(Received for publication, May 23, 1995; and in revised form, August 23, 1995)
From the
The myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from Dictyostelium discoideum has been implicated in the regulation of myosin II assembly in response to the chemoattractant, cAMP (Ravid, S., and Spudich, J. A.(1989) J. Biol. Chem. 264, 15144-15150). Here we report that elimination of MHC-PKC results in the abolishment of MHC phosphorylation in response to cAMP. Cells devoid of MHC-PKC exhibit substantial myosin II overassembly, as well as aberrant cell polarization, chemotaxis, and morphological differentiation. Cells overexpressing the MHC-PKC contain highly phosphorylated MHC and exhibit impaired myosin II localization and no apparent cell polarization and chemotaxis. The results presented here provide direct evidence that MHC-PKC phosphorylates MHC in response to cAMP and plays an important role in the regulation of myosin II localization during chemotaxis.
When cells of Dictyostelium are starved, they acquire
the ability to bind cAMP to specific cell surface receptors and to
respond to this signal by chemotaxis. The process of chemotaxis
involves phosphorylation and reorganization of myosin
II(1, 2, 3, 4, 5) . In
response to cAMP stimulation, myosin II, which exists as thick
filaments, translocates to the cortex(5) . This translocation
is correlated with a transient increase in the rate of myosin II heavy
chain (MHC) ()as well as light chain
phosphorylation(3, 4, 6) . In addition, in vitro studies strongly suggest that MHC phosphorylation
plays an important role in the regulation of myosin II filament
formation(7, 8, 9, 10) . The
importance of MHC phosphorylation in the regulation of myosin II in
vivo was demonstrated by Egelhoff et al.(11) .
They found that elimination of the MHC phosphorylation sites allows in vivo contractile activity, but this myosin II shows
substantial overassembly. Mimicking the negative charge state of
phosphorylated myosin II eliminates filament formation in vitro and renders the myosin II unable to drive any tested contractile
event in vivo(11) .
We previously reported the
isolation of a MHC-specific PKC (MHC-PKC) from Dictyostelium that phosphorylates Dictyostelium MHC specifically and is
homologous to ,
, and
subtypes of mammalian
PKC(9, 12) . This kinase, which is membrane-associated
and is expressed during Dictyostelium development, was
implicated in the increase in MHC phosphorylation during chemotaxis in
this species(9) . In vitro phosphorylation of MHC by
MHC-PKC results in inhibition of myosin II thick filament
formation(9) , by inducing the formation of a bent monomer of
myosin II whose assembly domain is tied up in an intramolecular
interaction that precludes intermolecular interaction, which is
necessary for thick filament formation(10) . The findings that
MHC-PKC is a member of the PKC family and that it regulates myosin II
assembly suggest a link between the extracellular chemotactic signal
and subsequent intracellular events.
A considerable amount of information is now available regarding the regulation of myosin II by MHC phosphorylation, and a picture is beginning to emerge in which the molecular changes in myosin II that are involved in MHC phosphorylation are related to cAMP-induced directed cell movement. Nevertheless, the molecular mechanism by which a cAMP signal is transmitted to myosin II, resulting in myosin II localization and chemotaxis, remains unclear. In an attempt to throw some light on this issue, we studied the role of MHC-PKC in vivo by eliminating and by overexpressing the MHC-PKC protein in Dictyostelium cells. Analysis of these cell lines allowed us to address directly the role of MHC-PKC in vivo and consequently the role of MHC phosphorylation in the regulation of myosin II. The results presented here indicate that MHC-PKC is a key modulator in the regulation of myosin II localization in response to the chemoattractant, cAMP.
Figure 1:
Disruption of the MHC-PKC gene. A, integration of pThy1-MHC-PKCcat into the MHC-PKC gene.
