Department of Biochemistry, Robert Wood
Johnson Medical School, Piscataway, New Jersey 08854
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INTRODUCTION |
Myxococcus xanthus is a
gram-negative soil bacterium which undergoes spectacular multicellular
development and cellular differentiation. Upon starvation at high cell
density (2 × 109 cells/ml) on a semisolid
surface, cells move to aggregation centers, where they form
mounds called fruiting bodies. Within the fruiting bodies, many cells
lyse while others differentiate to become sonication- and
heat-resistant spores (for a recent review, see reference 3). Its social behavior and morphogenesis resemble
those of eukaryotic slime molds like Dictyostelium
discoideum, in which a signal transduction system consisting of a
receptor, a G-protein, an effector, and protein serine/ threonine
kinases is involved in sensing starvation and regulating gene
expression for development. Due to the close resemblance of the life
cycles of Dictyostelium and M. xanthus, it
has been proposed that a similar signal transduction system might exist
in this developmental bacterium. In particular, the recent discovery of
a large family of eukaryote-like protein serine/threonine kinases in
M. xanthus (3, 5, 29, 31, 32) provided an
intriguing opportunity to examine the role of these kinases in
prokaryote development.
The first eukaryote-like protein serine/threonine kinase found in
bacteria was discovered in M. xanthus (20)
and found to be required for normal development. Subsequently,
M. xanthus was found to contain a family of at least 13 eukaryote-like protein serine/threonine kinases (31).
The cloning and sequencing of these 13 protein serine/threonine
kinases have revealed that all of them retain the conserved structural
features of eukaryotic protein kinases (11). Many of these
protein kinases are transmembrane proteins (5, 28, 32). It
seems very likely that these transmembrane protein kinases sense
certain environmental signals and are involved in various signal
transduction pathways leading to regulation of growth and development.
Because of the sequence similarity between eukaryotic and
M. xanthus protein serine/threonine kinases, it
is possible that known inhibitors for eukaryotic kinases affect the activity of protein kinases of M. xanthus. In this
study, we have examined the effect of some of these protein kinase
inhibitors on fruiting-body formation as well as spore formation by
M. xanthus.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The M. xanthus strain used was DZF1. The cells were grown vegetatively in
CYE medium (1% Casitone, 0.5% yeast extract, 0.1% MgSO4), and development was studied on CF agar (10 mM
Tris HCl [pH 7.6], 8 mM MgSO4, 0.02% Casitone,
0.2% NH4SO4, 1 mM potassium phosphate buffer
[pH 7.6], 0.2% sodium citrate, 0.1% sodium pyruvate, 1.5% agar),
supplemented with protein kinase inhibitors in 48-well microtiter
plates (Falcon Inc). For quantitation of 
-galactosidase activity,
strain DK6620 carrying 
4521 (kindly provided by H. Kaplan,
University of Texas Medical School, Houston, Tex.) was used
(12).
Protein kinase inhibitors.
Staurosporine and genistein were
purchased from Sigma (St. Louis, Mo.); K252c was purchased from
Calbiochem (San Diego, Calif.); and chelerythrin, KN-62,
bisindolylmaleimide, daidzein and tyrphostin B52 were from Alexis Co.
(Woburn, Mass.).
Inhibition of development of M. xanthus by
various inhibitors.
To study the development of M. xanthus under starvation conditions, cells were grown in CYE
medium until they reached a turbidity of 100 Klett units, at which time
they were harvested, washed once with TM buffer (10 mM Tris HCl
[pH 7.6], 8 mM MgSO4), and resuspended in TM buffer at
4,000 Klett units. Cell suspension (2 µl) was spotted on each
well of a 48-well microtiter plate containing 300 µl of CF agar and
the individual protein kinase inhibitors at 5 µM. The inhibitors
were dissolved in dimethyl sulfoxide (DMSO), and the final
concentration of DMSO in CF agar wells was 0.5%. The control plates
contained 0.5% DMSO only. The plates were incubated at 30°C, and
development of M. xanthus was monitored every 8 h
under a dissecting microscope. To study the effect of inhibitors during
vegetative growth, cells were grown to a turbidity of 100 Klett units
in CYE medium, 2 µl of growing culture was spotted onto each well of
a 48-well microtiter plate containing 300 µl of CYE agar and the
individual protein kinase inhibitors at 5 µM, and the plates were
incubated at 30°C. The effect on growth and motility of cells was
assessed by the ability of cells to grow and move away from the growing
spot. To study the effect of addition of protein kinase inhibitor
5 h after the onset of development, the cells were allowed to
develop on CF agar in a 24-well microtiter plate. After 5 h, the
agar was gently lifted from one end and protein kinase inhibitor was
added at the bottom of the agar to a final concentration of 5 µM.
