(Received for publication, August 13, 1996, and in revised form, October 16, 1996)
From the Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel
Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP. cAMP stimulation of Dictyostelium cells leads to translocation of MHC-PKC from the cytosol to the membrane fraction, as well as causing an increase in both MHC-PKC phosphorylation and its kinase activity. MHC-PKC undergoes autophosphorylation with each mole of kinase incorporating about 20 mol of phosphate. The MHC-PKC autophosphorylation sites are thought to be located within a domain at the COOH-terminal region of MHC-PKC that contains a cluster of 21 serine and threonine residues. Here we report that deletion of this domain abolished the ability of the enzyme to undergo autophosphorylation in vitro. Furthermore, after this deletion, cAMP-dependent autophosphorylation of MHC-PKC as well as cAMP-dependent increases in kinase activity and subcellular localization were also abolished. These results provide evidence for the role of autophosphorylation in the regulation of MHC-PKC and indicate that this MHC-PKC autophosphorylation is required for the kinase activation in response to cAMP and for subcellular localization.
We have previously reported the isolation of a
MHC1-specific PKC (MHC-PKC) from the ameba,
Dictyostelium that phosphorylates Dictyostelium
MHC specifically and is homologous to ,
, and
subtypes of
mammalian PKC (1, 2). In vitro phosphorylation of MHC by
MHC-PKC results in inhibition of myosin II thick filament formation (1)
by inducing the formation of a bent monomer of myosin II, whose
assembly domain is tied up in an intramolecular interaction that
precludes the intermolecular interaction necessary for thick filament
formation (3).
The MHC-PKC which is expressed during Dictyostelium development has been implicated in the increase in MHC phosphorylation observed in response to cAMP stimulation (1). We have recently found that elimination of MHC-PKC abolishes this cAMP-induced MHC phosphorylation, indicating that MHC-PKC is the enzyme which phosphorylates MHC in response to cAMP stimulation (4). MHC-PKC null cells exhibit a substantial myosin II overassembly in vivo, as well as aberrant cell polarization, chemotaxis, and morphological differentiation. Cells that overexpress MHC-PKC contain highly phosphorylated MHC. They show no apparent cell polarization and chemotaxis, and exhibit impaired myosin II localization (4). These findings establish that, in Dictyostelium, the MHC-PKC plays an important role in regulating the cAMP-induced myosin II localization required for cell polarization and, consequently, for efficient 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, which requires phosphorylation and reorganization of myosin II (5, 6, 7, 8, 9). That is, the myosin II, which exists as thick filaments, translocates to the cortex (9) in response to cAMP stimulation. This translocation is correlated with a transient increase in the rate of MHC as well as light chain phosphorylation (5, 6, 10). We have recently shown that cAMP exerts its effects on myosin II via the regulation of MHC-PKC (11). cAMP stimulation of Dictyostelium cells results in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing MHC-PKC phosphorylation and its kinase activity (11). We could also show that MHC is phosphorylated by MHC-PKC in the cell cortex, and this leads to myosin II dissociation from the cytoskeleton (11).
Members of the PKC family are composed of a single polypeptide
consisting of an NH2-terminal lipid binding regulatory
domain and an ATP-binding catalytic domain located in the COOH-terminal region of the protein (12, 13). PKC is phosphorylated on multiple serine and threonine residues located in both domains (14, 15, 16, 17). Three
clusters of autophosphorylation sites have been mapped in
vitro in the PKC II isoenzyme at the NH2 terminus, the COOH-terminal tail, and the hinge region between the regulatory and
catalytic domains (14). However, in vivo studies have
indicated that the autophosphorylation sites are all localized at the
COOH-terminal tail of PKC (18, 19). Bond and co-workers (20) have
replaced the three clusters of the autophosphorylation sites found
in vitro by alanine residues. Altering the
autophosphorylation sites in this way at the NH2-terminal
region or at the hinge region did not affect the activity or
subcellular localization of the kinase. However, replacing the
autophosphorylation sites at the COOH-terminal tail with alanine
residues resulted in inactive, Triton X-100-insoluble enzyme (20).
