ABSTRACT
Recent studies have demonstrated that
Ca
/calmodulin-dependent protein kinase IV (CaM-kinase
IV) can mediate Ca
-dependent regulation of gene
expression through the phosphorylation of transcriptional activating
proteins. We have previously identified and purified a 68-kDa rat brain
CaM-kinase kinase that phosphorylates and increases total and
Ca
-independent activities of CaM-kinase IV
(Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and
Soderling, T. R.(1994) J. Biol. Chem. 269, 28640-28647).
Using a partial amino acid sequence of the purified brain kinase, a
CaM-kinase kinase cDNA was cloned from a rat brain cDNA library.
Northern blot analysis showed that CaM-kinase kinase mRNA (3.4
kilobases) was expressed in rat brain, thymus, and spleen. Sequence
analyses revealed that the cDNA encoded a 505-amino acid protein, which
contained consensus protein kinase motifs and was 30-40%
homologous with members of the CaM-kinase family. Expression of the
cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein,
which catalyzed in vitro activation in the presence of
Mg
/ATP and Ca
/CaM of CaM-kinases I
and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase
with CaM-kinase IV gave a 14-fold enhancement of cAMP-response
element-binding protein-dependent gene expression compared with
CaM-kinase IV alone. These results are consistent with the hypothesis
that CaM-kinases I and IV are regulated through a unique signal
transduction cascade involving CaM-kinase kinase.
The Ca
/calmodulin-dependent protein kinase
family (CaM-kinases I-V) (
)is involved in many
cellular responses that are triggered by elevated intracellular
Ca
concentration(1) . CaM-kinase IV was first
identified as a multifunctional Ser/Thr protein kinase that has two
monomeric isoforms;
(63 kDa) and
(67 kDa)(2) .
Although several in vitro substrates for CaM-kinase IV have
been reported(3, 4) , the physiological function(s) of
CaM-kinase IV remains to be established. Three groups have reported
that CaM-kinase IV is involved in Ca
-dependent
transcriptional activation through the phosphorylation of cAMP-response
element-binding protein (CREB) (5, 6, 7) and
of the serum response factor(4, 32) , and this would
be consistent with its nuclear localization(8) . However, the
specific activity of recombinant CaM-kinase IV for phosphorylation of
CREB or other substrates is about 20-fold lower than that of other
protein kinases(5) . This observation prompted studies of
mechanisms, in addition to Ca
/CaM, for activating
CaM-kinase IV.
The original studies on purified brain CaM-kinase IV
indicated that its autophosphorylation results in 20-fold increases in
Ca
-independent and total kinase
activities(9) . However, since recombinant CaM-kinase IV
exhibits very slow autophosphorylation, which at best increases total
activity only 2-fold(4, 10) , this suggested that the
purified brain CaM-kinase IV may contain a contaminating activator
protein. Indeed, it has been reported that rat brain extract contains a
protein, which increases the total activity of bacterially expressed
CaM-kinase IV(11) . More recently, we (10) and another
group (12) purified a 66-68-kDa protein from rat brain,
which in the presence of Mg
/ATP and
Ca
/CaM gives time-dependent increases in total and
Ca
/CaM-independent activities of recombinant
CaM-kinase IV. This activation of CaM-kinase IV correlates with
P incorporation and could be reversed by subsequent
treatment with protein phosphatase 2A(10) , so the activator
protein is referred to as CaM-kinase kinase. CaM-kinase IV, which has
been activated by CaM-kinase kinase, exhibits kinetics for the in
vitro phosphorylation of Ser
in CREB that are
comparable with CREB phosphorylation by cAMP-kinase(13) . These
results suggest that CaM-kinase IV may be involved in transcriptional
regulation through phosphorylation of CREB and perhaps other
transcriptional activators as part of a unique kinase cascade pathway
involving CaM-kinase kinase. Interestingly, a 52-kDa activating factor,
which is probably a protein kinase, for CaM-kinase I has been highly
purified from rat brain(14) . We therefore set out to clone and
characterize the 68-kDa CaM-kinase kinase and to test its specificity
for activating CaM-kinases.