Effect of inhibitors on sporulation.
To count the number of
spores produced in the presence of protein kinase inhibitors on CF
medium, M. xanthus cells were allowed to develop in
48-well microtiter plates in the presence of the individual inhibitors
at 5 µM, as described above. After 5 days, all the cells (containing
well-developed fruiting bodies in some cases) were scraped off the
surface of the agar, washed once with 200 µl of TM buffer,
resuspended in 200 µl of TM buffer, and sonicated to disrupt the
fruiting bodies. Sonication-resistant refractile spores were counted
under the microscope. The numbers listed in Table 1 reflect the yield
of spores from 2 µl of cell suspension that was spotted on the
surface of CF agar.
Effect of inhibitors on activity of protein kinases in
vitro.
To study the inhibition of protein kinases of M. xanthus in vitro by protein kinase inhibitors, the inhibitors were
added to 0.1 µg of purified protein at a final concentration of
5 µM, in a total volume of 20 µl. The reaction was carried out
in kinase buffer (50 mM Tris HCl [pH 7.6], 50 mM KCl, 10 mM
MgCl2, 5 mM NaF, 0.1 mM ATP, 1 mM -mercaptoethanol) in
the presence of 10 µCi of [
-32P]ATP at room
temperature for 15 min. For Pkn2, MgCl2 was replaced by
MnCl2, and for Pkn11, 5 mM CaCl2 was included
in the reaction mixture. The reaction was terminated by addition of
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
loading buffer and heating at 90°C for 10 min. The autophosphorylated protein was subjected to SDS-PAGE and visualized by autoradiography.
Assay of -galactosidase activity.
To study the expression
of 4521, at the indicated times 40 µl of cells was scraped off the
surface of CF agar containing different protein kinase inhibitors and
washed once with TM buffer, and the cell pellet was stored at 
20°C.
-Galactosidase activity was assayed by the method of Kroos et al.
(15).
In vitro phosphorylation during development.
To study the
pattern of phosphorylated proteins during development, at the indicated
times 40 µl of cells was scraped off the surface of agar containing
different protein kinase inhibitors and sonicated to disrupt the cells
(the sonication buffer contained 10 mM Tris HCl [pH 7.6], 1 mM EDTA,
and 5 mM phenylmethylsulfonyl fluoride). Unbroken cells were removed by
centrifugation, and total-cell extract was used in an in vitro
phosphorylation reaction as described above. After the proteins were
separated by SDS-PAGE, they were transferred to a polyvinylidene
difluoride membrane. The membrane was washed with 10% trichloroacetic
acid (TCA) at 55°C for 2 h to remove acid-sensitive
phosphorylation and autoradiographed.
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RESULTS AND DISCUSSION |
When M. xanthus cells are spotted on CF agar at a
high cell density (2 × 109 cells/ml), they
aggregate to form fruiting bodies. This process is completed within
48 h. To elucidate the roles of protein serine/threonine kinases
during vegetative growth and the developmental cycle of M. xanthus, a series of single and multiple deletions
of protein kinases have been constructed by positive-negative KG
cassettes (11). These deletions include a quadruple deletion
mutant with pkn6, pkn7, pkn11, and
pkn13. Examination of fruiting-body formation and
sporulation by this mutant strain revealed that fruiting-body formation
was initiated earlier in the mutant than in the wild-type strain but
the fruiting bodies showed normal morphology and could produce normal
numbers of spores. These results indicated a redundancy in the
functions of protein kinases of M. xanthus. Therefore, to investigate the overall function of protein kinases during the
development and sporulation of M. xanthus, we tested
the effects of several different protein kinase inhibitors on
development of M. xanthus. We found that in the
presence of protein kinase inhibitors, which included both
serine/threonine and tyrosine protein kinase inhibitors, the formation
of fruiting bodies was either delayed or completely inhibited (Table
1).
