(Received for publication, November 20, 1995)
From the
We have previously purified and cloned rat brain
Ca/calmodulin-dependent protein kinase kinase
(CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen,
H., and Soderling, T. R.(1995) J. Biol. Chem. 270,
19320-19324). In the present study we have determined that
activation of CaM-K IV through phosphorylation of Thr
by
CaM-KK is triggered by elevated intracellular Ca
in
intact cells and requires binding of Ca
/CaM to both
enzymes. An expressed fragment of CaM-K IV (CaM-K
IV
), which contains the activating
phosphorylation site (Thr
) but not the autoinhibitory
domain or the CaM-binding domain, still required
Ca
/CaM for phosphorylation by wild-type CaM-KK. A
truncated mutant of CaM-KK (CaM-KK
)
phosphorylated CaM-K IV
in a
Ca
/CaM-independent manner, but this constitutively
active CaM-KK
required Ca
/CaM
for phosphorylation and activation of wild-type CaM-K IV. These results
demonstrate that binding of Ca
/CaM to both CaM-K IV
and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and
CaM-K IV appear to have similar Ca
/CaM requirements
with EC
values of approximately 100 nM. Studies
using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated
that CaM-KK rapidly activated both total and
Ca
/CaM-independent activities of wild-type CaM-K IV,
but not the Thr
Ala mutant, upon ionomycin
stimulation.
Ca/calmodulin-dependent protein kinase IV
(CaM-K IV)(
)(1, 2, 3) , a member
of the CaM-kinase family, mediates Ca
-dependent
transcriptional activation through phosphorylation of transcription
factors such as cAMP response element binding protein (4, 5, 6, 7) and serum response
factor(8, 9) . Originally, it was reported that
purified CaM-K IV from rat brain undergoes regulatory
autophosphorylation which enhances Ca
/CaM-dependent
(total) and -independent activities of the
enzyme(10, 11) . However, since recombinant CaM-K IV
exhibits little autophosphorylation and activation (4, 8) , it was likely that the purified brain CaM-K
IV contained a contaminating activator. Recently several groups have
identified and purified brain CaM-kinase kinases (CaM-KK) that
phosphorylate and activate not only CaM-K IV (12, 13) but also CaM-K I(14) . These results
suggested the existence of a CaM-kinase cascade analogous to other
kinase cascades such as cAMP-dependent protein kinase/phosphorylase
kinase(15) , MAP-kinase(16) , and AMP-kinase (17) .
The cDNA for a 68-kDa rat brain CaM-KK has been
cloned, and the expressed recombinant CaM-KK in vitro activates CaM-K I and CaM-K IV but not CaM-K II(18) .
CaM-KK is abundant in brain and detectable in thymus and spleen,
similar to the tissue distribution of CaM-K IV (19) but more
restricted than the distribution of CaM-K I (20) . CaM-KK binds
Ca/CaM by the gel overlay technique, and its
activation of CaM-K IV and CaM-K I requires
Ca
/CaM(18) . However, it is not clear whether
the required Ca
/CaM is binding to CaM-KK, CaM-K IV or
both. Since there are no known substrates of CaM-KK which do not also
bind Ca
/CaM, we decided to examine the
phosphorylation of a fragment of CaM-K IV that contains its activating
phosphorylation site. Both CaM-K I and IV, but not CaM-K II, contain
Thr residues in putative ``activation loops'' similar to the
MAP-kinases and cdc kinases which are also activated by kinase
cascades(21, 22) . Thus, we suspected that Thr
in this activation loop of CaM-K IV may be the phosphorylation
site for CaM-KK. Recently it has been shown that purified 52-kDa
porcine brain CaM-K I kinase increases CaM-K IV total activity through
phosphorylation of Thr
(23) , but effects of the
Thr
Ala mutation on the generation of
Ca
-independent activity of CaM-K IV were not
reported. Since the 52-kDa CaM-K I kinase has not been cloned, it is
difficult to compare it with our 68-kDa CaM-KK. The 52-kDa CaM-KK may
represent the porcine brain isoform of rat brain 68-kDa CaM-KK.
