From the
We have isolated a protein from Dictyostelium with a
molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can
stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein
incorporated phosphate into itself and into the heavy chain, but not
the light chain, of MyoD. Phosphorylation to 0.5 mol of
P
Myosins comprise a superfamily of motor proteins characterized
by a conserved amino-terminal
Five genes coding for
myosin I isozymes (myoA-E) have so far been identified in the
lower eukaryote Dictyostelium discoideum and the complete
sequences of four of these isozymes (A, B, D, and E)
have been reported
(6, 7, 8, 9) . Recent
evidence suggests additional Dictyostelium myosin I isozymes
probably also exist
(8, 10, 11) . The myosin I
isozymes from Dictyostelium can be divided into two
subfamilies; MyoB,
It has been
proposed that the Dictyostelium myosin I isozymes may be
involved in vesicle movement and in the extension and contraction of
pseudopods and filopodia
(16, 17) . Immunofluorescent
studies have shown that MyoB, MyoC, and MyoD are localized mainly in
the actin-rich regions at the leading edge of migrating
cells
(8, 11, 18) but similar studies for MyoA
and MyoE have not yet been performed. Attempts have been made to
directly ascertain the role of the myosin I isozymes by analysis of
cells in which myosin I genes have been rendered nonfunctional by
homologous recombination. Cells lacking a functional myoA or
myoB gene exhibit normal morphology and can complete
development but display subtle abnormalities including slower
translocation rates, an increase in the frequency of turning and
lateral pseudopod formation and a slight delay in chemotactic
aggregation
(19, 20, 21) . A preliminary analysis
of MyoD null cells similarly did not reveal any striking behavorial
defects (8). One explanation of these results is that there is
considerable functional overlap between the multiple Dictyostelium myosin I isozymes, so that deletion of any one isozyme has only a
minimal effect on cellular processes.
At present, little is known
concerning the mechanisms that regulate the motile activities of the
Dictyostelium myosin I isozymes in vivo. Some
evidence is available, though, to suggest that heavy chain
phosphorylation, which stimulates the actin-activated MgATPase activity
of the Acanthamoeba myosin I isozymes
(22, 23) ,
and is required for these enzymes to support movement in in vitro motility assays
(24, 25) , may also play a role in
regulating the properties of some of the Dictyostelium myosin
I isozymes. The strongest direct evidence is derived from a study
showing that the purified Acanthamoeba myosin I heavy chain
kinase (26) can stimulate the actin-activated MgATPase activity of one
of the Dictyostelium myosin I isozymes
(27) (later
identified as MyoB
(8) ). The studies described in this paper
were undertaken in order to identify and isolate an endogenous
Dictyostelium factor that could promote the actin-activated
MgATPase activity of Dictyostelium myosin I. By assaying
fractions for their ability to stimulate the actin-activated MgATPase
activity of MyoD we have purified a protein with a molecular mass of
110 kDa that displays intrinsic protein kinase activity, is activated
by autophosphorylation, and phosphorylates the MyoD heavy chain.
The pooled
material from the phosphocellulose column was subsequently
chromatographed over a hydroxylapatite column (Fig. 2) and then a
Mono Q column (Fig. 3). In both cases single peaks of an activity
capable of greatly stimulating the actin-activated MgATPase activity of
MyoD were obtained. An SDS-PAGE analysis of fractions collected
throughout the purification procedure (Fig. 4) indicated that the
hydroxylapatite pool consisted primarily of two proteins with molecular
masses of 110 and 55 kDa (Fig. 4, lane d).
Chromatography over Mono Q removed the 55-kDa protein, which eluted in
the flow-through, while the 110-kDa protein bound to the column and
eluted with those fractions capable of stimulating the actin-activated
MgATPase activity of MyoD (Fig. 4, lane e). The
approximate degree of purification of the 110-kDa band at each
chromatographic step in the purification procedure was estimated by
densitometry of Coomassie Blue-stained SDS gels (). The
110-kDa band represented less than 1% of the total protein in the
phosphocellulose pool and about 10% of the total protein in the
hydroxylapatite pool. The recovery of the 110-kDa protein from the Mono
Q column was quite low (about 10%) and reflects, in part, the fact that
the Mono Q column was pooled very narrowly. Usually only the two most
active fractions from the column were retained for further study. The
110-kDa band comprised 95% of the total protein in the Mono Q pool
(average of five separate preparations), while no other band
represented more than 1% of the total protein.