Effect of staurosporine and other protein serine/threonine kinase
inhibitors.
Staurosporine is the most potent inhibitor of
kinases in vitro that has been found to date (26). It has
been found to inhibit a variety of serine/threonine and tyrosine
kinases, e.g., protein kinase C (PKC) (50% inhibitory concentration
[IC50] = 2.7 nM), PKA (IC50 = 8.2 nM),
tyrosine kinase p60v-src (IC50 = 6.4 nM) (21), and tyrosine kinase epidermal growth factor
receptor (IC50 = 630 nM) (16). It has been found
to interact with the catalytic domain of protein kinases and prevent
the binding of ATP to protein kinases. In in vitro experiments,
staurosporine inhibits the autophosphorylation activity of Pkn2, a
serine/threonine protein kinase of M. xanthus
(28). At 5 µM, staurosporine completely blocked the
formation of fruiting bodies in M. xanthus (Fig.
1a); however, at 1 µM, fruiting-body
formation, although delayed by 24 h, could occur. Interestingly,
even though staurosporine could completely inhibit the formation of
fruiting bodies at 5 µM, it did not inhibit the formation of
spores (Table 1). It has been known for a long time that the pathways
leading to fruiting-body formation and differentiation into spores are
not always coupled. Many nonfruiting mutants of M. xanthus are able to form normal numbers of spores (18).
Staurosporine and other inhibitors tested did not inhibit vegetative
growth and motility of M. xanthus, as indicated by the
presence of a normal "flare" on CYE plates (Fig. 1b).
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FIG. 1.
Effect of protein kinase inhibitors on the development
(a) and vegetative growth (b) of M. xanthus.
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K252c is another indole carbazole protein kinase inhibitor isolated
from microorganisms (13). Like staurosporine, K252c has a
broad specificity for inhibiting protein kinases, although it is less
potent than staurosporine. It inhibits
Ca2+/calmodulin-dependent protein kinase (CaM kinase)
(Ki = 18 nM), myosin light-chain kinase
(Ki = 17 nM), PKA (Ki = 25 nM), PKC (Ki = 25 nM), and PKG
(Ki = 20 nM). K252c at 5 µM also could inhibit fruiting-body formation and sporulation of M. xanthus.
It is interesting that although staurosporine and K252c have very
similar structures, as shown in Fig. 1a, K252c could inhibit both
sporulation (Table 1) and fruiting-body formation (Fig. 1a)
whereas staurosporine could not inhibit sporulation.
Chelerythrin chloride inhibits PKC (IC50 = 660 nM) but does
not inhibit PKA or CaM kinase (7). It is not competitive
with ATP, suggesting that it binds to a different region of the
catalytic domain than staurosporine and its analogs. As shown in Fig.
1a and Table 1, 5 µM chelerythrin chloride could inhibit
fruiting-body formation and sporulation.
KN-62 selectively inhibits CaM kinase II (Ki = 900 nM) by binding directly to the calmodulin-binding site of the
enzyme (27). KN-62 at 5 µM could delay the formation of
fruiting bodies in M. xanthus by 16 h (Fig.
1a). The fruiting bodies formed in the presence of KN-62 contained
nearly normal numbers of spores (Table 1).
Bisindolylmaleimide is known to be a selective inhibitor of PKC
(Ki = 10 nM) (19, 27). It is
structurally similar to staurosporine and is a competitive inhibitor
with respect to ATP. However, 5 µM bisindolylmaleimide showed only a
weak inhibition of M. xanthus development (Fig. 1a).
The spores produced in the presence of various inhibitors were checked
for their ability to germinate on CYE medium, and all of them were
found to be viable (data not shown).