The
above studies suggest the existence of a unique protein kinase cascade
regulating CaM-K I and IV, but this system needs further confirmation
in intact cells. Although we showed that co-transfection of CaM-KK with
CaM-K IV gave a 14-fold enhancement of transcriptional activation of a
reporter gene(18) , the direct activation of CaM-K IV by
transfected CaM-KK has not been demonstrated in intact cells. In this
report, we demonstrate in COS-7 cells the activation of CaM-K IV by
CaM-KK and its requirement for elevation of intracellular
Ca. Furthermore, we establish in vitro that
binding of Ca
/CaM to both CaM-K IV and CaM-KK is
required for the CaM-K activation cascade.
Figure 1:
Activation of CaM-K IV by purified
recombinant CaM-KK in vitro. A, Purified E.
coli-expressed CaM-KK (2 µg, right lane),
Sf9-expressed CaM-K IV (1.5 µg, center lane), and M standard proteins (left lane) were
separated on SDS-10% PAGE and stained with Coomassie Brilliant Blue
R250. B, CaM-K IV (5.4 µM) or kinase buffer was
incubated at 30 °C for 5 min with either purified, recombinant
CaM-KK (36 nM) or buffer in a kinase cascade activation
reaction (see ``Experimental Procedures'') with either 1
mM EGTA or 1 mM CaCl
, 10 µM CaM as indicated. After terminating the reaction, CaM-K IV
activity was measured using 40 µM syntide-2 in the
presence of 1 mM EGTA (open bars) or 1 mM CaCl
, 1 µM CaM (closed bars)
under standard assay conditions. CaM-KK activity toward syntide-2 was
also measured in the absence of exogenous CaM-K IV under the same
conditions, but it was negligible (right four bars). The mean
± S.E. of three independent experiments is
shown.
In
order to determine whether the required Ca/CaM was
binding to CaM-KK, CaM-K IV, or both, we needed a substrate of CaM-KK
which itself does not bind Ca
/CaM. We constructed a
poly(His)
-tagged fragment of CaM-K IV (residues
178-246) which contains the activating phosphorylation site (i.e. Thr
) (23) but not the AID or
CaM-binding domain (residues 304-328). The His/CaM-K
IV
was expressed in E. coli and
purified for in vitro phosphorylation by CaM-KK. Using this
fragment of CaM-K IV eliminates any ambiguity due to
autophosphorylation by activated CaM-K IV. As shown in Fig. 2A (lower panel), His/CaM-K IV
was phosphorylated by recombinant CaM-KK in a completely
Ca
/CaM-dependent manner. Phosphoamino acid analysis
(not shown) showed that this phosphorylation was exclusively on Thr,
and mutation of Thr
to Ala abolished approximately 90% of
P incorporation into His/CaM-K IV
by CaM-KK (Fig. 2A). These results demonstrate
that recombinant CaM-KK is Ca
/CaM-dependent and
directly phosphorylates Thr
of CaM-K IV. The His/CaM-K
IV
was not phosphorylated by CaM-K IV itself
(data not shown).
Figure 2:
Ca/CaM-dependent
phosphorylation of His/CaM-K IV
by CaM-KK and
of His/CaM-K IV
by CaM-K IV. A, wild-type
or Thr
Ala mutant of His/CaM-K
IV
was expressed, purified, and incubated (40
µg/ml) at 30 °C for 15 min with 36 nM of recombinant
CaM-KK (or buffer) and 100 µM [
-
P]ATP in the presence of either 1
mM CaCl
/1 µM CaM or 1 mM EGTA as indicated. Reactions were analyzed by SDS-18% PAGE
(Tricine system) and either Coomassie Blue stain (top panel)
or autoradiography (bottom panel). B, His/CaM-K
IV
was expressed, purified, and incubated with
either CaM-KK (36 nM, lanes 1 and 2), CaM-K
IV (22 nM, lanes 3 and 4) activated by
CaM-KK as in Fig. 1B, or nonactivated CaM-K IV (22
nM, lanes 5 and 6) for 15 min at 30 °C
in the presence of either 1 mM CaCl
, 1 µM CaM or 1 mM EGTA as indicated. Samples were analyzed by
SDS-PAGE and autoradiography as in A.