Gel
filtration chromatography of the 110-kDa protein on a Bio-Gel A-0.5m
column resulted in the elution of the protein at a Stokes radius
estimated as 3.6 nm (standards used were myoglobin, ovalbumin,
Assays performed over a 4-fold range of
MyoD concentrations in 5 mM KCl showed that the initial rate
of phosphorylation of MyoD by the autophosphorylated 110-kDa protein
kinase increased in almost direct proportion to the increase in MyoD
concentration (Fig. 6D). At the highest MyoD
concentration employed (0.56 µM) the specific activity for
the autophosphorylated 110-kDa kinase protein was close to 0.5
µmol/min/mg. Unfortunately, higher concentrations of MyoD could not
be tested since purified MyoD could not be obtained at a concentration
greater than about 1 µM. The results indicate that at the
concentration of MyoD used in most experiments (0.25-0.5
µM) the measured activity of the 110-kDa protein kinase is
probably well below its V
The present report
establishes the existence of an endogenous Dictyostelium myosin I heavy chain kinase and demonstrates that it is competent
to promote the actin-activated MgATPase of MyoD. It is noteworthy that
only a single peak of activity able to stimulate the actin-activated
MgATPase rate of MyoD was detected through all three chromatography
steps. This suggests that the 110-kDa protein kinase is likely to be
the major, and perhaps the only, endogenous factor that has a
significant ability to stimulate the actin-activated MgATPase activity
of MyoD. However, we have found, by assaying column fractions obtained
during the course of these studies for MyoD heavy chain kinase
activity, that the 110-kDa protein kinase is not the only
Dictyostelium protein kinase that can phosphorylate the MyoD
heavy chain.
The 110-kDa protein kinase
apparently exists as a monomer as judged by size exclusion
chromatography and requires autophosphorylation to display a high level
of MyoD phosphorylation activity (Fig. 6B). It is
interesting to note that the Acanthamoeba myosin I heavy chain
kinase, which has a molecular mass of 97 kDa on SDS gels, is also
monomeric and activated by autophosphorylation. However, the 110-kDa
protein kinase incorporated only a single mol of phosphate/mol via
autophosphorylation, while the Acanthamoeba myosin I kinase
autophosphorylates to a level of 7-8 mol of
phosphate/mol
(41) . When maximally autophosphorylated the
mobility of the Acanthamoeba kinase on SDS gels is retarded,
yielding an apparent molecular mass of 107 kDa. The single mol of
phosphate incorporated into the 110-kDa protein kinase caused no
detectable change in its mobility.
Early in the development of the
purification procedure it was found that significant stimulation of the
MyoD actin-activated MgATPase rate could be observed only if column
fractions were first preincubated with Mg
The
phosphorylated residue responsible for activation of the
Acanthamoeba myosin I isozymes has been identified by sequence
analysis as Ser-315 in the heavy chain of myosin IB, as Ser-311 in
myosin IC, and a threonine at the corresponding position in myosin
IA
(44) . It is noteworthy that Dictyostelium MyoA,
MyoB, MyoC, and MyoD all have a homologous phosphorylation site
(RXS/TXY) at the appropriate location in the heavy
chain
(4, 39) . MyoD contains a serine at this position
(8) and although it seems reasonable to suppose that this is the
serine phosphorylated by the 110-kDa protein kinase, a synthetic
peptide substrate based on this portion of the MyoD sequence was not a
good substrate for the kinase. In contrast, the Acanthamoeba myosin I kinase exhibits excellent activity when assayed with
short peptides based on the consensus phosphorylation sites of
Acanthamoeba myosin I. The Acanthamoeba and
Dictyostelium protein kinases have both been assayed using the
PC9 peptide (representing the phosphorylation site of Acanthamoeba myosin IC) and exhibit V
Starting material was 200 g of wet packed Dictyostelium.