It is also interesting that compounds like calphostin C, sphingosine,
and hypericin, which are known to specifically inhibit PKC by
competitively inhibiting the binding of diacylglycerol to the
regulatory domain of PKC (6, 14, 25), failed to show
any effect on development of M. xanthus (data not
shown). Sphingosine has also been thought to inhibit phospholipase
A2 and phospholipase D by acting as the biosynthetic
precursor of sphingolipids. This could mean that a signal transduction
pathway involving phospholipases, diacylglycerol, and inositol
triphosphate (22), similar to that found in eukaryotes,
probably does not operate during development of M. xanthus. However, although Benaissa et al. (2) have
shown that synthesis and degradation of inositol phospholipids occur
during clumping in Stigmatella aurantiaca and suggested that
a phosphoinositol cycle might operate, the relation between protein
kinases and a phosphatidylinositol cycle in M. xanthus
remains to be investigated, although the role of Ca2+ in
clumping of M. xanthus is well known (24,
30). In this respect, it is interesting that several
serine/threonine protein kinases identified in M. xanthus require Ca2+ for their activity
(32). It is possible that Ca2+ influxes,
along with starvation, activate certain protein kinases and trigger a
signal transduction pathway to initiate development.
The effect of the H-7 series of compounds (which are
isoquinolinesulfonamide derivatives) (8, 9) on fruiting-body
formation and sporulation by M. xanthus was also
tested (data not shown). These compounds inhibit PKA
(Ki = 1.2 to 3.0 µM), PKC
(Ki = 6.0 to 40 µM), PKG (Ki = 480 nM to 5.8 µM), and myosin light-chain kinase
(Ki = 3.6 to 150 µM). The compounds tested are
HA-100, HA-1004, H-7, Iso H-7, H-8, and H-9. None of these compounds
could inhibit fruiting-body formation and sporulation in M. xanthus, probably because their Ki values
are too high (their inhibitory activity is 103-fold lower
than that of K252c) and so at 5 µM they failed to show any effect.
Olomoucine is a specific inhibitor of the cdc/cdk family of
cyclin-dependent protein kinases, which are involved in
regulation of the cell cycle. Olomoucine did not have any
effect on vegetative growth or development of M. xanthus (data not shown).
Effect of tyrosine kinase inhibitors.
In general, the tyrosine
kinase inhibitors can be divided into two groups: genistein (and its
analogs) and tyrphostins.
Genistein competitively inhibits the binding of ATP and thus inhibits
protein tyrosine kinase (1). However, since genistein itself
bears no structural resemblance to ATP, it has been suggested that the
inhibition might not be due to true competition but the binding sites
of genistein and ATP might overlap. Genistein is almost inactive
against protein serine/threonine kinases and so is used to distinguish
between the two groups of kinases (1). In the present
experiments, no effect of genistein on the development of M. xanthus was observed; however, a genistein analog, daidzein, could
delay fruiting-body formation by 24 h in M. xanthus, suggesting that all the features of eukaryotic tyrosine
kinases might not be conserved in protein kinases of M. xanthus. The number of spores produced in the presence of daidzein
were close to normal, and these spores were viable.
Interestingly, genistein has been shown to inhibit histidine kinases
(10, 23), with an IC50 of 110 µM, which is
much higher than the concentration of genistein used in the present
experiments (see Materials and Methods). Moreover, all the kinase
inhibitors used in the present experiments were tested for inhibition
of autophosphorylation of purified EnvZ, a histidine kinase of
Escherichia coli; however, at 5 µM none could inhibit
the autophosphorylation activity of purified EnvZ (data not
shown). These results indicate that inhibition of the development of
M. xanthus by the kinase inhibitors used in the present
experiments is not due to inhibition of histidine kinases.
Tyrphostins are synthetic compounds which structurally resemble
tyrosine and thus act as specific inhibitors of tyrosine kinases. Many
of them have broad specificities, while some are selective (16). We found that 5 µM tyrphostin B52 was able to
inhibit fruiting-body formation and sporulation of M. xanthus on CF plates. This indicates the existence of tyrosine
phosphorylation, which may play important roles during the development
of M. xanthus. However, a protein tyrosine kinase has
not been identified in M. xanthus, although the
presence of phosphotyrosine has been reported (4), and the
patterns of tyrosine-phosphorylated proteins were shown to vary during
development. There is no evidence at present to show that
phosphorylation of tyrosine occurs by a tyrosine-specific kinase,
although its presence in M. xanthus has been speculated (4). It is also possible that some of the M. xanthus serine/threonine kinases possess dual specificity and
phosphorylate on tyrosine residues in addition to serine/threonine and
that these kinases are inhibited by tyrphostins as well as genistein analogs.