It has also been reported that phosphorylation of
Ser residues in the NH terminus of CaM-K IV by purified rat
brain CaM-KK is responsible for activation of CaM-K IV(31) . We
tested the ability of CaM-KK to phosphorylate this domain by expressing
a His/CaM-K IV
construct. However, His/CaM-K
IV
was not significantly phosphorylated by CaM-KK,
but it was phosphorylated by CaM-K IV, especially after its activation
by CaM-KK (Fig. 2B). These results suggest that the
observed phosphorylation of the NH
-terminal Ser residues in
CaM-K IV may have been due to autophosphorylation subsequent to
activation of CaM-K IV by CaM-KK.
Figure 3:
Phosphorylation and activation of CaM-K IV
by truncated CaM-KK. A,
CaM-KK
(36 nM) or buffer were assayed
(30 °C, 15 min) for their abilities to phosphorylate wild-type
His/CaM-K IV
(lanes 1-4), the
Thr196Ala mutant of His/CaM-K IV
(lanes 5 and 6) or buffer (lanes 7 and 8) in the
presence of either 1 mM EGTA or 1 mM CaCl
, 1 µM CaM as indicated. Reactions
were analyzed by SDS-PAGE and either stained with Coomassie Blue (top panel) or subjected to autoradiography (bottom
panel). B, CaM-K IV (5.4 µM) was incubated
(30 °C, 5 min) with either 36 nM of partially purified
recombinant CaM-KK
or buffer in a kinase
activation reaction (see ``Experimental Procedures'') with
either 1 mM EGTA or 1 mM CaCl
, 10
µM CaM as indicated and either 400 µM ATP or
100 µM [
-
P]ATP (inset). After terminating the activation, the reaction
mixture was analyzed by SDS-PAGE autoradiography (inset) or
CaM-K IV activity was measured using 40 µM syntide-2 in
the presence of 1 mM EGTA (open bars) or 1 mM CaCl
, 1 µM CaM (closed bars)
under standard assay conditions. Kinase activity toward syntide-2 of
the recombinant CaM-KK was also measured in the absence of exogenous
CaM-K IV under the same conditions, but it was negligible (right
four bars). The mean ± S.E. of three independent
experiments is shown. For the insert: a and b = CaM-K IV - CaM-KK
-/+ Ca
/CaM, respectively; c and d = CaM-K IV + wild-type CaM-KK
-/+ Ca
/CaM, respectively; e and f = CaM-K IV + CaM-KK
-/+ Ca
/CaM, respectively. The arrow denotes the position of CaM-K
IV.
Since binding of Ca/CaM to both
CaM-KK and CaM-K IV was required for the CaM-kinase cascade to operate,
we tested whether CaM-KK and CaM-K IV had different requirements for
Ca
/CaM. If CaM-KK needed much higher concentrations
of Ca
/CaM than CaM-K IV, it was possible that small
elevations of intracellular Ca
might selectively
activate CaM-K IV, and higher elevations of Ca
would
be required to trigger the CaM-kinase cascade. However, it appears that
the Ca
/CaM requirement (EC
) of both
CaM-KK and CaM-K IV is approximately 100 nM (Fig. 4).
