reported that
calmodulin-dependent protein kinase IV (CaMKIV) is also regulated in a
Ca/calmodulin-dependent manner by an activator
protein kinase (CaMKIV kinase). In this case, it is not clear whether
the effect of Ca
/calmodulin to promote CaMKIV
phosphorylation is due to binding to CaMKIV kinase, to CaMKIV, or both,
although it is intriguing that CaMKIV kinase, like CaMKIK, binds to
calmodulin-Sepharose(36, 37) . Moreover, CaMKIK also
activates CaMKIV and does so by phosphorylating the equivalent
threonine residue (Thr-196) to that phosphorylated in CaMKI (Thr-177) (14, 27) . The precise relationship between CaMKIV
kinase and CaMKIK remains to be established.
It is also interesting
to compare AMPK and CaMKI with calmodulin-dependent protein kinase II
(CaMKII), which is regulated by autophosphorylation on Thr-286.
Autophosphorylation of the latter is an intersubunit reaction occurring
between neighboring subunits of the multimeric enzyme.
Ca/calmodulin appears to play a role in CaMKII
phosphorylation analogous to its role in CaMKI (and AMPK)
phosphorylation in that it is required not only to initially activate
the CaMKII subunit, which is acting as the kinase, but also must be
bound to the neighboring subunit in order for it to act as a substrate (38) . An important difference between CaMKI and CaMKII is that
phosphorylation occurs at nonequivalent sites(14) . Another
major difference is that autophosphorylation of CaMKII on Thr-286
converts it from an active and
Ca
/calmodulin-dependent form to a
Ca
/calmodulin-independent form, whereas
phosphorylation of CaMKI by CaMKIK converts it from an inactive form to
an active and Ca
/calmodulindependent
form(10, 15) . CaMKIV incorporates both forms of
regulation, since its phosphorylation by CaMKIK or CaMKIV kinase
activates and imparts partial
Ca
/calmodulinindependence(27, 35, 36) .
In conclusion, studies described here indicate that the apparent
relatedness of the calmodulin-dependent protein kinase kinases
described to date extends beyond the immediate calmodulin-dependent
protein kinase subfamily to that regulated by a distinct allosteric
activator (AMP). This relationship is seen both in the ability of AMPKK
and CaMKIK to cross-regulate the heterologous downstream kinases, as
well as in the remarkable similarity of the roles of the allosteric
activators (AMP and Ca/calmodulin) in the activation
process. Despite our findings that the upstream protein kinases have a
limited ability to activate the alternative downstream protein kinases in vitro, we suspect that they are likely to be dedicated to
their respective downstream protein kinases in vivo, and would
thus constitute elements in separate signal transduction cascades akin
for example, to the MAP kinase kinase/MAP kinase signaling module. For
both the AMP-regulated and Ca
/calmodulin-regulated
pathways, an important question for future consideration is the
quantitative significance of the direct activation of the upstream
protein kinases by the respective allosteric activators, relative to
the dual effects of the activators on the downstream protein kinases.
Studies of analogues that differentially activate or inhibit the
upstream and downstream protein kinases or of mutant kinase kinases or
kinases that do not respond to the activators will be necessary to
address these questions.