When M. xanthus cells were spotted on CF agar plates
containing 1 µM staurosporine or tyrphostin, their development was
delayed by 24 h (data not shown). However, when other inhibitors
were tested at 1 µM, the cells could develop normally.
It is also important to note that the inhibitory activities of
different protein kinase inhibitors also depend on the permeability of
the cell membrane and their stability.
Effect of addition of protein kinase inhibitors after the onset of
development.
The process of development is accompanied by the
expression of several developmentally regulated genes. The products of
these genes are probably required for development. By analyzing 36 different developmentally expressed genes, Kroos et al. (15)
have divided them into three groups: group I, the
initiation/aggregation group, which is expressed from h 0 to 5; group
II, the postaggregation group, which is expressed from h 9 to 15; and
group III, the sporulation group, which is expressed from h 18 to 24.
We tried to determine if protein kinases of M. xanthus
affect the early or late stages of development. If these inhibitors affect late stages of development, their addition after the onset of
starvation should be able to inhibit development. However, if they
inhibit early stages of development, their addition after the onset of
starvation might fail to show any effects. Thus, protein kinase
inhibitors were added to developing cells 5 h after the onset of
development, to allow the expression of group I genes, as described in
Materials and Methods. The results are shown in Fig.
2. We found that most of the protein
kinase inhibitors failed to inhibit the development of M. xanthus if they were added 5 h after the onset of
development, except for tyrphostin B52, which was able to delay the
development. KN-62 also failed to inhibit development; however, the
fruiting bodies produced were larger than those produced in the absence
of any inhibitor. These results show that most of the protein kinase
inhibitors inhibit steps occurring during the early stages of
development.
Effect of kinase inhibitors on gene expression during early
development.
One of the earliest genes to be expressed during
development has been identified as 4521 (15), a commonly
used marker for development of M. xanthus. 4521 was
originally identified by Tn5 lac insertion in this gene
(15). The Tn5 lac transposon carries a
promoterless lacZ gene, which can be expressed under the
transcriptional control of any transcriptionally active gene into which
the transposon is inserted in the right orientation. Strain DK6620
(12) carries such an insertion, called 4521, and was used
for this experiment. The expression of 4521 requires both an
A-signal, which is a mixture of amino acids and their peptides, and
nutritional starvation during development (17). The
expression of 4521 is detectable as early as 1.5 h after the
onset of starvation and reaches a peak at 21 h of development (15). Since most inhibitors seem to act during the early
stages of development, we attempted to examine whether any of the
kinases could block the signal transduction pathways leading to
induction of 4521. The results are shown in Table
2 (the numbers shown are the averages of
the results of two independent experiments). The -galactosidase
activity values obtained in the absence of inhibitors are in good
agreement with that reported by Kroos et al. (15).
It is interesting that genistein, which had no effect on the
development of M. xanthus, was a potent inhibitor of
4521 expression during the early stages of development, indicating
the involvement of tyrosine phosphorylation in the signal transduction
pathway leading to expression of 4521. Other kinase inhibitors also
showed inhibition of 4521 to various degrees; e.g., K252c and
tyrphostin B52, which inhibited fruiting-body formation and sporulation
(Fig. 1), showed inhibition of 4521 expression to the same degree as genistein did. Interestingly, chelerythrin, although completely inhibiting development, had no effect on expression of 4521 but, rather, seemed to enhance the expression of 4521. This supports the
previous observation by Kroos et al. (15) that development of M. xanthus does not completely depend on expression
of 4521, so that a strain carrying a Tn5 lac insertion in
this gene can develop normally. In other words, the process of
development does not absolutely require the gene product of 4521 and
the expression of 4521 does not guarantee that development can
occur. That is why, even though expression of 4521 is found to be
normal in some cases, development is inhibited, probably by inhibition
of pathways other than the one leading to expression of 4521.
Another possibility is that the kinases inhibited by these inhibitors, e.g., staurosporine and chelerythrin, are situated downstream of
4521 in the developmental pathway.
