(Received for publication, August 3, 1995; and in revised form, October 1, 1995)
From the
Ca/calmodulin-dependent protein kinase IV
(CaM-kinase IV), a member of the CaM-kinase family involved in
transcriptional regulation, is stimulated by Ca
/CaM
but also requires phosphorylation by a CaM-kinase kinase for full
activation. In this study we investigated the physiological role of a
CaM-kinase cascade in Jurkat T human lymphocytes through antigen
receptor (CD3) signaling. Total and Ca
-independent
CaM-kinase IV activities were increased 8-14-fold by anti-CD3
antibody. This CD3-mediated activation involved phosphorylation since
the immunoprecipitated CaM-kinase IV from stimulated Jurkat cells could
be subsequently inactivated in vitro by protein phosphatase
2A. CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells or
CD3-negative mutant Jurkat cells could be activated in vitro 10-40-fold by CaM-kinase kinase purified from rat brain or
thymus, whereas CaM-kinase IV from CD3-stimulated wild-type Jurkat
cells was only activated to 2-3-fold by exogenous CaM-kinase
kinase. CaM-kinase IV activation was triggered by Ca
acting through calmodulin since activation could also be elicited
by ionomycin treatment, and CD3-mediated activation was blocked by the
calmodulin antagonist calmidazolium. These data are consistent with a
CaM-kinase cascade in which CaM-kinase IV is activated by a CaM-kinase
kinase cascade triggered by elevated intracellular calcium in Jurkat
cells.
Ca/calmodulin-dependent protein kinase IV
(CaM-kinase IV), (
)a member of the CaM-kinase family, was
first named CaM-kinase-Gr due to its abundance in rat cerebellar
granule cells(1) . Subsequently, CaM-kinase IV was found in
thymus, spleen, and testis(2) , and it was localized to both
the cytoplasm and nucleus(3) . The substrate specificity of
CaM-kinase IV has not been well established, but it can phosphorylate in vitro the synthetic peptide syntide-2, synapsin
I(1) , Rap-1b(4) , and several transcription factors
including serum response factor (5) and cAMP-responsive element
binding protein(6, 7) . Recently, it was shown that
oncoprotein 18 (Op 18) is a major cytosolic target for CaM-kinase IV in
Jurkat cells(8) . Although the function of Op 18 is not clear,
its involvement in signal transduction and/or cell cycle control has
been suggested(9, 10, 11, 12) .
The primary structure of CaM-kinase IV has been resolved by cDNA cloning from rat (1, 13, 14) and mouse brain (15) and human Jurkat T lymphocytes(16) . CaM-kinase IV is structurally related to CaM-kinase II in its catalytic and regulatory CaM-binding domains(1) . However, the COOH terminus of CaM-kinase IV is highly acidic with polyglutamate stretches, a characteristic of many chromatin-associated proteins(17) , which probably accounts for its monomeric structure. Truncation of CaM-kinase IV at residue 313 produces a CaM-independent kinase(18) , and an autoinhibitory domain within residues 305-321 has been characterized by site-specific mutagenesis(19) .
It was originally thought that CaM-kinase
IV is activated by autophosphorylation since the enzyme purified from
rat brain and thymus exhibits 10-fold or greater increases in both
total (i.e. assayed in the presence of
Ca/CaM) and Ca
-independent (assayed
in the presence of EGTA) activities upon incubation in the presence of
Ca
/CaM and
ATP/Mg
(2, 20, 21) .
However, recombinant CaM-kinase IV expressed in Escherichia coli(16, 22) or baculovirus/Sf9 cells(18, 19) exhibits very slow and substoichiometric
autophosphorylation associated with only a 2-fold activation even after
incubation for 1-2 h. It has been demonstrated recently that
recombinant CaM-kinase IV can be activated by preincubation with brain
extract in the presence of ATP/Mg
and
Ca
/CaM(16, 19, 22) , and a
66-68-kDa CaM-kinase kinase has been purified from rat
brain(19, 23) . After activation by CaM-kinase kinase,
the ratio of V
/K
for CaM-kinase IV phosphorylation of the transcriptional
activator cAMP-responsive element binding protein is increased
30-fold(7) . Although transfected CaM-kinase IV weakly
stimulates transcription of a reporter gene through phosphorylation of
cAMP-responsive element binding protein, co-transfection of CaM-kinase
IV with the recently cloned CaM-kinase kinase potentiates
transcriptional activation by 14-fold(24) . This is good
evidence in transfected cells for a CaM-kinase cascade involving
CaM-kinase IV, which can regulate transcription.