EXPERIMENTAL PROCEDURES
Materials
CaM-kinase IV kinase was purified from
rat brain as described previously(10) . Recombinant wild type
and double mutant (F316D,N317D) of CaM-kinase IVs and
CaM-kinase
II were expressed in Sf9 cells and purified as described
previously(10, 15) . Recombinant CaM-kinase I, which
was expressed in Sf9 cells and purified, was kindly provided by Drs. P.
Sun and R. A. Maurer (Oregon Health Sciences University). Rat brain
gt10 cDNA libraries were provided by Dr. A. Yamakawa (Dana Farber
Cancer Institute). Calmodulin was purified from bovine brain.
Protein Sequencing of CaM-kinase Kinase
Purified
rat brain CaM-kinase kinase (10) was separated by 7.5% SDS-PAGE
and electrotransferred onto PVDF membrane. After staining with Amido
Black, the protein band (100 pmol) was excised and digested with
trypsin (2 µg). Digested peptides were separated by C18 reverse
phase chromatography (Applied Biosystems 130A high pressure liquid
chromatography) and subjected to automatic gas-phase protein sequencing
(Applied Biosystems 470A equipped with 120A PTH analyzer).
Cloning of CaM-kinase Kinase cDNA
Two degenerate
primers corresponding to tryptic peptide 1 (IADFGVSNQFEGNDAQLSST) of rat brain CaM-kinase kinase
were synthesized: sense primers, 5`AT(TCA)GC(TCAG)GA(TC)TT(TC)GG3`
based on IADFG, and antisense primers,
5`A(AG)(TC)TG(TCAG)GC(AG)TC(AG)TT3` based on NDAQL. Forty cycles of PCR
amplification were performed using rat brain random-primed,
first-stranded cDNA as a template. The 50-bp PCR product was cloned
into pBluescript, confirmed by sequencing, and used as the authentic
probe. About 4.8
10
plaques from a rat brain
oligo(dT)-primed cDNA library in
gt10 were screened with the 50-bp
probe labeled with [
-
P]dCTP. The 5` end EcoRI-StuI 0.3-kbp fragment of clone 7AL was used as
a hybridization probe for screening a rat brain random-primed
gt10
cDNA library (4.7
10
plaques) to obtain clone R5.
Amplification of CaM-kinase Kinase cDNA 5` End
Rat
brain mRNA (0.94 µg) was reverse transcribed with 10 pmol of a
gene-specific primer (5`AGCATCATTCCCCTCAAACTGGTT3`) using the
Invitrogen Fast Track cDNA kit. After removing excess primer by
Centricon 100, the tailing reaction was carried out with 15 units of
terminal deoxytransferase (Life Technologies, Inc.) and dCTP (0.2
mM). The PCR reaction was performed using poly(dC)-tailed
first stranded DNA, a nested primer 1 (5`TGCTGACACCAAAGTCGGCGATCTT3`,
0.5 µM), and an anchor primer
(5`ATCGAATTCCGGGIIGGGIIGGGIIG3`, 0.5 µM) under the
following conditions: 1st cycle, 94 °C for 4 min, 42 °C for 2
min, 72 °C for 3 min; 2nd to 36th cycles, 94 °C for 1 min, 52
°C for 1 min, 72 °C for 2 min. After digestion with XhoI and EcoRI, the PCR fragment was subcloned into
pBluescript. Another set of PCR reactions was performed using a nested
primer 2 (5`GCGAGGAAAGCCATACT3`, 0.5 µM) and an anchor
primer under conditions as described above. The PCR product was
digested with XbaI and EcoRI and then subcloned into
pBluescript. We sequenced both strands of cDNAs using a Sequenase
version 2 kit (U. S. Biochemical Corp.). Sequence data are being
submitted to GenBank.
Northern Blot Analysis
Total cellular RNA from rat
tissues was isolated by the guanidine thiocyanate method, and rat brain
poly(A
) RNA was selected by an oligo(dT)-cellulose
column. The total RNAs (30 µg) were electrophoresed on a
formaldehyde-containing 1% agarose gel and then blotted onto Hybond-N
(Amersham Corp.). The blot was hybridized in rapid hybridization buffer
(Amersham Corp.) with a [
-
P]dCTP random
primed XbaI-XhoI fragment (540 bp) from P32 clone as
a probe and washed in 0.2
SSC, 0.1% SDS at 65 °C. The
blotted membrane was dried and autoradiographed.