The circle represents the transformation plasmid
pThy1-MHC-PKC
cat, which contains the MHC-PKC regulatory domain
coding region (R), the Thy1 gene, and the plasmid
sequence of the Bluescript vector. In the center of the diagram is the
chromosomal region of the MHC-PKC gene(12) . Integration of one
copy of pThy1-MHC-PKC
cat into the MHC-PKC locus would result in
the map illustrated. The horizontal arrows indicate the size
in kilobases of the restriction fragments that can be detected with a
1.0-kb probe (thick line) directed against the catalytic
domain coding portion. B, Southern analysis of the MHC-PKC
gene in Dictyostelium JH10 (wild type) and MHC-PKC
cell lines. Restriction maps were generated using a probe that
hybridized only to the C domain of MHC-PKC. This region is not
represented in the transformation plasmid pThy1-MHC-PKC
cat, and
thus serves as a specific probe for the MHC-PKC locus. The map obtained
by hybridizing the C domain probe to the restriction digest of DNA of
wild type cells revealed a 10-kb BglII and a BglII/PstI fragment (lanes 1 and 3), as predicted in Fig. 1A. This restriction
pattern was altered in MHC-PKC
cells, in that the BglII fragment had shifted to a fragment of about 18 kb, and
the BglII/PstI digest revealed a 4.5-kb fragment (lanes 2 and 4). Total DNA from the different cell
lines was digested with the enzymes indicated above, separated on 0.6%
agarose gel, transferred to a nylon membrane, and hybridized with the
probe indicated above. C, immunoblot analysis of Dictyostelium JH10 (wild type) and MHC-PKC
cells. Cells were developed for 4 h in suspension, and whole cell
lysates were prepared as described under ``Experimental
Procedures.'' Samples (100 µg of total protein) were subjected
to 10% SDS-PAGE and blotted on nitrocellulose. The immunoblots were
stained with affinity-purified anti-MHC-PKC
antibodies(12) .
pDRE106 (see Fig. 2A) was produced by fusing an actin-15 promoter from pSC79 (15) to the cDNA clone encoding the MHC-PKC (12) , and ligating this fused product into the extrachromosomal vector pnDeI(16) . The resulting plasmid pDRE106 contains the entire MHC-PKC coding region fused in phase to actin-15 promoter and in an antisense orientation relative to the neomycin gene (Fig. 2A). Full details of the construct are available upon request.
Figure 2:
Expression of MHC-PKC. A,
expression vector pDRE106 (see ``Experimental Procedures''
for details). B, Western blot analysis of whole cell lysates
and of membrane fractions from wild type and from MHC-PKC cells at the vegetative stage and after 4 h of development.
Samples (40 µg of total protein) were subjected to 10% SDS-PAGE and
blotted on nitrocellulose. Immunoblots were stained with
affinity-purified anti-MHC-PKC antibodies(12) . C,
membrane-associated MHC-PKC specific activity of wild type and
MHC-PKC
cells at the vegetative stage and after 4 h of
development. MHC-PKC activity was assayed by incubating purified LMM58
with the MHC-PKC samples in 10 mM Tris (pH 7.6), 6 mM
MgCl
, 0.2 mM [
-
P]ATP,
1 mM dithiothreitol for 10 min at 22 °C. The results are
mean values of five different
determinations.
For MHC-PKC disruption experiments, we
used the Dictyostelium JH10 strain that is an auxotroph for
thymidine. Cells were grown in HL5 medium and 50 µg/ml thymidine
(Sigma). To express the MHC-PKC we used the Dictyostelium Ax2
strain, that were grown in HL5 medium. To express the MHC-PKC along
with 3X ALA myosin II(11) , we co-transformed the Dictyostelium myosin II null cells (HS1 strain) with the
pDRE106 and pBIG-ALA (11) expression vectors. Transformation
was carried out using the calcium phosphate procedure(17) .
