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(Received for publication, February 22, 1996) From the
This communication reports the specific induction of calmodulin
kinase IV by the thyroid hormone 3,3`,5-triiodo-L-thyronine
(T
As one of the intracellular second messengers, calcium plays a
central role in cell growth and differentiation(1) . The
primary receptor protein mediating the calcium signal inside the cell
is calmodulin (CaM), ( In comparison to CaMKII, the substrate specificity of
CaMKIV seems to be more restricted. Apart from synapsin I, which is a
substrate for both CaM kinases, the only other substrates reported for
CaMKIV are the Ras-related GTP-binding protein Rap-1b(17) , the
cAMP regulatory element-binding protein CREB(11, 18) ,
the serum response factor SRF (19) , and members of the Ets
family of transcription factors(20, 21) . The recent
report of a substantial localization of CaMKIV in the nucleus (18, 22) permits the enzyme direct access to these
transcription factors to regulate their function in a
Ca Since thyroid hormones have been shown to be required for normal
growth and differentiation of the mammalian
brain(26, 27, 28) , we initiated studies to
investigate the influence of the thyroid hormone
3,3`,5-triiodo-L-thyronine (T
Aggregating cell cultures prepared from 15-day fetal rat
telencephalon and grown for 5 days in the presence or absence of the
thyroid hormone 3,3`,5-triiodo-L-thyronine (T
Figure 1:
Identification of CaMKIV in fetal rat
telencephalon cell culture extracts grown in the presence or absence of
T
Since the apparent molecular
weight and autophosphorylation kinetics showed similarities between p64
and CaMKIV we attempted to purify p64 from a pool of aggregate cultures
grown for 5 days in the presence of 3
Figure 2:
Purification of p64 and identification as
CaMKIV by specific antibodies (A) and by autophosphorylation (B). A, protein extracts of fetal rat telencephalon
cell cultures grown in the absence (lane 1) or presence (lane 2) of 3
It was noted that autophosphorylation
of pure p64 was rather slow, as described by Cruzalegui and Means (11) for a recombinant CaMKIV. On the other hand, slow
phosphorylation of p64 could also be indicative for the presence of
contaminating amounts of the recently described CaMKIV
kinase(13, 14, 15, 16) . The other
CaM-binding proteins exhibiting M The influence of T
Figure 3:
Figure 4:
Figure 5:
A further observation supporting the view that the
expression of CaMKIV was a T
Figure 6:
Recent reports suggested that CaMKIV is responsible for the
Ca
Volume 271,
Number 19,
Issue of May 10, 1996 pp. 11055-11058
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) in a time- and concentration-dependent manner at a very
early stage of brain differentiation using a fetal rat telencephalon
primary cell culture system, which can grow and differentiate under
chemically defined conditions. The induction of the enzyme that can be
observed both on the mRNA and on the protein level is
T-specific, i.e. it cannot be induced by retinoic
acid or reverse T, and can be inhibited on both the
transcriptional and the translational level by adding to the culture
medium actinomycin D or cycloheximide, respectively. The earliest
detection of calmodulin kinase IV in the fetal brain tissue of the rat
is at days E16/E17, both on the mRNA as well as on the protein level.
This is the first report in which a second messenger-dependent kinase
involved in the control of cell regulatory processes is itself
controlled by a primary messenger, the thyroid hormone.
)which regulates, among others,
protein kinases and protein phosphatase(s). Next to the well studied
multifunctional CaMKII (see (2) for a recent review), CaMKIV
recently received considerable attention. In contrast to the ubiquitous
CaMKII, CaMKIV is more restricted in its expression in different
tissues. The highest levels of the enzyme in mammalian tissues can be
found in brain, thymus, and, to a somewhat lower extent, in testis and
spleen, whereas in other tissues the enzyme remained undetectable (3, 4, 5, 6, 7) . The amino
acid sequence of CaMKIV, which exists in two monomeric isoforms of M
65,000 (
) and M 67,000
(
) due to alternative splicing, has been deduced from rat, mouse,
and human brain
cDNAs(4, 5, 8, 9, 10) ,
demonstrating less than 50% homology to the corresponding regions of
CaMKII. Next to the rather poor sequence homology between the two CaM
kinases, CaMKII and IV, the two enzymes also seem to differ in their
activation mechanism. Whereas CaMKII can be efficiently activated by
autophosphorylation(2) , the activation of CaMKIV by
autophosphorylation is rather slow and
inefficient(11, 12) . Recent reports seem to indicate
that CaMKIV is activated by a CaMKIV kinase (13, 14, 15, 16) , reminiscent of
the regulation of mitogen-activated protein kinase activity by a kinase
cascade.