CaM-kinase IV might
be involved in T cell development since it is more abundant in immature
thymocytes compared with mature thymocytes and circulating T
lymphocytes, and it is not expressed in B lymphocytes or
monocytes(25) . In Jurkat T lymphocytes, both
Ca-independent and total activities of CaM-kinase IV
are maximally increased within 1 min of stimulation of the CD3
receptor(25) . Stimulation of the CD3 receptor is known to
activate multiple cellular signaling systems including protein tyrosine
kinases, phospholipase C, protein kinase C, and mobilization of
IP
-sensitive Ca
stores (see review in (26) ). Therefore, Jurkat cells seem to provide a good system
to study the regulation of CaM-kinase IV in situ. In this
paper, we demonstrate that 1) CaM-kinase IV activated through
CD3-receptor stimulation or in vitro by CaM-kinase kinase can
be inactivated by type 2A protein phosphatase but not by type 1
phosphatase, 2) CaM-kinase kinase purified from rat brain and thymus
activates CaM-kinase IV from unstimulated Jurkat cells, and 3)
ionomycin activates CaM-kinase IV whereas a calmodulin antagonist
blocks activation. We conclude that activation of CaM-kinase IV in
response to CD3 receptor stimulation is mediated by
Ca
/CaM, probably through activation of CaM-kinase
kinase.
For immunoprecipitation of CaM-kinase IV, supernatant was precleared for 1 h with pansorbin coated with rabbit control IgG (Sigma), and the supernatant was then incubated with 9.6 µg of CaM-kinase IV antibody for 1 h at 4 °C. Antigen-antibody complex was precipitated with pansorbin for 30 min on ice. The immunoprecipitates were washed three times with TBS plus 0.3 M NaCl followed by one wash with 50 mM HEPES and 0.1 mM EDTA. Final immunoprecipitates were resuspended in 40 µl of kinase dilution buffer consisting of 50 mM HEPES, pH 7.5, 1 mg/ml bovine serum albumin and 10% ethylene glycol, and 5 µl were used for syntide-2 kinase assay as described below.
For CaM-kinase kinase
treatment of Jurkat cell CaM-kinase IV, 5 µl of immunoprecipitated
CaM-kinase IV was incubated with purified rat brain or thymus
CaM-kinase kinase for 10 min at 30 °C in a 25-µl reaction
mixture containing 50 mM HEPES, 2 mM DTT, 0.2 mg/ml
bovine serum albumin, 2% ethylene glycol, 1 mM
CaCl, 5 µM CaM, 10 mM magnesium
acetate, 0.4 mM ATP, and 1 µM microcystin-LR. The
reaction was terminated with 500 µl of the stop buffer followed by
immediate centrifugation in a microfuge for 5 min at 4 °C. The
CaM-kinase IV immunoprecipitates were resuspended in 25 µl of
kinase dilution buffer. Five microliters were used for kinase assay.
All assays were performed in duplicate.
CaM-kinase IV activity was
measured by P-incorporation into syntide-2 in a 25-µl
reaction mixture containing 50 mM HEPES, pH 7.5, 2 mM DTT, 40 µM syntide-2, 0.2 mM
[
-
P]ATP (500-1,000 cpm/pmol), 10
mM magnesium acetate, 5 µM PKI 5-24, 2
µM PKC 19-36 inhibitor peptides, 1 µM microcystin-LR, and either 1 mM EGTA (for
Ca
/CaM-independent activity) or 0.8 mM CaCl
and 1 µM CaM (for total activity).
The reaction proceeded for 10 min (unless otherwise indicated) at 30
°C and was then terminated by spotting a 15-µl aliquot onto
phosphocellulose P81 paper (Whatman) followed by washing with 75
mM phosphoric acid(29) .