Assays contained 12 nM autophosphorylated 110-kDa protein kinase and were performed as
described under ``Experimental Procedures'' with at least a
100-fold range of peptide concentration. Aliquots of 10 µl were
taken at 1, 2, and 3 min and spotted onto squares of P-81
phosphocellulose paper (34). Over this time period rates of phosphate
incorporation were linear for all peptide concentrations. The
K
We thank Quintus G. Medley for helpful discussions.
/mol increased the MyoD actin-activated MgATPase rate from
0.2 to 3 µmol/min/mg. Renaturation following SDS-gel
electrophoresis demonstrated that the 110-kDa protein contained
intrinsic protein kinase and autophosphorylation activity.
Autophosphorylation to 1 mol of P
/mol enhanced the rate at
which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The
rate of autophosphorylation was strongly dependent on the 110-kDa
protein kinase concentration, indicating an intermolecular reaction.
Synthetic peptides of 9-11 residues corresponding to the heavy
chain phosphorylation site of Acanthamoeba myosin IC and the
homologous sites in Dictyostelium myosin IB (MyoB) and MyoD
were poor substrates for the 110-kDa protein kinase. The 110-kDa
protein kinase was unable to phosphorylate the MyoB isozyme suggesting
that it may be specific for MyoD.
80-kDa head domain that binds to
actin filaments in an ATP-dependent manner. Phylogenetic analysis based
on a sequence comparison of the head domains divides the currently
known myosins into at least nine classes
(1, 2) . The
members of the myosin I class are single-headed and have been
identified in organisms ranging from amoebae to mammals. Myosin I
isozymes are monomeric and do not assemble into filaments but have
non-helical tail domains that can interact with phospholipids and, in
some cases, actin filaments in an ATP-independent
manner
(3, 4, 5) .
(
)
MyoC, and MyoD have heavy
chains of
125 kDa in size
(7, 8, 11) , while
MyoA and MyoE are smaller with heavy chains of
115
kDa
(6, 9) . The tail regions of MyoB, MyoC, and MyoD
contain a basic domain (TH1) at the head/tail junction that is
implicated in membrane binding
(12) , a domain consisting of a
repetitive GPX motif (TH2) that is responsible for ATP-independent
actin binding
(11, 13) and a domain with homology to SH3
domains (TH3)
(14, 15) . The shorter MyoA and MyoE tails
consist of only the TH1 membrane-binding domain.
Materials
ATP (grade I), Tes,
diisopropylfluorophosphate, bovine serum albumin, and histone 2A were
obtained from Sigma; pepstatin, leupeptin, and antipain were supplied
by Peptides International; and okadaic acid was from Calbiochem.
[-
P]ATP was from DuPont NEN. The PC9
peptide was a gift from H. Brzeska (National Heart, Lung, and Blood
Institute, NIH, Bethesda, MD) and the MyoB peptide was a gift from M.
A. L. Atkinson (University of Texas Health Science Center, Tyler, TX).
Dictyostelium myosin II
(28) and skeletal muscle actin
(29) were prepared as described previously.
Purification of Dictyostelium Myosin I
Isozymes
MyoD and MyoB were purified as described
previously
(30) , except that the actin-MyoD precipitate was
resolubilized in 0.25 M KCl, 4 mM MgATP, 1
mM dithiothreitol, 20 mM Tes, pH 7.5. Soluble
material obtained following centrifugation was immediately loaded onto
an FPLC Mono S HR 5/5 column (Pharmacia LKB Biotech Inc.) and eluted
with a 30-ml linear KCl gradient (0.25-0.55 M). The peak
of MyoD activity was pooled, dialyzed against 30% glycerol, 10
mM KCl, 1 mM dithiothreitol, 20 mM Tes, pH
7.5, and stored at -20 °C. Under these conditions MyoD was
stable in terms of enzymatic activity and SDS-PAGE
(31) profile
for several months. The MyoD was routinely >90% pure as judged by
SDS-PAGE (see Fig. 6A) and displayed a
KEDTA-ATPase of 0.5 µmol/min/mg when assayed as
described
(30) . The MgATPase activity of the MyoD used in these
studies was in the range of 0.06-0.07 µmol/min/mg and
increased to no more than 0.20 µmol/min/mg in the presence of 10
µM F-actin.