-dependent manner. Thus, it has been reported that
CaMKIV is involved in the Ca-dependent regulation of
expression of immediate early genes either through CREB (Refs. 12, 18,
23, and 24; see also (25) ) or through SRF(19) . ) on the expression
of a number of neuronal and glial membrane markers during development
in rat brain cell cultures(29, 30, 31) . Here
we report the specific induction of CaMKIV by T in a time-
and concentration-dependent manner at a very early stage of brain
differentiation using a fetal rat telencephalon primary cell culture
system, which can grow and differentiate under chemically defined
conditions(32) . The induction is T-specific, i.e. the expression of the enzyme cannot be induced by either
reverse T or retinoic acid. The expression of CaMKIV is
regulated on both the transcriptional and the translational level,
since both the addition of actinomycin D as well as cycloheximide to
the cultural medium can prevent the T-dependent induction
of the enzyme. In addition, the T-specific induction can be
observed both on the mRNA and on the protein level. This is the first
report in which a second messenger-dependent kinase involved in the
control of cell regulatory processes is itself controlled by a primary
messenger, the thyroid hormone. Preliminary accounts of part of the
data presented here have been given
elsewhere(33, 34) .
Cell Culture
Serum-free, rotation-mediated
aggregating cell cultures were prepared from fetal (15 days of
gestation) rat (OFA/Ico/Ibm strain, Biological Research Laboratories
Ltd., Basel, Switzerland) telencephalon, as described in detail
previously(32) . Aliquots of 6 
10
cells
were transferred to DeLong flasks and maintained under constant
gyratory agitation at 37 °C, in an atmosphere of 10% CO
and 90% humidified air. The culture medium used was
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.), supplemented with nutritional factors, vitamins, trace elements,
transferrin (1 mg/liter), insulin (800 nM), and hydrocortisone
21-phosphate (20 nM). Gentamicin sulfate (25 mg/liter) was
used as an antibiotic. For analyses, the aggregates of each flask were
washed twice with 5 ml of phosphate-buffered saline (PBS).Preparation of Cell Extracts
Proteins were
extracted, usually from the pellet of whole aggregates (4-5
10 cells), by adding 200 µl of 25 mM HEPES, pH 7.5, 5 mM EGTA, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptine, 1 mML-1-tosylamido-2-phenylethyl chloromethyl ketone, 1
mMN
-p-tosyl-L-lysine
chloromethyl ketone, 1,10-phenanthroline, 1 mMp-aminobenzamidine (buffer A), and incubated on ice for
10 min after extensive vortexing. After centrifugation at 25,000
g for 5 min, proteins in the supernatant were stored
at -70 °C.SDS-PAGE and Electrophoretic Blotting
To 40 µl
of supernatants of cellular extracts, 20 µl of loading buffer (35) was added and the proteins were separated on a 10%
SDS-PAGE(35) . After electrophoretic transfer onto
nitrocellulose sheets for 2 h using 240 mM glycine, 30 mM Tris, 20% methanol, and 0.02% SDS as transfer buffer, the blots
were incubated overnight at 4 °C with 2% milk powder in PBS as
blocking buffer, afterward incubated at room temperature for 3 h with 
I-CaM (36) (2 10
cpm/ml) in
2% milk powder/PBS containing either 1 mM CaCl or
5 mM EGTA, washed once with 2% milk powder/PBS containing
either CaCl or EGTA, then with PBS containing either
CaCl or EGTA, and third with PBS. Each washing was done for
15 min. After drying the blots under cold air, the sheets were exposed
either to x-ray sensitive films (Kodak XAR-5) at -70 °C or to
PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA) at room
temperature and developed. Quantitation was obtained using the
PhosphorImager software as recommended by the manufacturers. CaM was
purified from bovine brain as described previously(37) .Preparation and Characterization of Total RNA
For
RNA preparation, cell culture aggregates (6-8 10
cells) were grown for 5 days either in the absence or in the
presence of 3 10
M T,
washed three times with PBS, and quickly frozen in liquid nitrogen.