Transformants for the disruption of MHC-PKC were selected on the basis
of their ability to grow in the absence of thymidine. Transformants for
MHC-PKC and 3X ALA expression were selected on the basis of their
resistance to the antibiotic G418 (Boehringer Mannheim), and their
ability to grow in suspension since HS1 cells are unable to grow in
suspension(11) . Clones were initially screened with G418 at a
concentration of 10 µg/ml, which was increased to 50 µg/ml.
Because MHC-PKC cells were found to express the
maximum amount of MHC-PKC in the presence of 50 µg/ml G418, all
subsequent experiments were performed using this concentration.
G418-resistant clones were screened by Western blot analysis using
MHC-PKC polyclonal antibodies(12) .
Dictyostelium cell lines were developed on MES plates (20 mM MES (pH
6.8), 0.2 mM CaCl, 2 mM MgSO
,
2% agar) as described by Berlot et al.(3) .
For Western blot analysis, cells
were developed for 4 h in shaking flasks as described(3) .
Samples were prepared from whole cell lysates (18) or from the
insoluble fraction(9) . Protein was determined by the method of
Peterson(19) , and lysates were electrophoresed on 10% sodium
dodecyl sulfate-polyacrylamide gels (SDS-PAGE)(20) . The
Western blots were probed with affinity-purified MHC-PKC polyclonal
antibody (12) . This antibody recognizes the full-length
MHC-PKC as well as the R domain. ()
In the second method, MHC-PKC was immunoprecipitated from the soluble and insoluble fractions described above. The supernatants of these fractions were added to preadsorbed MHC-PKC antibody-Staphylococcus A cell mixture prepared as described by Berlot et al.(3) and incubated for at least 1 h at 4 °C with rotation. The immunoprecipitated MHC-PKC from the soluble and insoluble fractions was assayed for kinase activity as described above, except that the reaction was stopped by spinning out the Staphylococcus A cell mixture containing the MHC-PKC and adding of 5% trichloroacetic acid to the supernatant to precipitate the LMM58.
For cAMP stimulation of Dictyostelium cell lines, cells were overlaid with agar sheets and the excess buffer was removed with a filter paper. When the cells had flattened out, 5 µl of 1 µM cAMP solution was added to one corner of the agar sheet, and after 1 min of incubation at room temperature the cells were fixed as described(22) .
To investigate the in vivo role of MHC-PKC in Dictyostelium, we produced two Dictyostelium cell lines that represent contrasting extremes with respect to the presence of MHC-PKC. In one cell line the endogenous copy of MHC-PKC was inactivated by gene disruption, and in the other the MHC-PKC protein was overexpressed.
Several
independent disrupted MHC-PKC clones (MHC-PKC cells)
and Dictyostelium JH10 cells were subjected to Western blot
analyses (Fig. 1C). JH10 cells did not express the
MHC-PKC protein during their vegetative stage (data not shown), as was
reported for Ax3 cells (12) , but did express it after 4 h of
development (Fig. 1C). We had expected that the MHC-PKC
disruption construct would result in the expression of R domain, but we
could not detect it in MHC-PKC
cells either in the
vegetative stage (data not shown) or in the developmental stage (Fig. 1C). Our results may indicate either that the R
domain is not expressed at all or that it is expressed but is unstable
and degraded.
Western and Southern analyses confirmed that the
MHC-PKC gene was disrupted. To ensure that disruption of the MHC-PKC
gene also results in elimination of its activity, we assayed JH10 and
MHC-PKC cells for the presence of MHC-PKC activity in
the membrane fraction, having shown previously that MHC-PKC is a
membrane-associated protein(9) . The JH10 cells were found to
contain MHC-PKC activity (20 pmol/min/mg) that is comparable to the
activity published previously for Dictyostelium Ax3 cells (9) . In contrast, the MHC-PKC
cells
displayed negligible amounts of kinase activity (0.01 pmol/min/mg)
associated with the membrane fraction. These results suggest that
disruption of the MHC-PKC gene resulted in elimination of its activity
and that MHC-PKC is the only MHC kinase (MHCK) activity in the membrane
of Dictyostelium.