Figure 1:
Activation of Jurkat cell CaM-kinase IV
via CD3 receptor stimulation. A, Jurkat E6-1 cell lysate
supernatants were subjected to Western blot analyses using antibody to
CaM-kinase IV before (lane 1) and after (lane 2)
immunoprecipitation with anti-CaM kinase IV. B, Jurkat cells
(3 10
) were treated for the indicated times with
0.3 µg/ml anti-CD3 (Leu-4) in the presence of 25 µg/ml rabbit
anti-mouse IgG. Cells were centrifuged and lysed, and CaM-kinase IV was
immunoprecipitated with anti-CaM kinase IV. CaM-kinase IV activity was
assayed in the presence (black bars) and absence (gray
bars) of Ca
/CaM (see ``Experimental
Procedures''). C, Jurkat E6-1 and J.RT3-T3.5 cells
were treated with 25 µg/ml rabbit anti-mouse IgG in the presence or
absence of 0.3 µg/ml Leu-4 for 1 min, and CaM-kinase IV activity
was measured in the presence of EGTA (gray bars) or
Ca
/CaM (black bars). Error bars indicate standard deviation from five independent experiments in
duplicate.
When immunoprecipitates from unstimulated Jurkat
cells were assayed for CaM-kinase IV activity, there were very low
total and Ca-independent activities (Fig. 1, B and C). Stimulation with anti-CD3 resulted in
elevated total and Ca
-independent CaM-kinase IV
activities, which were maximal within 1 min of stimulation (Fig. 1B) and at 0.2 µg/ml of anti-CD3 (data not
shown). In CD3-stimulated cells the Ca
-independent
activity was enhanced approximately 20-fold, and the total activity was
enhanced about 8-fold (Fig. 1C, left panel).
The ratio of Ca
-independent to total activity was
increased from <0.2 in unstimulated cells to 0.3-0.4 in
stimulated cells. To demonstrate that CaM-kinase IV was activated
through the T cell receptor (TCR)-CD3 complex, a mutant Jurkat clone
J.RT3-T3.5 (CD3-negative) was also treated with anti-CD3 (Fig. 1C, right panel). This mutant Jurkat
clone lacks the
-subunit of TCR; therefore, no TCR-CD3 complex is
assembled on the cell surface(36) . CaM-kinase IV activity was
not increased after anti-CD3 treatment of this mutant clone, and this
was not due to a lower level of CaM-kinase IV protein in these cells,
since Western immunoblotting revealed a similar level of CaM-kinase IV
in this Jurkat cell line (data not shown). These results demonstrate
that CD3-induced activation of CaM-kinase IV in Jurkat required
signaling through TCR-CD3.
To test this
hypothesis, CaM-kinase IV was immunoprecipitated from both control and
CD3-stimulated Jurkat cells and then incubated with or without purified
rat brain CaM-kinase kinase (19) prior to the assay for
CaM-kinase IV activities. In vitro incubation with CaM-kinase
kinase enhanced both Ca-independent and total
activities of CaM-kinase IV from unstimulated cells by 40- and 8-fold,
respectively (Fig. 2A, Control). This in vitro increase in total CaM-kinase IV activity was about 2-fold greater
than the activation due to CD3-treatment in this particular experiment (Fig. 2A; Stimulated, CaM KK -). When CaM-kinase IV from CD3-stimulated Jurkat cells was
treated in vitro with rat brain CaM-kinase kinase, the final
activity was approximately the same as achieved by the in vitro activation of CaM-kinase IV from control cells. The fact that
CD3-stimulation and subsequent in vitro treatment by
CaM-kinase kinase gave nonadditive activation of CaM-kinase IV suggests
that CD3-activation in the Jurkat cells may use the same mechanism(s)
as activation by CaM-kinase kinase in vitro. Note that there
was little if any activation by in vitro incubation in the
absence of added CaM-kinase kinase, consistent with a lack of Jurkat
CaM-kinase IV activation by autophosphorylation.