Total RNA was isolated by using the Ultraspec RNA kit (Biotecx
Laboratories, Inc., Houston, TX) as described by the manufacturer. For
the identification of CaMKIV-specific mRNA, 40 µg of total RNA was
denatured and size-fractionated on a 1.5% agarose, 2.2 M formaldehyde gel. Electrophoresis was carried out at 30 V for 16 h
with circulating NaPO
buffer at 4 °C. Prior
to blotting the gels were stained with ethidium bromide to identify 18
and 28 S rRNA as markers. The RNA was transferred onto Biotrans nylon
membranes (ICN Biomedicals, Inc., Costa Mesa, CA) and stabilized by
using UV cross-linking. Northern blots were prehybridized and
hybridized at 37 °C in 50% formamide as described
previously(38) . The probe was a 1-kilobase pair (BamHI-KpnI) fragment of the rat CaMKIV cDNA (4) and was labeled with [-
P]dCTP
according to the random oligonucleotide priming method of Feinberg and
Vogelstein(39) .Purification and Characterization of
p64
Aggregates of a total of 20 culture flasks (
10
cells), grown for 5 days in the presence of T (3
10M), have been homogenized at 4
°C in three volumes of ice-cold buffer A, basically following the
purification protocol for CaMKIV described in (40) . Briefly,
the lysates were centrifuged at 100,000 g for 30 min,
and the supernatants were pooled and applied to a 15-ml DEAE-cellulose
column equilibrated with 25 mM Hepes, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, and 1 mM DTT (buffer B). The column was washed with
10 bed volumes of buffer B, and bound proteins were eluted with a
stepwise gradient using buffer B and increments of 100 mM NaCl
at each step. p64 eluted between 150 and 300 mM NaCl. The
fractions containing p64 were pooled and applied onto a prepacked 1-ml
hydroxylapatite column from Bio-Rad equilibrated with buffer B
containing 200 mM NaCl (buffer C). The column was washed with
five bed volumes of buffer C, and bound proteins were eluted with a
linear 0-200 mM NaHPO gradient.
p64-containing fractions eluting between 75 and 100 mM NaHPO were pooled, dialyzed overnight
against 5 liters of 25 mM Hepes, pH 7.5, 2 mM CaCl, 2 mM MgCl, 1 mM DTT (buffer D), and applied to a 5-ml CaM column (36) equilibrated with buffer D. After washing the column with
10 bed volumes of buffer D containing 10% glycerol (buffer E), followed
by a wash of five bed volumes with buffer D containing 0.5 M NaCl, p64 was eluted by a buffer containing 25 mM Hepes,
pH 7.5, 2 mM EDTA, 5 mM EGTA, 1 mM DTT, 10%
glycerol. Purified p64 was identified as a calmodulin-dependent kinase
by autophosphorylation using the assay as described by Cruzalegui and
Means(11) . Autophosphorylated CaMKII and were
identified by specific antibodies, kindly provided by Dr. H. Schulman.
Identification of p64 as CaMKIV by specific antibodies was carried out
by Western blotting using 2% milk powder/PBS as blocking buffer.
) in
a chemically defined medium were collected and extracted with an
EGTA-containing buffer. The extracted proteins were separated by
SDS-polyacrylamide gel electrophoresis and electrophoretically
transferred onto nitrocellulose, and CaM-binding proteins were
identified by incubation with I-labeled CaM in the
presence or absence of Ca. As shown in Fig. 1A, a number of different CaM-binding proteins
were present in the M range between 40,000 and
160,000, which could not be detected in the presence of EGTA (data not
shown). By comparing cultures that were grown for 5 days either in the
presence or absence of 3 10M T, it was obvious that one CaM-binding protein with a M of 64,000 (later identified as CaMKIV; see
below) appeared only in cultures grown in the presence of
T, suggesting that its expression depended on this hormone.
This result could be corroborated by the observation that in Northern
blots of total RNA isolated from similar cell cultures a mRNA of 2
kilobases could be clearly identified by a probe specific for CaMKIV (Fig. 1B). This band could only be observed in the
presence of T, but not in its absence (even at higher RNA
concentrations; data not shown). On the other hand, a band of 3.5
kilobases, which has been described before as a much less abundant mRNA
of CaMKIV in adult rat brain(3, 4) , could be observed
as a weak band even in the absence of T-induced cultures.
The difference between the two mRNAs could derive from differences in
the 3`-untranslated region by use of alternate poly(A) sites as
discussed before(4) , but the reason for the apparent
difference in the T-dependent expression between the two
mRNAs needs further investigation.