Effect of protein kinase inhibitors on autophosphorylation of
protein kinases of M. xanthus.
The autophosphorylation
of purified Pkn2, Pkn5, and Pkn11 was examined in the presence of
different protein kinase inhibitors, as described in Materials and
Methods. It was found that Pkn5 was inhibited only by
bisindolylmaleimide (Fig. 3) whereas Pkn2 was inhibited by both staurosporine and bisindolylmaleimide (Fig. 3).
The IC50 for inhibition of Pkn2 by staurosporine is 440 nM (29). Pkn11 was not inhibited by any of the protein kinase
inhibitors that we have tried so far (Fig. 3).
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FIG. 3.
Effect of protein kinase inhibitors on
autophosphorylation of serine/threonine protein kinases of
M. xanthus.
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Effect of protein kinase inhibitors on the pattern of
phosphorylated proteins during development.
The pattern of
phosphorylated proteins was examined as described in Materials and
Methods. As shown in Fig. 4, no
significant difference in the phosphorylation pattern was detected in
the cells which were developing on CF agar plates containing different protein kinase inhibitors. However, at the onset of development, several phosphorylated bands disappeared, indicating downregulation of
some kinases or activation of some specific phosphatases. The lack of
any obvious difference in the pattern of phosphorylation of proteins in
cells developing on different inhibitors could be due to the targets of
phosphorylation being minor proteins which could not be detected in the
experimental system we used.
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FIG. 4.
Effect of protein kinase inhibitors on the pattern of in
vitro-phosphorylated proteins of M. xanthus during
development. (A) Before the filter is washed with TCA. (B) After the
filter is washed with TCA. Lanes: 1, no inhibitor; 2, staurosporine; 3, K252c; 4, chelerythrin; 5, KN-62; 6, bisindolylmaleimide; 7, genistein;
8, daidzein; 9, tyrphostin B52. The differences between lane 1 and the
other lanes seen at 2 and 4 h of development were not consistently
observed.
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In conclusion, we have shown that general eukaryotic protein kinase
inhibitors can inhibit the development of M. xanthus. A
wide variety of protein kinase inhibitors can inhibit the development of M. xanthus to various degrees, suggesting that they
have different specific targets in the cell. The fact that protein
kinases of M. xanthus can be directly inhibited by
inhibitors of eukaryotic protein kinases indicates that
serine/threonine protein kinases of M. xanthus have
catalytic domains similar to those of PKC and PKA and also that these
kinases are involved in developmental pathways. Interestingly,
inhibitors with different known specificities could inhibit the
development of M. xanthus, suggesting the involvement of more than one kinase in the regulation of development. Most of the
protein kinase inhibitors showed only a partial inhibition of 4521
expression, indicating that the kinases might act in a different
pathway leading to development. Nutritional starvation and an A-signal
could activate many different pathways, including elevation of
intracellular levels of guanosine tetraphosphate and cyclic AMP, all of
which contribute to the process of fruiting-body formation and
sporulation; the expression of 4521 is just one of the effects of
these pathways. Each of the protein kinase inhibitors could affect one
or more of these steps. For example, it is possible that genistein
inhibits one or more steps leading to the synthesis of 4521, while
allowing the other steps to go on. This might result in inhibition of
expression of 4521 although development can proceed. On the other
hand, some other inhibitors, such as staurosporine, might inhibit the
pathways leading to development while allowing the expression of
4521. Moreover, inhibitors such as K252c and tyrphostin B52 might
inhibit many different pathways, which leads to inhibition not only of
development and sporulation but also of expression of 4521.
It is important to note that these inhibitors did not inhibit
vegetative growth (data not shown), indicating that they are not toxic
to the cell and supporting the previous hypothesis that kinase activity
is required for the development of M. xanthus.
We thank Heidi Kaplan, University of Texas Medical School,
Houston, Tex., for providing strain DK6620 and William Hanlon, Merck
Co., Rahway, N.J., for helpful discussions. We also thank Masayori
Inouye, UMDNJ, Piscataway, N.J., for critical reading of the manuscript.
This work was supported by Public Health Service grant GM26843 from the
National Institutes of Health.
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Abstract
Introduction