Figure 2:
Activation of Jurkat CaM-kinase IV by rat
brain CaM-kinase kinase. Five microliters of CaM-kinase IV
immunoprecipitated from wild-type (panel A) or CD3-negative (panel B) Jurkat cells, which had been treated with 25
µg/ml of rabbit anti-mouse IgG alone (Control) or with 0.3
µg/ml Leu-4 (Stimulated) as described in the Fig. 1legend were preincubated for 10 min at 30 °C with
Ca/CaM and ATP/Mg
in the
absence(-) or presence (+) of purified rat brain CaM-kinase
kinase (0.26 µg/ml). CaM-kinase IV activity was measures as
described under ``Experimental Procedures.'' Dotted bars indicate Ca
/CaM-independent activity, and filled bars indicate total
activity.
CaM-kinase IV from CD3-negative Jurkat cells was also fully activated in vitro by CaM-kinase kinase (Fig. 2B), indicating that the J.RT3-T3.5 cells contained normal CaM-kinase IV and that the inability of CD3-stimulation to activate CaM-kinase IV in these cells is due to lack of proper signaling from the cell surface. Therefore, these results suggest that CaM-kinase IV might be directly activated by CaM-kinase kinase in vivo.
Figure 3:
Inactivation of activated CaM-kinase IV by
protein phosphatase treatment. A, CaM-kinase IV
immunoprecipitated from CD3-stimulated (0.3 µg/ml for 1 min) Jurkat
cells was incubated with PP2A (3.85 milliunits/ml) and PP1C (100
milliunits/ml) in the absence or presence of 0.1 µM calyculin A for 20 min at 30 °C. Reactions were stopped with
0.1 µM calyculin and assayed for syntide-2 kinase
activity. Dotted bars indicate
Ca/CaM-independent activity, and filled bars indicate total activity. Total activity of each
non-phosphatase-treated sample was equated to 100% (average value
= 2.34 ± 0.8 pmol/min). The results show means and
standard deviations from four experiments. B, recombinant
CaM-kinase IV (5 µM) was activated with recombinant rat
brain CaM-kinase kinase expressed in COS cells(24) , and the
samples were incubated with protein phosphatases and assayed for
CaM-kinase IV as in panel A. 100% activity = 8.5
pmol/min. For details see ``Experimental
Procedures.''
Figure 4:
Phosphorylation and activation of
CaM-kinase IV by rat thymus CaM-kinase kinase. A,
Sf9-expressed CaM-kinase IV (0.38 µg) was phosphorylated with
partially purified rat thymus CaM-kinase kinase (0.14 µg) for 60
min at 30 °C in the presence of 1 mM EGTA (lanes
1, 3, and 5) or 4 mM CaCl and 2 µM CaM (lanes 2, 4, and 6). The reactions were stopped by adding 5
SDS-sample
buffer, and the reactions were then separated by 10% SDS-polyacrylamide
gel electrophoresis. Top panel, Coomassie stain. Bottom
panel,
P-incorporation was visualized by
autoradiography. Lanes 1 and 2, CaM-kinase kinase (CaM KK) alone; lanes 3 and 4, CaM-kinase IV
and CaM-kinase kinase; lanes 5 and 6, CaM-kinase IV
alone. B, CaM-kinase IV immunoprecipitated from unstimulated
Jurkat E6-1 cells was incubated without(-) or with (+)
0.96 µg/ml CaM-kinase kinase partially purified from rat thymus in
the presence of Mg
/ATP and Ca
/CaM
as in Fig. 2, and CaM-kinase IV activity was then measured. Open bars indicate Ca
/CaM-independent
activity, and filled bars indicate total activity. The data
represent means and standard errors from four
experiments.
Next, we tested whether thymus CaM-kinase kinase could activate CaM-kinase IV in the immunoprecipitates from unstimulated and CD3-stimulated Jurkat cells. As shown in Fig. 4B, preincubation of CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells with thymus CaM-kinase kinase led to strong activation of CaM-kinase IV.