The expressed MHC-PKC protein was active. Fig. 2C shows the MHC-PKC activity associated with
membrane fractions of Ax2 and MHC-PKC cells, in the
vegetative stage as well as in cells developed for 4 h. We detected
negligible amounts of MHC-PKC activity associated with the membrane
fraction in vegetative Ax2 cells (Fig. 2C). This
activity appeared upon starvation of the cells. In contrast, vegetative
MHC-PKC
cells contained MHC-PKC activity comparable to
that detected in Ax2 cells developed for 4 h. Upon cell starvation this
activity was increased 5-fold (Fig. 2C).
The
MHC-PKC cells expressed comparable amounts of MHC-PKC
protein during the vegetative and development stages (Fig. 2B), but the activity of MHC-PKC in
MHC-PKC
vegetative cells was 5-fold lower than in
MHC-PKC
cells developed for 4 h. To resolve this
apparent discrepancy, we quantified the amount of MHC-PKC protein
associated with cell membranes in MHC-PKC
cells at
both stages. As shown in Fig. 2B, the amount of MHC-PKC
protein associated with the membrane fraction in vegetative
MHC-PKC
cells was lower than in developed
MHC-PKC
cells. These results may indicate that a much
smaller part of the MHC-PKC protein translocates to the membrane in the
former case than in the latter. We found that translocation of MHC-PKC
to membranes is in response to the extracellular signal, cAMP. (
)Since in vegetative MHC-PKC
cells the
cAMP signal is missing, it is likely that most of the expressed MHC-PKC
resides in the cell soluble-fraction in inactive state. To test this
hypothesis, we compared the activity of the cytosolic and
membrane-associated MHC-PKC. Since Dictyostelium contains
several soluble MHCKs (for review see (26) ), we first
immunoprecipitated the MHC-PKC from the soluble and insoluble fractions
and assayed it for kinase activity as described under
``Experimental Procedures.'' We compared the
P
incorporated into LMM58 by soluble and by insoluble MHC-PKC. The
soluble MHC-PKC possessed only 0.5% of the kinase activity present in
the membrane-associated MHC-PKC. These results suggested that the
MHC-PKC that resided in the cytosol is inactive and MHC-PKC
translocation to the cortex resulted in activation of the enzyme.
In
the following experiments, we compared MHC-PKC cells
to JH10 cells and MHC-PKC
cells to Ax2 cells that were
transformed with the vector pnDeI(16) ; we refer to both cell
lines as wild type cells.
Figure 3:
In vivo levels of MHC
phosphorylation after cAMP stimulation in wild type and MHC-PKC cell
lines. A developed cell suspension was labeled with P (0.5
mCi/ml) for 30 min, after which a stimulus of 2 µM cAMP
was applied. Samples were taken at various time points and
immunoprecipitated for myosin II and subjected to SDS-PAGE
electrophoresis. Relative phosphorylation was quantified by
densitometry and phosphorimaging, as described under
``Experimental Procedures.''
Overexpression of the MHC-PKC resulted in highly phosphorylated MHC.
cAMP stimulation of these cells resulted in a slight increase in MHC
phosphorylation. To find out whether the expressed MHC-PKC in
MHC-PKC cells phosphorylates the same sites on MHC as
the native MHC-PKC in wild type cells, we compared the phosphorylation
levels of MHC after cAMP stimulation of cells expressing the 3X ALA MHC (17) and in cells that co-expressed MHC-PKC and the 3X ALA MHC.