Similar results have been reported by Su et al. (21), who
found that deletion of 23 amino acids from the COOH terminus of PKC
,
which includes the autophosphorylation sites, caused total loss of
catalytic activity. These results indicate that the PKC
autophosphorylation sites located at the COOH-terminal tail play an
important role in the regulation of the kinase in vivo.
MHC-PKC also undergoes phosphorylation in vivo, which consists of both autophosphorylation and phosphorylation by another kinase, presumably cGMP-dependent protein kinase. These two kinds of phosphorylation involve different phosphorylation sites (11). Here we report that deletion of 33 amino acids from the COOH-terminal tail of MHC-PKC, which includes a cluster of 21 serine and threonine residues, abolished both MHC-PKC autophosphorylation in vitro and the cAMP-dependent MHC-PKC autophosphorylation. This indicates that this portion of the protein contains the in vitro and in vivo MHC-PKC autophosphorylation sites. We further present data showing that MHC-PKC autophosphorylation plays an important role in the kinase activation and subcellular localization.
Dictyostelium discoideum strain amebas were grown in HL-5 medium (22), harvested at a density of 2 × 106 cells/ml, washed twice in MES buffer (20 mM MES, pH 6.8, 0.2 mM CaCl2, 2 mM MgSO4), and resuspended at a density of 2 × 107 cells/ml to initiate development. Cells were shaken at 100 rpm at 22 °C for 3.5 h. 5 mM caffeine was added to the suspension 30 min prior to the addition of cAMP.
Expression of MHC-PKCAll DNA manipulations were
carried out using standard methods (23). We used the expression vector
pDXA-HY which contains the actin-15 promoter and allows the expression
of proteins carrying a NH2-terminal His tag (24).
pDXA-MHC-PKCST was constructed as follows. The vector pBS-MHCK (2),
containing a 2.6-kilobase pair MHC-PKC cDNA clone, was digested
with SmaI and SwaI, which deleted a fragment of
309 base pairs from the 3
of the MHC-PKC clone. The deleted fragment
contains 99 base pairs of coding region and 210 base pairs of noncoding
region. The deleted coding region is 33 amino acids in length and
contains a cluster of 21 serine and threonine (ST) residues which are
thought to be the MHC-PKC autophosphorylation domain (Fig. 1, ST
domain). The resulting MHC-PKC fragment was named MHC-PKC
ST.
This MHC-PKC
ST fragment was sequenced to confirm the deletion of the
ST domain. It was cloned into pDXA-HY digested with SmaI.
pDXA-MHC-PKC
ST was used for the transformation of
MHC-PKC
cells (4) using calcium phosphate precipitate
(25). Clones were selected on the basis of their resistance to G418
(Boehringer Mannheim) and screened using Western blot analysis (see
below).
Purification of His-tagged MHC-PKC
50 ml of 2 × 106 cells/ml expressing MHC-PKCST were washed twice in
20 mM phosphate buffer (pH 6.5), and the cells were lysed in 1 ml of lysis buffer containing 20 mM HEPES (pH 7.5),
1% Triton X-100, 0.2% Nonidet P-40, 200 mM KCl, 5 mM
-mercaptoethanol, and protease inhibitor mix (2 mM phenylmethylsulfonyl fluoride, 200 µM
leupeptin, and 200 µM pepstatin). The extracts were
centrifuged in a microcentrifuge for 15 min at 4 °C, and the
supernatant was incubated with 50 µl of a slurry of
Ni+-agarose beads (Qiagen) in 20 mM phosphate
buffer, pH 6.5, and 200 mM KCl for 1 h at 4 °C. The
bead-protein complex was washed three times with lysis buffer, twice
with lysis buffer containing 20 mM imidazole, and twice
with lysis buffer containing 50 mM imidazole. The
MHC-PKC
ST was eluted with 100 µl of lysis buffer containing 150 mM imidazole and then eluted with 100 µl of lysis buffer
containing 250 mM imidazole.