Transfection of COS Cells
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium containing
10% fetal calf serum. Cells were subcultured in 10-cm dishes 12 h
before transfection. The cells were then transferred to serum-free
medium and treated with a mixture of either 20 µg of pME18s plasmid
DNA (DNAX Research Institute) (mock transfection) or CaM-kinase kinase
full-length cDNA containing plasmid DNA (20 µg, pMECaMKK) and 80
µg of LipofectAMINE Reagent (Life Technologies, Inc.) in 10 ml of
medium. After a 20-h incubation, the cells were collected and
homogenized with 1 ml of lysis buffer (50 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.1
mM PMSF, 1 mM benzamidine, 5 mg/liter leupeptin, and
5 mg/liter pepstatin A) using a Potter-Elvehjem homogenizer at 4
°C.
CaM-kinase Activation
The kinase activation
reaction contained 5.7 µM CaM-kinase IV (wild-type or
double mutant(10) ) or CaM-kinase IV buffer (control blank) and
50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1
mM dithiothreitol, 0.4 mM ATP, and 1 µM
microcystin-LR plus EGTA, Ca
, and/or CaM as indicated
in Fig. 3legend. CaM-kinases I or II were used at 2
µM. The activation reactions were terminated by dilution
in buffer containing EDTA, and kinase activity (46 nM of
CaM-kinase IV or 15 nM of either CaM-kinases I or II) was
determined at 30 °C for 1-5 min using standard assay
conditions as described previously (10) with 40 µM syntide-2 as substrate and either 1 mM EGTA
(Ca
-independent activity) or 1 mM CaCl
plus 1 µM CaM (total activity).
Figure 3:
Expression of CaM-kinase kinase and its
activation of CaM-kinase IV. A, COS-7 cells were transfected
(see ``Experimental Procedures'') with plasmid encoding
CaM-kinase kinase (CaMKK) or plasmid alone (Mock),
the cells were lysed, and lysate (6 µg) was separated by 10%
SDS-PAGE and electrotransferred onto PVDF membrane. The membrane was
subjected to Western blotting using CaM-kinase II peptide 132-146
antiserum (1/500 dilution, leftpanel) or to a
biotinylated CaM (0.5 µg/ml) overlay (rightpanel). Molecular mass markers are shown to the left. B, Sf9 cell-expressed and purified wild-type or
double mutant (F316D,N317D (10) ) CaM-kinase IV or kinase
buffer (K-buffer) was incubated at 30 °C for 5 min with
either the lysate buffer (Buffer) or lysate from COS-7 cells
(180 ng) transfected with plasmid (Mock) or plasmid-expressing
CaM-kinase kinase (CaMKK) in a kinase activation reaction
(see ``Experimental Procedures'') with either 1 mM
EGTA, 1 mM CaCl
, or 10 µM CaM, as
indicated. After terminating the activation, CaM-kinase IV activity was
measured using 40 µM syntide-2 in the presence of either 1
mM EGTA (Ca
-independent activity, openbar) or 1 mM CaCl
plus 1 µM CaM (total activity, closedbar) under standard
assay conditions. Kinase activity toward syntide-2 of the lysate from
COS-7 cells transfected with CaM-kinase kinase was also measured in the
absence of exogenous CaM-kinase IV under the same conditions, but it
was negligible (rightsixlanes). The mean
± S.E. of three experiments using three independent
transfections is shown.
Transcriptional Activation
COS-7 cells (8
10
cells/10-cm dishes) were transfected with 2 µg of
GAL4 or a GAL4-CREB
fusion protein (16) and 5 µg of a reporter plasmid (5) and a
combination of plasmids (pME18 s) encoding CaM-kinase IV (1 µg)
and/or CaM-kinase kinase (5 µg). After incubation for 36 h, the
cells were treated with 5 µM ionomycin and 10 mM
CaCl
for 2 h. The cells were collected and lysed, and
luciferase activity was measured by the luciferase assay kit (Promega).
Luciferase activity for the mock transfected cells has been subtracted
as a blank, and transfection with GAL4 without the CREB fusion protein
gave insignificant luciferase activities.