. Soluble proteins (A) or total RNA (B)
extracted from cultures grown for 5 days in the presence (+) or
absence(-) of 3 10M T were separated on either SDS-PAGE (A) or agarose gels (B), and after electroblotting onto appropriate membranes
identified by either I-CaM (A) or by a
CaMKIV-specific cDNA probe (B), as described in detail under
``Experimental Procedures.'' Molecular size standards (in
kDa) are indicated at the left of panel A; the arrow denotes the location of CaMKIV (p64). The numbers on the right of panel B (right panel)
indicate the calculated sizes of the hybridizing bands. Migration of
nucleic acid size standards are shown at the left of panel
B (left panel), which shows the ethidium bromide staining
of the same gel prior to blotting. 28S and 18S mark
the levels of the corresponding ribosomal RNA
species.
10M T by applying a modification of the
purification protocol of Hanissian et al. ((40) ; see
also ``Experimental Procedures''). As shown in Fig. 2,
p64 eluted from the DEAE-cellulose column at a concentration of NaCl
between 200 and 300 mM, typical for CaMKIV. Further
purification was obtained by using a hydroxylapatite column, and
finally a CaM affinity column. After extensive washing with a
calcium-containing buffer, p64 was eluted using an EGTA-containing
buffer (Fig. 2). The purified protein was identified as a
CaM-dependent kinase by CaM-dependent autophosphorylation (Fig. 2B) and as CaMKIV by specific antibodies (Fig. 2A). Using immunoprecipitation CaMKIV could be
identified only in cells that had been grown in the presence of T (data not shown). With respect to its M of
64,000-65,000 and its prenatal appearance, p64 most likely
represents the -isoform of CaMKIV since the -polypeptide has
a slightly higher M, i.e. 67,000, is
specifically expressed in brain only in cerebellum, and can be observed
only postnatally(6) .
10M T were separated by SDS-PAGE, blotted onto nitrocellulose, and
incubated with I-CaM as described under
``Experimental Procedures.'' Lane 3, aliquot of the
starting material for p64 purification. Lanes 4-6,
fractions from the DEAE-cellulose eluate containing 100 mM NaCl (lane 4), 200 mM NaCl (lane 5),
and 300 mM NaCl (lane 6). p64 was purified to
homogeneity by hydroxylapatite column and CaM affinity chromatography (lane 7) and identified as CaMKIV by specific antibodies (lane 8). B, identification of p64 as a CaM kinase by
autophosphorylation. Purified p64 and CaMKII and were
autophosphorylated by using [P-
]ATP in a
Ca/CaM-dependent manner as described in (11) and separated by SDS-PAGE (lane 3).
Identification of p64 in fetal rat telencephalon cell cultures grown in
the absence (lane 1) or presence (lane 2) of 3
10M T. p64 is
indicated by the arrow. Molecular size standards (in kDa) are
indicated at the left of panels A and B;
CaMKIV and CaMKII and are indicated at the right of panel B.
values between
60,000 and 63,000 were identified as CaMKII and calcineurin A,
respectively, by using monoclonal antibodies against CaMKII and by
comparison with purified calcineurin. It appeared that in contrast to
CaMKIV, the expression of both CaMKII and calcineurin was independent
of T (data not shown). was studied further in a dose- and time-dependent expression of
CaMKIV. As shown in Fig. 3, the enzyme was detectable already at
very low concentrations of T (3 10
M; Fig. 3) and increased in intensity with
increasing concentrations of T in the culture medium,
indicating that the induction of CaMKIV by T was
dose-dependent. In addition, when cultures received T (3
10M) for various lengths of time,
CaMKIV was already detectable after 6 h of stimulation (Fig. 4),
and the amount of CaMKIV increased as a function of the duration of the
stimulus until maximal expression was reached between 24 and 48 h,
suggesting that protein synthesis played a role in the induction of
this gene. This interpretation was corroborated by incubating the cell
cultures with either actinomycin D or cycloheximide, respectively, to
prevent transcription or protein synthesis of the inducible gene. As
can be seen from Fig. 5, CaMKIV was clearly induced after
exposure to 3 10M T for 24 h (Fig. 5, lane 3; see also Fig. 4, lane 4), but the protein was not detectable if the cultures
had been preincubated for 1 h with either 1 µM actinomycin
D (Fig. 5, lane 5) or 5 µM cycloheximide (Fig. 5, lane 4), respectively, before T was added. This is in contrast to the other detectable
calmodulin-binding proteins, which during the time period of
observation (i.e. 24 h) were independent of transcription or
translation.
I-CaM overlay of extracts of
fetal rat telencephalon cell cultures grown in the absence (lanes 1 and 5, control) or in the presence of 3
10M T (lane 2), 3
10
M T, (lane
3), and 3 10M T (lane 4). The arrow indicates
CaMKIV.