In the 3X ALA MHC mutant, the phosphorylation sites previously shown to
be a target of several MHCKs (27, 28) were converted
to alanine residues, thus eliminating phosphorylation at these
positions. These phosphorylation sites are localized within the region
of MHC that is phosphorylated by MHC-PKC. (
)The rationale
for this experiment was that if the highly phosphorylated MHC in
developed MHC-PKC
cells (Fig. 3) reflects
additional phosphorylation sites on MHC phosphorylated by the expressed
MHC-PKC, then overexpression of MHC-PKC with 3X ALA MHC should result
in phosphorylated MHC, while cells expressing 3X ALA MHC alone will
contain nonphosphorylated MHC. We found that cAMP stimulation of 3X ALA
cells or cells co-expressing MHC-PKC and 3X ALA MHC do not respond to
this stimulation by increasing the phosphorylation level of MHC. The
MHC from both cell lines contained the same basal level of
phosphorylation as was found for wild type cells (data not shown).
These results indicate that MHC-PKC overexpression did not result in
phosphorylation of new sites on MHC.
To find out whether the
expressed MHC-PKC in vegetative MHC-PKC cells also
resulted in highly phosphorylated MHC as was found for developed
MHC-PKC
cells (Fig. 3), we compared the
phosphorylation level of MHC isolated from vegetative
MHC-PKC
and wild type cells. We found that the
phosphorylation levels of MHC in both cell lines were similar (data not
shown). These results indicate that although the MHC-PKC in vegetative
MHC-PKC
cell is active, it was incapable of
phosphorylating MHC in vivo.
Figure 4: Myosin II associated with Triton-insoluble cytoskeletal fraction. Triton-soluble and -insoluble fractions of cell lysates were subjected to SDS-PAGE electrophoresis. The MHC bands were scanned as described under ``Experimental Procedures,'' and the relative amount of MHC associated with the Triton-insoluble fraction was plotted.
Figure 5: Immunolocalization of myosin II in response to cAMP. Cells were allowed to attached to glass coverslips and were then fixed or stimulated with 1 µM cAMP before fixation. Cells were subjected to indirect immunofluorescence staining with antimyosin II.
In unstimulated developed MHC-PKC cells, as in wild type cells, myosin II was scattered throughout
the cytoplasm and the cells were round (Fig. 5). Strikingly,
upon cAMP stimulation MHC-PKC
cells did not undergo
myosin II localization or shape changes (Fig. 5). These results
suggest that MHC-PKC overexpression that leads to highly phosphorylated
MHC (Fig. 3) caused elimination of myosin II localization and
cell polarization.
Figure 6: Chemotaxis. Chemotactic activity was measured using the Zigmond chamber. A gradient of 1 µM cAMP was formed, and the percentage of cells that accumulated along the side of the bridge with the higher concentration of cAMP was plotted.
Figure 7:
Development on MES plates of wild type,
MHC-PKC, and MHC-PKC
cell lines. A, wild type; B, MHC-PKC
cells at
the slug stage (after starvation for 16 and 26 h, respectively). C, wild type cells after starvation for 26 h. D,
MHC-PKC
cells after starvation for 48 h. E,
MHC-PKC
cells after starvation for
26.
To determine whether the
distorted MHC-PKC fruiting bodies contain viable
spores, we collected spores from both wild type cells and
MHC-PKC
cells from fruiting bodies on bacterial lawns
and examined them using the spore viability assay (described under
``Experimental Procedures''). The viability of spores
generated by MHC-PKC
cells developed on bacterial
lawns was about 50% of that of wild type spores.
The most dramatic
effect was observed during development of MHC-PKC cells (Fig. 7E). These cells formed aggregates
and failed to complete the developmental cycle by forming fruiting
bodies. These findings, along with the failure of the MHC-PKC mutants
to exhibit normal chemotaxis, suggest that MHC-PKC is required for
proper development and may play an important role during the initial
step of development, namely chemotaxis to cAMP.
This study demonstrates that a member of the PKC family, MHC-PKC, is the enzyme that phosphorylates MHC in response to stimulation by the chemoattractant cAMP in Dictyostelium discoideum and is part of the chemotactic sensing mechanism in this species. Furthermore, MHC phosphorylation by MHC-PKC plays an important role in the regulation of myosin II localization and is required for proper chemotaxis and differentiation.