Cells were developed for 4 h in shaking flasks as described above. Cells were washed in 10 mM Tris-HCl, pH 8.0, and 150 mM KCl and lysed in 50 mM Tris-HCl, pH 8.0, 20 mM sodium pyrophosphate, pH 6.8, 5 mM EDTA, 5 mM EGTA, 0.5% Triton X-100, and protease inhibitor mix. Protein was determined by the method of Peterson (26), and lysates were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) (27). Western blots were probed with MHC-PKC polyclonal antibody (2), and the blots were developed using a horseradish peroxidase-coupled secondary antibody (Bio-Rad Laboratories). ECL was performed using a kit from Amersham Corp.
AutophosphorylationDeveloped Ax2 cell suspensions
containing 5 × 106 cells/ml were added to an equal
volume of ice-cold 2 × lysis buffer (40 mM Tris-Cl,
pH 7.5, 0.2% Nonidet P-40, 2 mM dithiothreitol, 10 mM EDTA, and protease inhibitor mix) and centrifuged for 5 min in a microcentrifuge at 4 °C. The supernatant was precleared by
incubation with 30 µl of rabbit preimmune serum at 4 °C for 1 h followed by incubation with Staphylococcus A cells at
4 °C for 30 min. Staphylococcus A cells were centrifuged,
and 10 µl of MHC-PKC antibody were added to the supernatant and
incubated at 4 °C for 1 h, followed by incubation with 50 µl
of protein A-conjugated agarose (100 mg/ml) at 4 °C for 1 h and
then centrifuged for 1 min. Pellets were washed twice in 1 × lysis buffer containing 1 mg/ml bovine serum albumin and once with
1 × lysis buffer only. The protein A-MHC-PKC complex was
resuspended in 200 µl of phosphatase mix (20 mM Tris-HCl,
pH 7.5, 200 µM MnCl2, 1 mM
dithiothreitol, 0.5 mM CaCl2, 0.04 mM EDTA, 300 mM KCl, and 5 units of alkaline phosphatase (Boehringer Mannheim)) and incubated at 37 °C for 30 min. The phosphatase-treated protein A-MHC-PKC complex was washed twice
in 1 × lysis buffer and incubated in 200 µl of phosphatase inhibitor mix (100 mM NaF, 200 µM
Na3VO4, 10 mM
KH2PO4 and protease inhibitor mix) for 30 min
at room temperature, followed by two washes in 1 × lysis buffer.
The phosphatase-treated protein A-MHC-PKC complex was resuspended in
100 µl of 20 mM Tris-HCl, pH 7.5, 8 mM
MgCl2, 0.2 mM [-32P]ATP and
incubated at 22 °C for 20 min and then centrifuged for 1 min. The
pellets were washed in 1 × lysis buffer and resuspended in SDS
sample buffer and boiled for 5 min. The supernatants from a
microcentrifuge spin were loaded on SDS-PAGE and analyzed using autoradiography and PhosphorImaging. Autophosphorylation of
MHC-PKC
ST was performed as described above using MHC-PKC
ST
purified with Ni+-agarose beads as described above.
Dictyostelium Ax2 and
MHC-PKCST cells were developed and treated with caffeine as
described above. Before and after the application of cAMP stimulus, 100 µl of developed cells were added to 200 µl of reaction mixture
containing 0.2% Triton X-100, 8 mM MgCl2, 10 mM Tris-HCl (pH 7.5), and 0.2 mM
[
-32P]ATP and incubated for 30 s at 22 °C. The
MHC-PKC and MHC-PKC
ST were immunoprecipitated and analyzed using
SDS-PAGE. Densitometric scanning of the Coomassie Blue-stained gels was
used to determine the relative amounts of immunoprecipitated MHC-PKC
and MHC-PKC
ST. The amounts of 32P incorporated into the
proteins were determined using the PhosphorImager. Relative
phosphorylation of MHC-PKC and MHC-PKC
ST was determined by dividing
the values obtained with the PhosphorImager by the values obtained by
scanning of the Coomassie Blue-stained gels. In vivo
phosphorylation of MHC was carried out as described previously (5,
6).