Others
Western blotting was performed using
anti-CaM-kinase II peptide(132-146) antiserum (1/500 dilution),
and immunoreactive protein was visualized by chemiluminescence (DuPont
NEN). Biotinylated CaM overlay was carried out as described
previously(10) .
RESULTS AND DISCUSSION
Isolation and Analysis of cDNA Clones Coding for
CaM-kinase Kinase
In order to clone CaM-kinase kinase cDNA, we
purified the 68-kDa rat brain enzyme and obtained an amino acid
sequence from two of its tryptic peptides. The amino acid sequence of
tryptic peptide 1 (IADFGVSNQFEGNDAQLSST) confirmed that this
protein contained a motif (DFG) that is conserved among the protein
kinase family. The PCR-amplified DNA fragment (50 bp) coding for
peptide 1 was prepared using degenerate oligonucleotide primers (see
``Experimental Procedures''), and this 50-bp fragment was
used as a probe to screen a rat brain oligo(dT)-primed cDNA library,
resulting in isolation of a 2.7-kbp clone 7AL (Fig. 1A). The 7AL clone contained sequences for
peptide 1, other highly conserved protein kinase motifs, a TGA stop
codon, and a 3` noncoding sequence including a poly(A) tail but no
sequence for peptide 2. Screening of a rat brain random primed
gt10 cDNA library yielded a 2.9-kbp clone (R5), which contained
the sequence for peptide 2 (Fig. 1B) but no consensus
ATP-binding motif (GXGXX(G/S)). To
complete the 5` sequence we used 5`-RACE PCR to obtain three
overlapping clones (P32, P122, and P22). Clones P32 and P122 contained
the ATP-binding motif (
GKGAYG), and clone P22 had an
initiation ATG that was consistent with a Kozak consensus
sequence(17) . These overlapping clones gave an open reading
frame of 1515 nucleotides coding for a protein of 505 amino acids with
a calculated molecular mass of 56 kDa (Fig. 1B).
Figure 1:
Cloning and tissue mRNA
analyses of rat brain CaM-kinase kinase. A, clone 7AL was
isolated from oligo(dT)-primed rat brain library using a PCR product
encoding tryptic peptide 1 of purified rat brain CaM-kinase kinase, and
clone R5 was obtained from a random-primed rat brain cDNA library in
gt10 using the 5` end EcoRI-StuI fragment of
clone 7AL as the probe. Clones P32, P122, and P22 were obtained as
5`-RACE PCR products (see ``Experimental Procedures'' for
cloning details). B, the nucleotide and deduced amino acid
sequences are shown for CaM-kinase kinase with the two tryptic peptides
(peptide 1 and peptide 2) obtained from purified rat brain CaM-kinase
kinase underlined; the ATP-binding site is indicated by a dashedunderline. Only part of the 3` noncoding
sequence is shown. C, total RNA (30 µg) from rat tissues
was hybridized with a XbaI-XhoI fragment (540 bp)
from clone P32. The estimated size of the major band (arrow)
is about 3400 nucleotides.
Northern Analysis
Northern analyses of rat tissues
were performed using a 0.54-kbp XbaI-XhoI fragment of
clone P32. This probe hybridized to a 3.4-kb RNA, which was very
abundant in forebrain, weaker in cerebellum, and detectable in thymus
and spleen. This tissue distribution is similar to that of CaM-kinase
IV except that CaM-kinase IV is more abundant in cerebellum than
forebrain(18) .
Homology to Other CaM-kinases
Homology analyses of
the deduced amino acid sequence of cloned CaM-kinase kinase cDNA
revealed it was a unique protein that had considerable sequence
identity (30-40%) between residues 121 and 408 with the catalytic
domains of Ser/Thr protein kinases, especially with the CaM-kinase
family (Fig. 2). Interestingly, CaM-kinase kinase had an insert
of 22 residues, which is rich in Pro, Arg, and Gly, between the
ATP-binding motif and the DGF kinase motif. An insert in this location
is present in a few other protein kinases such as STE11(19) , a
yeast homologue of Raf. Like CaM-kinase IV, the COOH terminus of
CaM-kinase kinase was quite acidic with residues 374-505 being
24% Asp/Glu and only 13% Arg/Lys. Other than the conserved protein
kinase region, no other conserved motifs were detected in CaM-kinase
kinase.