MHC from MHC-PKC cells exhibits the same basal level of phosphorylation as MHC
from wild type cells. This phosphorylation, which is cAMP-independent,
may represent serine phosphorylation carried out by other MHCKs, since
the cAMP-stimulated increases in phosphorylation occur primarily on
threonine(4) . In addition to MHC-PKC, several other MHCKs have
been identified in Dictyostelium(8, 29, 30) . Whether
these kinases regulate myosin II during different kinds of contractile
events or at different stages of the life cycle, or whether they
function in a redundant and overlapping fashion, is unknown. Our
results indicate that the different kinases respond to different
signals, i.e. MHC-PKC responds to cAMP stimulation while the
other MHCKs may not. In addition, MHC-PKC is expressed only during Dictyostelium development, while the other known Dictyostelium MHCKs are expressed during the vegetative stage
only, or during the vegetative stage as well as the development stage
(for review see (26) ). These findings indicate that the
different Dictyostelium MHCKs may function at different stages
of the life cycle of Dictyostelium and respond to different
signals. It is also possible that the localization within the cell of
the different MHCKs determines their role in the regulation of myosin
II. Support for this idea comes from the findings that, although the
MHC-PKC in vegetative MHC-PKC
cells is active, it did
not phosphorylate the MHC (see below).
Overexpression of MHC-PKC
resulted in highly phosphorylated MHC that does not represent new
phosphorylation sites. MHC-PKC phosphorylates four sites on MHC in
vitro(9) ; nevertheless, the in vivo levels of
phosphate incorporation into MHC are very low (0.05 mol of
phosphate/mol of MHC)(3, 31) . It was proposed that
incorporation of a single phosphate to a single myosin II filament is
enough for filament disassembly(31) . It is therefore
conceivable that in vivo MHC-PKC phosphorylates small fraction
of MHC and/or less then four sites on a single MHC, which is sufficient
for filament disassembly. In MHC-PKC cells the
overexpression of MHC-PKC may result in phosphorylation of larger
fraction of MHC and/or more than one site that results in the inability
of myosin II to form filaments.
The Triton-resistant cytoskeleton
analysis and the immunofluorescent microscopy study provide evidence
that MHC-PKC plays a key role in controlling myosin II assembly into
the cortical cytoskeleton. In MHC-PKC cells, myosin
II displayed substantial overassembly in these assays and cAMP
stimulation resulted in a gradual increase in myosin II association
with the cytoskeleton with no apparent dissociation. The simplest
explanation for these results is that, in unstimulated
MHC-PKC
cells, the absence of MHC-PKC drives myosin
II molecules into filaments in vivo and that these filaments
have high affinity for the cortical cytoskeleton and therefore
accumulates as a cortical ring. Upon cAMP stimulation additional myosin
II translocates to the cytoskeleton and, subsequently, cannot
dissociate from it because of the absence of MHC-PKC. These results
also suggest that myosin II association and dissociation from the
cytoskeleton are two separate processes. Myosin II in
MHC-PKC
cells exhibits a low level of association with
the cytoskeleton; addition of cAMP results in a decrease in myosin II
insolubility. The interpretation of these results is that the
overexpression of MHC-PKC results in highly phosphorylated myosin II,
which is unable to form filaments and to accumulate at the cortex. The
myosin II observed in the cytoskeletal fraction in the Triton-resistant
cytoskeleton assay may represent small filaments, large enough to
associate with the cytoskeleton but not sufficient to drive cell
polarity and chemotaxis. Stimulation by cAMP results in dissociation of
myosin II from the cytoskeleton and increase in MHC phosphorylation,
possibly as a result of cAMP-induced MHC-PKC translocation to the
membrane and phosphorylation of the cortical myosin II. These results
are consistent with the results reported by Yumura and
Kitanishi-Yumura(31) , which indicate that during contraction,
the myosin II that have moved toward the foci are phosphorylated by a
specific MHCK that is localized at the foci, with the resultant
disassembly of filaments which are finally released from membrane
cytoskeleton.