This was assayed directly using the kinase
extracted from the insoluble cell fraction. Following resuspension of
1 × 107 developed Ax2 and MHC-PKCST cells in 1 ml
of sonication buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, and protease inhibitor mix), they were stimulated
with 1 µM cAMP and lysed by sonication using an
ultrasonic cell disruptor (Microson) model XL with a small sized tip at
50% output power. The extract was spun in a microcentrifuge for 20 min
at 4 °C. MHC-PKC and MHC-PKC
ST were extracted from the insoluble
fraction using sonication buffer containing 1% Triton X-100 and 0.5 M KCl. For kinase assay, the solubilized MHC-PKC or
MHC-PKC
ST were incubated with LMM58 (0.5-1 mg/ml), 6 mM
MgCl2, 0.2 mM [
-32P]ATP (500 cpm/pmol), 1 mM DTT for 10 min at 22 °C on a rotator. Reaction was initiated by the addition of ATP and stopped by the addition of 5% trichloroacetic acid. The precipitated LMM58 were pelleted in a microcentrifuge after incubation for 15 min on ice, washed twice with 5% trichloroacetic acid, resuspended in 20 µl of
SDS-PAGE sample buffer, and electrophoresed on 7% SDS-PAGE gels. To
determine incorporation of 32P into LMM58, the gels were
stained and destained, and the bands were cut out of the gels and
counted in a scintillation counter in 5 ml of scintillation fluid. The
amounts of MHC-PKC and MHC-PKC
ST in the cell extracts were
determined using densitometric scanning of Western blots and normalized
to the total amount of protein determined as described previously
(26).
Following resuspension of 1 × 107
developed Ax2 and MHC-PKCST cells in 1 ml of sonication buffer (10 mM Tris, pH 7.5, 50 mM KCl, and protease
inhibitor mix), they were lysed by sonication as described above, and
the extract was spun in a microcentrifuge for 20 min at 4 °C. The
soluble fraction was immunoprecipitated with MHC-PKC antibody as
described above. MHC-PKC and MHC-PKC
ST were extracted from the
insoluble fraction using sonication buffer containing 1% Triton X-100
and 0.5 M KCl. The extract was spun in a microcentrifuge
for 10 min at 4 °C, and the solubilized MHC-PKC and MHC-PKC
ST
were immunoprecipitated as described above. To quantify the amounts of
MHC-PKC and MHC-PKC
ST in the soluble and insoluble fractions, the
immunoprecipitated MHC-PKC and MHC-PKC
ST from both fractions was
electrophoresed on 7% SDS-PAGE gels and the Coomassie Blue-stained
gels were analyzed as described above.
Triton-insoluble cytoskeleton analysis was performed as described previously (28). Supernatant and cytoskeletal pellet fractions were resuspended in SDS-PAGE sample buffer, boiled for 5 min, and electrophoresed on 7% SDS-PAGE gels. The relative amounts of myosin II were determined by SDS-PAGE gel analysis as described above.
Phosphorylation of sites located at the COOH-terminal tail
of several PKC is important for the regulation of the kinase activity and subcellular localization (18, 20, 21, 29). MHC-PKC undergoes
autophosphorylation in vitro and phosphorylation in vivo in response to cAMP stimulation, and this phosphorylation coincides with the activation of the kinase (11). The MHC-PKC autophosphorylation sites are thought to be located within the COOH-terminal tail of MHC-PKC, which contains a cluster of 21 serine
and threonine residues (Fig. 1, ST domain)
(1, 2). To study the in vivo role of MHC-PKC
autophosphorylation, we engineered an MHC-PKC in which the putative
autophosphorylation domain was deleted (MHC-PKCST). The MHC-PKC
ST
was expressed in Dictyostelium cells that were previously
engineered to lack expression of MHC-PKC (so-called
MHC-PKC
cells) (4).
MHC-PKC cells transformed with the pDXA-MHC-PKC
ST
construct (see "Experimental Procedures") expressed MHC-PKC
ST at
125-150% of the level of MHC-PKC in Ax2 cells (Fig.