Figure 4:
Ca/CaM activation of
CaM-KK and CaM-K IV in vitro. Nonactivated CaM-K IV (22
nM,
), CaM-K IV (22 nM,
) activated by
CaM-KK, or CaM-KK itself (36 nM,
) were assayed for
their activities with 1 mM CaCl
and the indicated
concentrations of CaM (other conditions as given under
``Experimental Procedures''). The requirement for CaM of
activated CaM-K IV was determined by subtraction of its
Ca
-independent activity from total activity. For
nonactivated and activated CaM-K IV, 40 µM syntide-2 was
used as substrate, whereas His/CaM-K IV
(40
µg/ml) was the substrate for CaM-KK.
Figure 5:
Ca-dependent activation
of CaM-K IV by CaM-KK in COS-7 cells. A, CaM-K IV cDNA which
was fused with HIS-tag (4 µg of pME-His-CaM-K IV) was
co-transfected into COS-7 cells with either wild-type CaM-KK (0.8
µg of pME-CaM-KK-wild, circles) or plasmid alone (0.8
µg of pME18s, triangles). Cells were stimulated with 1
µM ionomycin and at the indicated times frozen with liquid
N
. Cells were lysed, expressed His/CaM-K IV was partially
purified by Ni
resin, and CaM-K IV activity was
measured either in the presence of 1 mM CaCl
, 1
µM CaM (closed symbols) or 1 mM EGTA (open symbols) using 40 µM syntide-2 as a
substrate. B, expressed His/CaM-K IV (15 µl of
Ni
column eluate) in the experiment of Panel A was quantitated at the indicated time points after ionomycin
stimulation by Western blot analysis (chemiluminescent detection) using
anti-CaM-K IV antibody.
It was recently reported that mutation of
Thr to Ala in CaM-K IV blocks the increase in its total
activity normally generated in vitro by purified procine brain
CaM-K I kinase(23) . The data of Fig. 2confirm that
Thr
in HIS/CaM-K IV
was
phosphorylated by CaM-KK. To test whether Thr
is also the
phosphorylation/activation site in intact cells, we introduced
His-tagged CaM-K IV
mutant into COS-7 cells with
CaM-KK. Fig. 6shows that the Thr
Ala
mutant had basal Ca
/CaM-dependent activity, but it
was not activated by CaM-KK in response to ionomycin treatment.
Figure 6:
Mutation of Thr
Ala
abolished CaM-K IV activation by CaM-KK in COS-7 cells. Either
wild-type or Thr
Ala mutant of CaM-K IV cDNA (4
µg), which are fused with HIS-tag, were transfected into COS-7
cells with wild-type CaM-KK (0.8 µg of pME-CaM-KK). After
stimulation with or without 1 µM ionomycin for 4 min,
cells were frozen with liquid N
and lysed. Expressed
His/CaM-K IV (wild-type or mutant) was partially purified by
Ni
resin, and CaM-K IV activity was measured in the
presence of either 1 mM CaCl
/1 µM CaM (solid bars) or 1 mM EGTA (open bars) using
40 µM syntide. The mean ± S.E. of three experiments
using three independent transfections is
shown.
Mutation of Thr to Ala not only prevents the increase
in total activity of CaM-K IV generated by CaM-KK(23) , but it
also blocked the increase in Ca
-independent activity (Fig. 6). It is possible that phosphorylation of Thr
is directly responsible for increasing both total and
Ca
-independent activities. Alternatively,
phosphorylation of Thr
may be a prerequisite for
phosphorylation of some other site, either by CaM-KK or through
autophosphorylation by activated CaM-K IV, which generates
Ca
-independent activity. Since Thr
of
CaM-K IV is analogous to the phosphorylation site (Thr
)
in CaM-K II which generates Ca
-independent activity,
Thr
was considered a likely candidate. Furthermore,
mutation of HMDT
to DEDD converts CaM-K IV into a
Ca
-independent species(13) . We therefore
made CaM-K IV mutants Thr
Ala and Ser
Ala and examined their in vitro activation by
CaM-KK. Both mutant CaM-K IV species showed normal increases in both
total and Ca
-independent activities upon
phosphorylation by CaM-KK (data not shown).