Figure 2:
Comparison of amino acid sequences of
catalytic domains of CaM-kinase kinase with other protein kinases.
Residues 121-408 of CaM-kinase kinase (CaMKK) are
aligned with the catalytic domains of rat CaM-kinase II
subunit
(CaMKII(28) ), rat CaM-kinase IV (CaMKIV (29)), rat
CaM-kinase I (CaMKI(30) ), and rat cAMP-dependent
protein kinase (cAPK(31) ). Identical residues are
shown in boldface. The ATP-binding site and catalytic motifs
are boxed. The peptide sequence in CaM-kinase II against which
an antibody was generated is doubleunderlined.
Characterization of Expressed CaM-kinase
Kinase
When the cDNA for CaM-kinase kinase was expressed in COS
cells, a new 68-kDa protein was detected by Western blotting (Fig. 3A) using an antibody against residues
132-146 of CaM-kinase II (underlined in Fig. 2).
This sequence in CaM-kinase II is highly conserved in Ser/Thr protein
kinases including CaM-kinase kinase (Fig. 2). We next tested
whether expressed CaM-kinase kinase bound Ca
/CaM
since the purified rat brain CaM-kinase kinase was purified by EGTA
elution from CaM-Sepharose, and activation of CaM-kinase IV by the
brain CaM-kinase kinase required Ca
/CaM(10) .
Gel overlay of the expressed CaM-kinase kinase with biotinylated CaM
revealed strong binding of CaM to the 68-kDa protein. Although no
obvious CaM-binding domain was detected by the homology search, it is
possible that residues 444-457, which contain hydrophobic and
basic residues, may constitute a basic amphipatic
-helix
characteristic of CaM-binding domains.
Activation of CaM-kinase IV
The most important
test of the cloned putative CaM-kinase kinase was whether the expressed
protein activated CaM-kinase IV. In Fig. 3B the COS
cell extract was incubated with Mg
/ATP for 5 min in
the presence of purified recombinant CaM-kinase IV and either EGTA,
Ca
, or Ca
/CaM. The reaction was
terminated by dilution and then assayed to determine the activity of
CaM-kinase IV in the presence of EGTA (Ca
-independent
activity, openbars) or Ca
/CaM
(total activity, solidbars). In the absence of
CaM-kinase kinase (i.e. buffer or mock-transfected COS cells)
the total CaM-kinase IV activity was about 2.5 pmol/min, and it
increased to about 4 pmol/min upon autophosphorylation in the presence
of Ca
/CaM. This small 1.5-2-fold activation
upon autophosphorylation is consistent with previous reports for Sf9
cell-expressed CaM-kinase IV(4, 10) . However, when
CaM-kinase kinase was expressed in the COS cells there was a 6-fold
increase in total CaM-kinase IV activity and a 100-fold increase in its
Ca
-independent activity (Fig. 3B).
Similar activation was observed with either wild-type CaM-kinase IV or
a double mutant in which the overlapping CaM-binding and autoinhibitory
domains were disrupted by mutation of Phe
and Asn
to Asp. This mutant no longer required Ca
/CaM
for activity, and it bound Ca
/CaM very
poorly(10) . The results of Fig. 3B, which were
essentially identical to those obtained with the purified brain
CaM-kinase kinase(10) , indicated that expressed CaM-kinase
kinase not only bound Ca
/CaM (Fig. 3A) but also required it for activity.
Activation of CaM-kinases I and II
A 52-kDa
activator of CaM-kinase I has been purified from rat
brain(14) , so it was of interest to determine whether our
expressed 68-kDa CaM-kinase kinase could also activate CaM-kinase I.
CaM-kinase I phosphorylates in vitro synapsin I (20) and the cystic fibrosis transmembrane conductance
regulator(21) . Fig. 4A shows that CaM-kinase
kinase catalyzed a 10-fold increase in the total activity of
recombinant CaM-kinase I; unlike with CaM-kinase IV, there was no
effect on Ca
-independent activity of CaM-kinase I.
CaM-kinase II is known to be regulated by autophosphorylation, which
only increases its Ca
-independent
activity(22) . There is no strong evidence that its total
activity can be increased by phosphorylation, and it was not activated
by CaM-kinase kinase (Fig. 4B).