2). The expressed MHC-PKC
ST migrated on SDS-PAGE with
an apparent molecular mass of about 80 kDa (Fig. 2), fitting well with
the predicted molecular mass of 80 kDa and indicating that the protein
was not phosphorylated. In contrast, MHC-PKC migrated on SDS-PAGE of
cell extracts of Ax2 with an apparent molecular mass of about 94 kDa
(Fig. 2), although the predicted molecular mass of MHC-PKC is 84 kDa
(2). These results are consistent with a migration of the
autophosphorylated form of MHC-PKC on SDS-PAGE (1), indicating that the
MHC-PKC was in its phosphorylated form, under these experimental
conditions. In addition to the 94-kDa band of MHC-PKC, another band
with an apparent molecular mass of 90 kDa was found (Fig. 2). This
protein could be either partially phosphorylated form of MHC-PKC or a degradation product of MHC-PKC.
MHC-PKC but Not MHC-PKC
To find out whether the ST domain of MHC-PKC is indeed the
kinase autophosphorylation domain, we studied the ability of
MHC-PKCST to undergo autophosphorylation in vitro. To do
this, we developed Ax2 and MHC-PKC
ST cells. The MHC-PKC was
immunoprecipitated and the MHC-PKC
ST purified using
Ni+-agarose beads as described under "Experimental
Procedures." To increase autophosphorylation, the proteins were
treated with phosphatase prior to the autophosphorylation reaction as
described under "Experimental Procedures." The phosphatase-treated
MHC-PKC immunocomplexed to protein A-Sepharose and the
MHC-PKC
ST-Ni+-agarose complex were incubated with
[
-32P]ATP and MgCl2, and the extent of the
autophosphorylation was analyzed using autoradiography as described
under "Experimental Procedures."
On addition of ATP and MgCl2 to MHC-PKC, it migrated with a
molecular mass of 94 kDa (Fig. 3A) which is
consistent with a molecular mass of the autophosphorylated form of
MHC-PKC (1). Autoradiography revealed that MHC-PKC indeed underwent
autophosphorylation (Fig. 3B), and similar results have been
previously reported (1). MHC-PKC appeared as a doublet on SDS-PAGE
(Fig. 3A), which may represent different extents of
autophosphorylation. In contrast, addition of ATP and MgCl2
to MHC-PKCST did not alter the migration pattern of the protein, and
it migrated with a molecular mass predicted for nonphosphorylated
truncated MHC-PKC protein (Fig. 3C). Autoradiography of
MHC-PKC
ST revealed that the protein was unable to undergo
autophosphorylation (Fig. 3D). These results indicate that
the ST domain is the in vitro MHC-PKC autophosphorylation domain.
MHC-PKC and Not MHC-PKC
We have previously reported that, in response to cAMP
stimulation, MHC-PKC undergoes a phosphorylation which is composed of both autophosphorylation and phosphorylation in a
cGMP-dependent manner (11). To address whether MHC-PKCST
is able to undergo in vivo phosphorylation in response to
cAMP stimulation, we stimulated Ax2 and MHC-PKC
ST cells with cAMP
and a total lysate of amebas labeled with [
-32P]ATP.
MHC-PKC and MHC-PKC
ST were immunoprecipitated and the levels of
their phosphorylation were determined as described under "Experimental Procedures."
MHC-PKC was transiently phosphorylated in response to cAMP stimulation
(Fig. 4, A and B), with peak
phosphorylation at about 40 s (Fig. 4B) (see also
Dembinsky et al. (11)). In contrast, addition of cAMP to
cells expressing the MHC-PKCST protein resulted in very low
phosphorylation levels (Fig. 4, A and B), whose
magnitude was similar to the basal level of MHC-PKC phosphorylation
determined 120 s after cAMP stimulation (Fig. 4B). This
low level of phosphorylation in the MHC-PKC
ST may be due to the
phosphorylation of the protein by another kinase possibly
cGMP-dependent protein kinase, as we recently suggested
(11). The inability of MHC-PKC
ST to undergo both autophosphorylation
in vitro (Fig. 3) and cAMP-dependent phosphorylation (Fig. 4), indicate that the in vitro and
in vivo autophosphorylation sites of MHC-PKC are the same
and are located within the ST domain. The finding of relatively high
phosphorylation levels of MHC-PKC prior to cAMP stimulation (Fig. 4,
A and B) is consistent with the results presented
in Fig. 2, in which MHC-PKC from Ax2 extract migrated on SDS-PAGE with
an apparent molecular mass consistent with migration of the kinase in
its autophosphorylated form (1).