Extensive in vitro studies have documented the
phosphorylation and activation of CaM-K IV by
CaM-KK(12, 13, 23) . This report extends
these studies by 1) establishing a requirement for binding of
Ca/CaM to both the CaM-KK and the substrate CaM-K IV
and 2) demonstrating in COS-7 cells with transfected CaM-KK the
Ca
-dependent activation of co-transfected CaM-K IV
through phosphorylation of Thr
.
The requirement for
binding of Ca/CaM to CaM-KK is consistent with the
previous observation that CaM-KK can be purified on CaM-Sepharose (13) and that the expressed CaM-KK binds
Ca
/CaM using the gel overlay technique(18) .
However, since both CaM-KK and CaM-K IV are apparently CaM-dependent,
it was not clear whether the requirement for Ca
/CaM
in the CaM-kinase cascade was due to binding of
Ca
/CaM to either CaM-KK, CaM-K IV, or both. By
constructing a His-tagged construct containing the phosphorylation site
in CaM-K IV (Thr
) but lacking the AID and CaM-binding
domain (residues 304-328), we demonstrated that phosphorylation
of Thr
in this CaM-independent substrate by wild-type
CaM-KK still required Ca
/CaM (Fig. 2). This
indicates a requirement for Ca
/CaM in this reaction
for the activation of CaM-KK. This conclusion was substantiated by
using a truncated form of CaM-KK, CaM-KK
, lacking
the putative AID and CaM-binding domain. This CaM-KK
phosphorylated the His/CaM-K IV
in a
Ca
/CaM-independent manner (Fig. 3), confirming
the requirement for Ca
/CaM of wild-type CaM-KK.
Furthermore, the requirement for Ca
/CaM in the
phosphorylation of wild-type CaM-K IV by CaM-KK
confirmed the requirement for Ca
/CaM-binding to
the substrate CaM-K IV. Thus, binding of Ca
/CaM to
both CaM-KK and CaM-K IV is required for this kinase cascade. The
binding of Ca
/CaM to CaM-KK is presumably required
for neutralization of an AID COOH-terminal of residue 434. Studies are
currently in progress to further define the AID and CaM-binding domain
in CaM-KK. Extensive studies have established the existence of adjacent
and sometimes overlapping AIDs and CaM-binding domains in numerous
other kinases activated by Ca
/CaM(32) .
Presumably Ca
/CaM must bind to CaM-K IV and remove
its AID to expose the activation loop Thr
which is
probably within the catalytic cleft.
Our studies confirm with
recombinant CaM-KK the observation made with purified porcine brain
CaM-K I kinase that mutation of Thr to Ala blocks the
increase in total activity of CaM-K IV(23) . We also observed
that the increase in Ca
-independent activity was also
absent in this mutant (Fig. 6). It is interesting that this
single site mutation blocks the increases in both total and
Ca
-independent activities of CaM-K IV, whereas
phosphorylation of the equivalent site (Thr
) in CaM-K I
produces only an increase in total activity(34) . Both of these
phosphorylation sites are within the ``activation loops''
that require phosphorylation for activation of numerous protein
kinases. One possibility is that the AID in CaM-kinase IV lies within
the catalytic cleft and makes an inhibitory interaction with the
activation loop. This could explain why removal of the AID through
binding of Ca
/CaM is required to expose Thr
for phosphorylation by CaM-KK. Phosphorylation of Thr
could prevent the inhibitory interaction of the activation loop
with the AID in the absence of Ca
/CaM, thereby
generating Ca
-independent activity. Another
possibility is that phosphorylation of Thr
in CaM-K IV
generates elevated total kinase activity which then allows
autophosphorylation on another site to account for the elevated
Ca
-independent activity. If such were the case, a
phosphorylation site in the AID would seem likely since introduction of
negative charge in the AID generates Ca
-independent
activity of CaM-K IV(13) . To test this possibility we mutated
Thr
, which is equivalent to the autophosphorylation site
in CaM-K II (Thr
) that generates
Ca
-independent activity. However, CaM-KK increased
both total and Ca
-independent activities of the
Thr
Ala mutant. Similar results were obtained with
the Ser
Ala mutant. Thus, it is still not clear
whether phosphorylation of Thr
alone is sufficient for
increasing Ca
-independent activity of CaM-K IV.