I-CaM overlay of extracts of
fetal rat telencephalon cell cultures grown in the absence (lane
1) or in the presence of 3 10M T. The cell cultures were harvested after 6 h (lane 2), 12 h (lane 3), 24 h (lane 4), or
48 h (lane 5). The arrow identifies
CaMKIV.
I-CaM overlay of extracts of
cell cultures grown in the presence of either cycloheximide or
actinomycin D. The proteins of the cell extracts were separated by
SDS-PAGE and blotted onto nitrocellulose as described under
``Experimental Procedures.'' Cell cultures were
grown for 5 days (lanes 1 and 2) either in the
absence (lane 1) or in the presence of 3
10 T (lane 2), for 24 h in the
presence of 3 10 T (lane
3, control) or preincubated for 1 h with either 5 µM cycloheximide (lane 4) or 1 µM actinomycin D (lane 5) prior to the incubation with 3
10 T for 24 h. Molecular size standards
(in kDa) are indicated at the left; the arrow denotes
the location of CaMKIV.
-specific process was the
finding that next to T only T was able to
induce CaMKIV (Fig. 6, lanes 2 and 3). On the
other hand, neither reverse T nor retinoic acid could
induce the expression of CaMKIV (Fig. 6, lanes 4 and 5). In addition, neither nerve growth factor nor epidermal
growth factor were able to induce the expression of the kinase (data
not shown), although both growth factors have been demonstrated to
regulate developmental processes in these
cultures(41, 42) . Also, CaMKIV may be specifically
expressed in neurons, since it was found at high levels in
neuron-enriched aggregate cultures (data not shown), in which highly
proliferating glial cells have been suppressed by the addition of
1--D-arabino-furanosylcytosine (Ara-C)(31) .
I-CaM overlay of extracts of
cell cultures grown in the presence of different hormones. The proteins
of the cell extracts were separated by SDS-PAGE and blotted onto
nitrocellulose as described under ``Experimental
Procedures.'' Cell cultures were grown for 5 days either in the
absence of T (lane 1) or in the presence (3
10M) of either T (lane 2), T (lane 3), reverse
T (lane 4), or retinoic acid (lane 5).
Molecular size standards (in kDa) are indicated at the left;
the arrow denotes the location of
CaMKIV.
-dependent regulation of expression of a number of
immediate early genes such as c-fos, due to the
phosphorylation of the cAMP-responsive element-binding protein (CREB) (12, 18, 23, 25) or the serum
response factor SRF(19) . Our results indicate that during rat
brain development the expression of CaMKIV, not detectable at the early
stages of ontogenesis (i.e. at E15; data not shown) is
regulated by the thyroid hormone in a time- and concentration-dependent
manner. Whether this T-dependent regulation is due to a
direct interaction of the T-receptor with a responsive
element of the CaMKIV gene (28) or whether the effect is
indirect remains to be determined, but since the T-specific
induction of CaMKIV could also be observed on the mRNA level, this
observation could be indicative for a T-receptor-dependent
regulation. In this respect it is of interest that in a recent abstract (43) , it was reported that in a mouse embryonic stem
cell-derived neuronal culture system the expression of CaMKIV was
strictly dependent on the presence of the thyroid hormone receptor. In
addition, it should be noted that Shakagami et al.(7) observed a rather late appearance of the mRNA of
CaMKIV during rat embryonal development, between days 15 and 18 of
gestation, i.e. at about the onset of the embryonal synthesis
of the thyroid hormone(44) . These findings strongly
corroborate our observation that the presence of the thyroid hormone is
essential for the expression of CaMKIV. It will be now of interest
whether the expression of the CaMKIV kinase is also regulated during
brain development and how it correlates with the expression of CaMKIV.
In addition, it will be important to know whether in the immune system
and in testis CaMKIV is also under the control of the thyroid hormone
or whether different signals regulate the expression. Preliminary
results indicate that the synthesis of CaMKIV in the rat embryonal
thymus is induced following the same time course as in the embryonal
brain. ()
)-CaM-dependent protein kinase II; CaMKIV,
Ca-CaM-dependent protein kinase IV or Gr; CREB, cAMP
response element-binding protein; DTT, dithiothreitol; PAGE,
polyacrylamide gel electrophoresis; SRF, serum response factor;
T, 3,3`,5-triiodo-L-thyronine; PBS,
phosphate-buffered saline.)
We are indebted to Dr. H. Schulman (Stanford
University, Stanford, CA) for kindly providing specific antibodies
against CaMKII and , and to Dr. C. B. Klee (National
Institutes of Health, Bethesda, MD) for kindly providing calcineurin.
We thank Dr. Peter James for critical reading of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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