Figure 4:
The effect of CaM-kinase kinase on
CaM-kinase I and II activities. Purified Sf9 cell-expressed wild-type
CaM-kinase I (2.0 µM, panelA) and
CaM-kinase II (2.0 µM, panelB) were
incubated with either the lysate buffer (Buffer) or COS-7 cell
lysate (180 ng) transfected with plasmid (Mock) or
plasmid-expressing CaM-kinase kinase (CaMKK) in the presence
of 1 mM CaCl
plus 10 µM CaM under the
same conditions as described in Fig. 3B. CaM-kinase I
and II activities were measured using 40 µM syntide-2 in
the presence of either 1 mM EGTA (openbar)
or 1 mM CaCl
plus 1 µM CaM (closedbar) under standard assay
conditions(10) . Syntide-2 phosphorylation activity of the
lysate from COS-7 cells transfected with pMECaMKK was also measured
under the same conditions (righttwolanes in each panel). The results indicate the mean ±
S.E. of three experiments using three independent
transfections.
Enhanced Gene Expression by Co-expression of CaM-kinase
Kinase and CaM-kinase IV
Considerable evidence exists that
CaM-kinase IV is involved in transcriptional regulation through the
phosphorylation of CREB(5, 6, 7) , but
CaM-kinase IV is less potent than protein kinase A and may need to be
activated by CaM-kinase kinase(5) . We further tested the
CaM-kinase cascade hypothesis by determining whether co-transfection of
COS cells with CaM-kinase kinase and CaM-kinase IV would enhance gene
transcription compared with CaM-kinase IV alone. COS-7 cells
transfected with either CaM-kinase IV or CaM-kinase kinase exhibited
little CREB-dependent luciferase gene expression under these conditions (Fig. 5). The weak effect of transfection by CaM-kinase IV alone
was consistent with the apparent lack of CaM-kinase kinase in COS-7
cells (Fig. 3). Co-transfection of CaM-kinase IV with CaM-kinase
kinase gave a 14-fold enhancement of luciferase expression compared
with transfection with either kinase alone (Fig. 5). However,
the dependence on ionomycin for the transcriptional enhancement by
CaM-kinase kinase was only about 2-fold (not shown). This small
Ca
effect could be due to
Ca
-independent activity of the overexpressed
CaM-kinase kinase, or it could indicate that factors in addition to
Ca
are required for full activation of CaM-kinase
kinase (see below). These possibilities are being explored.
Figure 5:
Demonstration of the CaM-kinase cascade in
CREB-mediated gene transcription. COS-7 cells were transfected with
plasmid alone (Mock) or plasmid(s) encoding CaM-kinase IV (CaMK
IV) and/or CaM-kinase kinase (CaMKK) as indicated with
plasmids encoding a fusion protein for GAL4-CREB and GAL4-luciferase as
described under ``Experimental Procedures.'' After 2 h in 5
µM ionomycin and 10 mM CaCl
,
luciferase activity was determined. Results are the mean ± S.E.
for three dishes per condition.
Conclusions
The results presented in this paper
are consistent with a signal transduction cascade involving CaM-kinase
kinase and CaM-kinase IV, which regulates gene expression. Protein
kinase cascades, such as the protein kinase A/phosphorylase kinase
cascade (23) and the mitogen-activated protein kinase
cascade(24) , are well established physiological pathways.
Agonists that trigger the CaM-kinase cascade are currently being
defined. Activation of the CD3 receptor in Jurkat cells, which
stimulates tyrosine and Ser/Thr protein kinases and Ca
mobilization via inositol trisphosphate formation(25) ,
gives a 10-20-fold activation of CaM-kinase IV (26) and
also initiates Ca
-dependent gene
transcription(27) . This CD3-mediated activation of CaM-kinase
IV is presumably due to CaM-kinase kinase since the CaM-kinase IV
activation is reversed by treatment in vitro with protein
phosphatase 2A but not phosphatase 1. (
)We are currently
investigating which agonists can activate CaM-kinase IV in various
cells and whether Ca
mobilization alone is sufficient
to maximally activate CaM-kinase kinase or whether other second
messengers are also required.