MHC-PKC
We reported previously that, on cAMP
stimulation, MHC-PKC translocates to the membrane, presumably as part
of the kinase activation mechanism. This translocation coincides with
the kinase phosphorylation (11). It was therefore of interest to study
the localization properties of MHC-PKCST on cAMP stimulation.
Ax2 and MHC-PKCST cells were developed and treated with caffeine,
stimulated with cAMP, and lysed using sonication, and the MHC-PKC and
MHC-PKC
ST were immunoprecipitated from the soluble and the insoluble
fractions using specific MHC-PKC polyclonal antibody (see
"Experimental Procedures"). Fig. 5 shows that, prior to cAMP stimulation, about 30% of the MHC-PKC resided in the insoluble fraction, whereas cAMP stimulation was followed by a rapid transient association of up to about 60% of the MHC-PKC with the membrane fraction, as reported previously (11). In contrast, about 70% of
MHC-PKC
ST remained in the membrane regardless of cAMP stimulation (Fig. 5). These results indicate that the MHC-PKC autophosphorylation mechanism is involved in the dissociation of the kinase from the membrane.
MHC-PKC, but Not MHC-PKC
To examine the possible role of
MHC-PKC autophosphorylation in the activation of the kinase, we studied
the specific activity of MHC-PKC and MHC-PKCST in response to cAMP
stimulation. Ax2 and MHC-PKC
ST cells were stimulated with cAMP, and
the kinase was solubilized from cell membranes and assayed for kinase
activity as described under "Experimental Procedures." Although
Dictyostelium contains several myosin heavy chain kinases,
all except MHC-PKC are located in the cytosol (for review, see Tan
et al. (30)). Furthermore, cells in which the MHC-PKC was
eliminated do not show MHC phosphorylation activity in their membranes
(4). Accordingly, all subsequent kinase assays were performed on
MHC-PKC or MHC-PKC
ST that were solubilized from the cell membrane
fraction.
Fig. 6 shows that cAMP stimulation of Ax2 cells resulted
in a transient increase in membrane-associated MHC-PKC kinase activity as reported previously (11). These cAMP-stimulated increases in MHC-PKC
activity coincided with the cAMP-stimulated membrane-association (Fig.
5) and phosphorylation (Fig. 4) of MHC-PKC, suggesting that these
processes are linked to each other and may be required for the
activation of MHC-PKC, as was proposed previously (11). In contrast,
cAMP stimulation of MHC-PKCST did not increase the kinase activity
(Fig. 6). MHC-PKC
ST showed a basal level of MHC kinase activity with
magnitude similar to that shown by MHC-PKC isolated from nonstimulated
Ax2 cells (Fig. 6). These results indicate that MHC-PKC
autophosphorylation plays a role in the cAMP-dependent
activation of the kinase. Interestingly, MHC-PKC
ST activity
decreased 60 s after cAMP stimulation, reaching a level of
activity similar to that of MHC-PKC determined 120 s after cAMP
stimulation. The decrease in MHC-PKC
ST activity indicates that, in
addition to autophosphorylation, there is another mechanism(s) that
regulate the activity of MHC-PKC. One such possible mechanism is a
cGMP-dependent phosphorylation (11).
MHC-PKC
We reported previously that MHC-PKC is
the kinase that phosphorylates MHC in response to cAMP stimulation (4).
We now tested how the cAMP-induced MHC phosphorylation is affected by
the inability of MHC-PKCST to undergo autophosphorylation and to
increase its activity in a cAMP-dependent manner. As shown
in Fig. 7A Ax2 cells, but not MHC-PKC
ST
cells, responded to cAMP stimulation by an increase in MHC
phosphorylation. These results indicate that deletion of the
autophosphorylation domain of MHC-PKC resulted in elimination of the
cAMP-dependent MHC phosphorylation. These results are
consistent with the finding that the MHC-PKC autophosphorylation plays
an important role in the regulation of MHC-PKC and, consequently in the
regulation of myosin II. Unstimulated developed Ax2 and MHC-PKC
ST
cells exhibited the same basal levels of MHC phosphorylation (Fig.