Both MHC-PKC mutants failed to exhibit normal chemotaxis toward cAMP. Wessels et al.(32) suggested that myosin II is required for fine tune locomotion during chemotaxis, and therefore cells lacking myosin II fail to exhibit efficient chemotaxis. On the basis of these results, we propose the following model for Dictyostelium chemotaxis. An unstimulated cell is rounded because of a contractile shell formed by an actin-myosin II network in the cortex. This network presumably inhibits events necessary for pseudopodial projection. Stimulation of one edge of the cell with the chemoattractant cAMP results in translocation of myosin II to the cortex and translocation of MHC-PKC to the site of stimulation, where it phosphorylates MHC and leads to local breakdown of myosin II thick filaments. Thus the barrier to pseudopodial extension is removed and the cell extends toward the source of the chemoattractant. The disassembled myosin II reassembles in the posterior portion of the cell, strengthening the existing cortical actin-myosin II network there and providing further inhibition of membrane ruffling in that area. In this way, the interaction between myosin II and MHC-PKC may play a major role in the generation of cell polarity for efficiently directed migration. In the absence of MHC-PKC, the cAMP-dependent mechanisms of myosin II phosphorylation and localization are disrupted. Instead of local breakdown of myosin II due to MHC phosphorylation by MHC-PKC, the myosin II forms a ring around the cell. As a result the cells are not properly polarized and exhibit inefficient chemotaxis. Cells overexpressing the MHC-PKC are unable to polarize since their myosin II is unable to assemble into filaments and to associate with the cortical cytoskeleton for the generation of cell polarization.
Our findings are consistent with those of Egelhoff et al.(11) , which suggested that MHC-PKC
phosphorylates only myosin II. In that study the phosphorylation sites
previously shown to be a target of two MHCKs (27, 28) were converted either to alanine residues (3X
ALA), or to aspartate residues (3X ASP), which mimics the negatively
charged state of the phosphorylated molecule. The 3X ALA mutant and the
MHC-PKC mutant exhibit similar behavior. In both cell
lines, myosin II exhibits substantial overassembly in vivo.
Myosin II in MHC-PKC
and 3X ASP mutants (11) is unable to form filaments, and this renders the myosin
II unable to drive development. The similarity of the myosin II mutant
cell lines and the MHC-PKC cell lines may indicate that MHC-PKC is
specific for myosin II and probably does not phosphorylate other
proteins in vivo.
MHC-PKC overexpression had a dramatic
effect on Dictyostelium development but no effect on growth,
even though the MHC-PKC was expressed to the same extent during the
vegetative and developed stages. Since MHC-PKC is not normally
expressed during the vegetative stage, we anticipated that its
overexpression in that stage would result in highly phosphorylated
myosin II, which would not be able to form filaments and to actuate
cytokinesis, as was reported for Dictyostelium hmm cells (18) . However, overexpression of MHC-PKC did not affect cell
growth, because of the incapability of MHC-PKC to phosphorylate MHC in
vegetative cells, possibly because of the different localization of
myosin II and active MHC-PKC. In vegetative MHC-PKC cells, most of the MHC-PKC is localized in the cytosol in
inactive form; it is conceivable that the myosin II is not accessible
to the active membrane-associated MHC-PKC. Another possible explanation
is that the target sites of MHC-PKC phosphorylation on MHC differ from
the sites that play a role in the regulation of myosin II during
cytokinesis, and that the later sites may be phosphorylated by other
MHCKs.
The results presented here provide a direct in vivo evidence that MHC-PKC plays a critical role in the regulation of myosin II localization within the cells in response to cAMP stimulation. Since PKC is a ubiquitous protein, and it has been shown to phosphorylate myosin II in other cell systems (for review see (26) ), it is possible that PKC in other systems plays a role similar to that played by MHC-PKC.