A
major purpose of this study was to test whether this CaM-kinase
cascade, which has been demonstrated in vitro, could also be
observed in intact cells. When COS-7 cells were transfected with
His-tagged CaM-K IV alone, there was no effect of
Ca-mobilization through ionomycin treatment on the
His/CaM-K IV activity subsequently assayed in vitro. However,
when CaM-KK was co-transfected with His/CaM-K IV, then
ionomycin-treatment resulted in a 3-6-fold increases in total and
Ca
-independent His/CaM-kinase IV activities (Fig. 5A). These changes in CaM-K IV activities were
not due to changes in amounts of expressed CaM-K IV (Fig. 5B). Interestingly, the activation of CaM-K IV by
ionomycin was transient, peaking at 4 min and returning to near basal
values at 10 min. This biphasic nature is consistent with
phosphorylation of CaM-K IV followed by dephosphorylation. That
phosphorylation was required for the activation was demonstrated by the
fact that the Thr
Ala mutant of CaM-K IV was not
activated by CaM-KK upon ionomycin treatment (Fig. 6). The fact
that only a 3-4-fold activation of CaM-K IV was observed in the
intact cells compared to the 10-fold or greater activation in vitro is probably due to the endogenous protein phosphatases in the
COS-7 cells that limit both the extent and duration of activation.
These results are similar to the activation of CaM-K IV in Jurkat cells
upon stimulation of the CD3 receptor(33) . The CD3-mediated
activation of CaM-K IV is due to its phosphorylation since this
activation can be reversed in vitro by treatment with protein
phosphatase 2A but not protein phosphatase 1(26) , the same
specificity as for reversal of in vitro activation of CaM-K IV
by CaM-KK. Furthermore, CD3-mediated activation of CaM-K IV is
transient, presumably due to endogenous protein phosphatase 2C which
can inactivated CaM-K IV(33) , and the in situ activation can be further augmented 2-3-fold by subsequent in vitro treatment with purified CaM-KK(26) . From
these studies we concluded that the CD3-dependent activation of CaM-K
IV in Jurkat cells is probably mediated by CaM-KK.
It is puzzling
why adjacent steps in a kinase cascade should both require the same
activator, i.e. Ca/CaM. Our initial thought
was that perhaps the CaM-KK would have a much lower affinity for
activation by Ca
/CaM. Thus, low levels of elevated
intracellular Ca
might selectively activate CaM-K IV
and CaM-K I, whereas much higher level of Ca
would be
required for activation of CaM-KK to initiate the cascade. However, the
data of Fig. 4show that both kinases have very similar
requirements for Ca
/CaM. Of course, the effect of
activation of different substrates of CaM-KK may confer differences in
their Ca
dependencies. CaM-K IV, which has been
activated by CaM-KK, can maintain sustained activity in the absence of
continued Ca
because of its considerable
Ca
-independent activity. This is not true for CaM-K I
which does not generate Ca
-independent activity upon
activation by CaM-KK. While this manuscript was under review, a paper
appeared which shows a requirement for binding of
Ca
/CaM to both CaM-KI and CaM-KI kinase and for
binding of AMP to both AMP-kinase and AMP-kinase kinase in those
cascades(35) . Lastly, it is possible there may be unidentified
CaM-KK substrates which themselves are not regulated by
Ca
/CaM. We are currently searching for additional
physiological pathways which may be regulated by CaM-KK.