7A).
MHC phosphorylation regulates the distribution of myosin II within the
cell (4, 5, 6, 11). In order to study what happens to the localization
properties of myosin II in the MHC-PKCST mutant cells when there is
no cAMP-dependent increase in the MHC-PKC
ST activity, we
isolated cAMP-stimulated actin-enriched Triton-insoluble cytoskeletons
(see "Experimental Procedures"). 33% of the myosin II was
insoluble in unstimulated developed Ax2 cells (Fig. 7B). Addition of cAMP resulted in a rapid accumulation of myosin II associated with the Triton-insoluble cytoskeleton (up to 65%), followed by an increase in myosin II solubility (Fig. 7B),
as found previously (5, 6, 11). However, about 50% of the myosin II
was already insoluble in unstimulated developed MHC-PKC
ST cells.
Addition of cAMP resulted in a gradual increase in myosin II
insolubility (up to 70%). In contrast to wild type cells, the increase
in myosin II insolubility was not followed by a decrease in its
insolubility. These findings are similar to those obtained for
MHC-PKC
cells (4).
Previous studies have suggested a correlation between PKC activation and its autophosphorylation (20, 31, 32, 33, 34). The autophosphorylation sites located at the COOH-terminal tail of several PKC are important in the regulation of the kinase activity and subcellular localization (18, 20, 21, 29). Here we have examined the functional role of autophosphorylation of MHC-PKC using deletion and biochemical analyses. Our results indicate that (i) it is the ST domain which is autophosphorylated in vitro and in vivo in response to cAMP stimulation and (ii) that MHC-PKC autophosphorylation plays a role in its partition into the cytosol and its activation in vivo.
We have previously shown that cAMP stimulation of Dictyostelium cells resulted in several changes in MHC-PKC behavior (11). First, cAMP stimulation causes MHC-PKC to translocate to the membrane. Second, translocation coincides with increases in MHC-PKC phosphorylation. A third change is an increase in MHC-PKC activity. The association of MHC-PKC with the membrane is necessary for MHC-PKC activation since the cytosolic MHC-PKC has a very low kinase activity. There are two types of MHC-PKC phosphorylation, which are different in their extent and their sites; autophosphorylation, which may occur at the membrane and accounts for most of the MHC-PKC phosphorylation, and cGMP-dependent phosphorylation, possibly via cGMP-dependent protein kinase which may take place in the cytosol. The two different phosphorylations occur on different serine and/or threonine residues in MHC-PKC (11).
Here, deletion of the ST domain abolished the in vitro
MHC-PKC autophosphorylation. However, in vivo MHC-PKCST
still exhibits a low level of phosphorylation which may represent
cGMP-dependent phosphorylation. These results are
consistent with our previous report that MHC-PKC and MHC-PKC
ST are
phosphorylated in vitro by cGMP-dependent
protein kinase (11). A similar phosphorylation of PKC by a heterologous
kinase and its involvement in the regulation of PKC has also been
reported for PKC
(35), however the identity of this kinase is
unknown. We suggested previously that a plausible candidate for a PKC
kinase is a cGMP-dependent protein kinase (11).
Phosphorylation of MHC-PKC by another kinase may be required for the
kinase translocation to the membrane which is required for the kinase
activation, whereas the MHC-PKC autophosphorylation may be required for
its activation and membrane dissociation.
Previous studies with mammalian PKC have shown that elimination of the
COOH-terminal autophosphorylation sites fully inactivates the kinase
(21, 29). In contrast, upon deletion of the ST domain the MHC-PKC
retained its basal kinase activity and lost the
cAMP-dependent increases in its activity. These findings
may indicate that the expressed protein is folded properly and the absence of cAMP-dependent increases in the kinase activity
was a result of the ST domain deletion, it further suggests that this domain plays a key role in the cAMP-dependent kinase
activation. It is noteworthy that the basal level of MHC-PKCST
activity decreased 60 s after cAMP stimulation. This may result
from phosphatases removing the phosphates that were incorporated into
the MHC-PKC
ST by cGMP mechanism thereby decreasing the kinase
activity.