Figure 5:
Activation of CaM-kinase IV by ionomycin
and inhibition of CD3-mediated activation by calmidazolium. A,
Jurkat cells (3 10
cells/ml) were preincubated for
10 min at 37 °C and then treated with increasing concentrations of
ionomycin for 2 min in duplicate. CaM-kinase IV was immunoprecipitated,
and Ca
/CaM-independent (open circles) and
total activities (closed circles) were measured as described
under ``Experimental Procedures.'' B, Jurkat cells
(2
10
cells/ml) were preincubated with the
indicated amount of calmidazolium for 20 min at 37 °C and then
stimulated with 0.3 µg/ml of Leu-4 for 1 min. CaM-kinase IV was
immunoprecipitated, and total activity was determined as described
above. Two independent experiments in duplicate are shown. 100%
activity = 5.2 pmol/min. *, CaM-kinase IV activity from
unstimulated cells.
If activation of
CaM-kinase IV by phosphorylation in CD3-stimulated Jurkat cells is
mediated by CaM-kinase kinase, then this activation should be blocked
by the calmodulin antagonist calmidazolium because CaM-kinase kinase is
a Ca/CaM-dependent enzyme(19, 24) .
Preincubation of the Jurkat cells with calmidazolium resulted in
complete inhibition of the CD3-mediated activation (Fig. 5B). Since CaM-kinase IV also binds
Ca
/CaM, one cannot conclude that the sole effect of
calmidazolium was on CaM-kinase kinase. It is possible that
phosphorylation of CaM-kinase IV requires binding of
Ca
/CaM to both CaM-kinase kinase and CaM-kinase IV.
However, this seems unlikely since a
Ca
/CaM-independent mutant of CaM-kinase IV still
requires Ca
/CaM for activation by CaM-kinase
kinase(19) , and the activating phosphorylation site in
CaM-kinase IV is located in the catalytic domain(38) , far
removed in the primary sequence from the autoinhibitory/CaM-binding
domains.
There is accumulating evidence suggesting that some members
of the CaM-kinase family, namely CaM-kinase Ia and CaM-kinase IV, are
regulated by upstream protein
kinases(19, 23, 39, 40) . Unlike
CaM-kinase II, whose total activity can be fully stimulated by
Ca/CaM (see review in (41) ), recombinant
CaM-kinase IV has a very low specific activity in the presence of
Ca
/CaM and requires phosphorylation by CaM-kinase
kinase for full activity. CaM-kinase kinases for CaM-kinase Ia (39, 40) and CaM-kinase IV (19, 23) have been purified from pig brain and rat
brain, respectively, and characterized. Both require ATP/Mg
and Ca
/CaM for their activities, but CaM-kinase
Ia kinase is a 52-kDa whereas CaM-kinase IV kinase is a 66-68-kDa
protein. Recently, a 68-kDa rat brain CaM-kinase kinase has been
cloned, and the expressed CaM-kinase kinase can catalyze
Ca
/CaM-dependent activation of both CaM-kinases Ia
and IV but not CaM-kinase II(24) . The recombinant CaM-kinase
kinase increased both Ca
-independent and total
activities of CaM-kinase IV, whereas it enhanced only total activity of
CaM-kinase Ia. Similar results have been obtained with the purified pig
brain CaM-kinase I kinase(38) . The present study was initiated
to determine if a CaM-kinase cascade involving CaM-kinase IV and
CaM-kinase kinase can be demonstrated in cultured cells. Jurkat cells
were chosen because they had previously demonstrated activation of
CaM-kinase IV by CD3 receptor stimulation(25) . We were able to
reproduce their results, and our study provides support for the
hypothesis that CD3 receptor stimulation activates CaM-kinase IV
through phosphorylation by CaM-kinase kinase.
Because there is no
specific assay for CaM-kinase IV, it was important to first establish
that our assay detected only CaM-kinase IV. The most likely
interference in the assay would come from CaM-kinase II since it
exhibits high specific activity for syntide-2 phosphorylation, but such
interference is unlikely since the antibody we used to
immunoprecipitate CaM-kinase IV does not show cross-reactivity with
either CaM-kinase I or II in the Jurkat cell extract (Fig. 1A). Furthermore, CaM-kinase II is not known to
increase its total activity upon phosphorylation, and its increase in
Ca-independent activity upon autophosphorylation is
reversed by both protein phosphatases 1 and 2A (42, 43, 44) .