Deletion of the ST domain results in kinase that is unable to partition into the cytosol, indicating that the autophosphorylation of the ST domain plays a role in the dissociation of the kinase from the membrane. The seemingly contradictory results that autophosphorylation is required for both kinase activation and membrane dissociation may be explained as follow: the cluster of the 21 serine/threonine residues is autophosphorylated in two steps. In the first step only a fraction of the sites is autophosphorylated and this autophosphorylation is required for the activation of the kinase in response to cAMP. In the second step the remaining sites are autophosphorylated and this phosphorylation is required for dissociation of the kinase from the membrane. We are currently attempting to express MHC-PKC proteins in which the autophosphorylation sites are randomly converted to alanine residues. Experiments with these altered MHC-PKC proteins will enable us to explore the in vivo role of the different autophosphorylation sites.
The myosin II Triton-solubility and phosphorylation characterization in
MHC-PKCST are similar to that in the mutant from which the MHC-PKC
was eliminated (4). The inability of MHC-PKC
ST to increase its
activity in response to cAMP stimulation is also reflected in the state
of MHC phosphorylation and its Triton-solubility; MHC in MHC-PKC
ST
cells is not phosphorylated in response to cAMP stimulation. These
results are consistent with the findings that the
cAMP-dependent MHC phosphorylation is carried out by
MHC-PKC (4). If the enzyme does not respond catalytically to cAMP
stimulation, the cell cannot respond to cAMP stimulation by
phosphorylating the MHC.
The aberrant cAMP-dependent myosin II Triton-solubility in
MHC-PKCST cells is consistent with the finding that it is through MHC phosphorylation that myosin II distributed throughout the cell in
response to cAMP stimulation. MHC-PKC
ST and MHC-PKC cells (4)
contain highly Triton-insoluble myosin II. This contrasts with wild
type cells in which cAMP stimulation resulted in translocation of
myosin II to the cortex, followed by dissociation of myosin II from the
membrane fraction (5, 6, 11). In the two mutant cell lines, 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
ST
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. cAMP stimulation, additional myosin II
translocates to the cytoskeleton and, subsequently, cannot dissociate
from it because of the presence of catalytically inactive MHC-PKC
ST.
The observed phenotype of MHC-PKC
ST cells described here is not a
result of the expression of MHC-PKC
ST per se but rather results from
the absence of the ST domain; this is indicated by previous experiments
in which we engineered cells expressing MHC-PKC under the same actin
promoter, and the resulting cells had a wild type phenotype.
The detailed mechanism by which autophosphorylation could control MHC-PKC activity remains to be elucidated. One possibility is that the autophosphorylation triggers a conformational change that is important in relieving inhibition of MHC-PKC pseudosubstrate prototope similar to PKC (12). Supporting this hypothesis, the putative substrate (and pseudosubstrate) binding site of PKC is thought to be located near the COOH-terminal autophosphorylation sites (36, 37). This model is further supported by the observation that the catalytic fragment of PKC retains histone kinase activity, even though it can no longer autophosphorylate (16). This suggests that removal of the regulatory domain eliminates the requirement for COOH-terminal autophosphorylation. Alternatively, this autophosphorylation may be critical for enzyme-substrate recognition or for interaction of MHC-PKC with regulatory factors.
A model consistent with both our data and previous data is that MHC-PKC is first synthesized as an inactive precursor that is cytosolic (11). The kinase is then recognized by a cGMP-dependent protein kinase, which phosphorylates it, possibly at the C-2 and C-4 domains. This phosphorylation may be required for the kinase translocation to the cell membrane. The membrane-bound enzyme is then stimulated by autophosphorylation at the ST domain. The first step of autophosphorylation may activate the MHC-PKC causing MHC phosphorylation, followed by a second step of autophosphorylation resulting in a decrease in the enzyme's membrane affinity so that it partitions into the cytosol. This returns the enzyme to its basal state.
We thank Dr. Dietmar Manstein for pDXA-HY vector.