Our results indicate that
activation of CaM-kinase IV upon CD3-stimulation of Jurkat cells is
mediated by CaM-kinase kinase. CaM-kinase kinase activity is present in
Jurkat cells, and it was purified from thymus (Fig. 4) and shown
to have properties similar to the purified rat brain enzyme. Second,
the activation of CaM-kinase IV by CD3-stimulation of Jurkat cells and
by in vitro activation with CaM-kinase kinase were nonadditive (Fig. 2), indicating that the same or overlapping activation
mechanisms are involved. Third, CaM-kinase IV activated in Jurkat cells
by CD3-stimulation (Fig. 3A) or recombinant CaM-kinase
IV activated in vitro with CaM-kinase kinase (Fig. 3B) was subsequently inactivated in vitro by PP2A but not by PP1. This unusual specificity for PP2A for
dephosphorylation is consistent with the same site being phosphorylated
by CaM-kinase kinase in vitro and by CD3-stimulation in the
Jurkat cells. Last, activation for CaM-kinase IV in Jurkat cells can be
stimulated by Ca mobilization (i.e. ionomycin, Fig. 5A) and blocked by a calmodulin
antagonist (Fig. 5B). These observations support the
hypothesis that CD3 receptor stimulation activates CaM-kinase IV
through an upstream cascade involving CaM-kinase kinase.
The
mechanism by which CD3 stimulation activates CaM-kinase kinase is
probably due to elevation of intracellular Ca(37) . Consistent with this hypothesis, CD3 stimulation does
not appear to generate a stable activation of CaM-kinase kinase, such
as phosphorylation, that survives cell lysis (data not shown).
Furthermore, CD3 stimulation does not fully activate CaM-kinase IV, as
subsequent in vitro phosphorylation by CaM-kinase kinase
results in an additional 2-3-fold increase in total CaM-kinase IV
activity (Fig. 2A). The most likely explanation is that
opposing protein phosphatases limit the extent and duration of
activation since activation is maximal around 1 min and returns to
basal values at 5 min (Fig. 1B). This temporal pattern
would be consistent with high activity of endogenous protein
phosphatases, but we have been unsuccessful in attaining further
activation or prolonging the duration of activation by treatment with
0.1-0.5 µM okadaic acid, a concentration that should
strongly inhibit protein phosphatase 2A. Whereas protein phosphatase 2A
reverses the activation of CaM-kinase IV in vitro (Fig. 3), it is possible that the okadaic acid-insensitive
phosphatase 2C may reverse the activation in Jurkat cells(45) .
In summary, all of our data is consistent with the hypothesis that
CD3 receptor stimulation activates CaM-kinase IV through
phosphorylation by CaM-kinase kinase. Further studies will be required
to determine whether this is simply the consequence of elevated
intracellular Ca or if more complicated signaling
pathways are also involved. The latter possibility would be appealing,
since it is puzzling why two consecutive steps in a kinase cascade
should both be controlled by Ca
/CaM. One possibility
is that activation of CaM-kinase IV by CaM-kinase kinase requires
convergence of signaling pathways in addition to elevated intracellular
Ca
. Another possibility is that activation of
CaM-kinase kinase requires either higher concentrations of
Ca
or a different pool of Ca
than
does simple activation CaM-kinase IV by Ca
/CaM alone.
This would allow selective and transient partial activation of
CaM-kinase IV by Ca
/CaM alone under some
circumstances and stronger activation through phosphorylation by
CaM-kinase kinase in response to other agonists. Furthermore,
activation of CaM-kinase IV by CaM-kinase kinase has the potential for
prolonged activation if the appropriate protein phosphatase activity is
low, since the phosphorylated CaM-kinase IV has significant
Ca
-independent activity. Of course, it is possible
that CaM-kinase kinase might regulate currently unidentified substrates
that themselves are not Ca
/CaM-dependent. Their
phosphorylation by CaM-kinase kinase would make them
Ca
-responsive. These speculative possibilities will
require further experimentation.