Figure 6:
Phosphorylation of the MyoD heavy chain by
the 110-kDa protein kinase. A, lane a shows a
Coomassie Blue-stained 15% SDS-polyacrylamide gel of purified MyoD and
lane b shows an autoradiogram of MyoD following a 40-s
incubation with the 110-kDa protein kinase in the presence of
[-
P]ATP. Phosphorylation was performed as
described under ``Experimental Procedures'' with final MyoD
and the 110-kDa protein concentrations of 0.35 and 0.1 µM,
respectively. A 40-µl aliquot of the reaction was loaded on the
gel. The 110-kDa protein was autophosphorylated for 40 min prior to the
assay at a concentration of 0.23 µM as described under
``Experimental Procedures.'' B, time courses of
phosphate incorporation into the MyoD heavy chain by the 110-kDa
protein kinase. Assays were carried out using the 110-kDa protein
kinase as isolated (
) or after autophosphorylation (
). The
kinase was autophosphorylated and the MyoD phosphorylation reactions
were performed as described in A. Incorporation of
P into the MyoD heavy chain was determined following
SDS-PAGE of 10-µl aliquots as described under ``Experimental
Procedures.'' At the time indicated by the arrow, an
additional 0.1 µM of the appropriate 110-kDa kinase was
individually added to both reactions. The inset shows an
autoradiogram of the phosphoamino acid analysis of maximally
phosphorylated MyoD performed as described in the legend to Fig.
5C. C, the MgATPase activity of unphosphorylated MyoD
(
) or MyoD maximally phosphorylated by the 110-kDa protein kinase
(
) as described in B, was measured at varying actin
concentrations as described under ``Experimental
Procedures.'' Assays contained 40 nM phosphorylated or
unphosphorylated MyoD in a volume of 200 µl. D, the
dependence of the specific activity of MyoD phosphorylation on the
concentration of MyoD. Phosphorylation assays contained 30 nM
autophosphorylated 110-kDa protein kinase and were performed as
described under ``Experimental Procedures.'' Aliquots of 10
µl were taken from the reactions at 20 and 40 s and
P
incorporation into the MyoD heavy chain was measured following
SDS-PAGE. In all reactions phosphate incorporation into the MyoD band
was linear over this time period.
Purification of the 110-kDa Protein
Kinase
Approximately 200 g of D. discoideum AX-3 was grown and harvested as described
(32) . All
subsequent procedures were performed at 0-4 °C. The cell
pellet was resuspended in 2 volumes of extraction buffer consisting of
12 mM sodium pyrophosphate, 1 mM EGTA, 2 mM
EDTA, 1 mM dithiothreitol, 1.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 2 µg/ml
antipain, 2 µg/ml leupeptin, and 30 mM Tes, pH 7.5, and
disrupted by 20 strokes in a tight-fitting Dounce homogenizer (Kontes).
Diisopropylfluorophosphate and solid KCl were then added to give final
concentrations of 1 and 100 mM, respectively. The homogenate
was stirred for 30 min and centrifuged at 100,000 g for 1 h (Beckman type Ti-60 rotor). The clear supernatant was
collected, diluted with 2 volumes of Buffer A (1 mM EGTA, 1
mM
-mercaptoethanol, 10 mM Tes, pH 7.5),
adjusted to pH 7.5 with 1 M Tris, pH 11, and mixed with 200 ml
of packed P-11 phosphocellulose (Whatman BioSystems) previously
equilibrated with Buffer A containing 40 mM KCl. The slurry
was gently stirred for 2 h, packed into a 5-cm diameter column, washed
extensively with Buffer A containing 40 mM KCl and 0.4
mM ATP, and eluted using a 1.5-liter linear KCl gradient
(0.04-0.85 M) in Buffer A containing 0.4 mM ATP
(Fig. 1). Fractions were assayed for the ability to stimulate the
actin-activated MgATPase activity of MyoD (as described below), and the
most active fractions, eluting at a KCl concentration of 0.2
M, were pooled. The pooled material was immediately applied to
a 2.5
4-cm column of hydroxylapatite HT (Bio-Rad) equilibrated
in 5% sucrose, 0.15 M KCl, 1 mM EGTA, 1 mM
-mercaptoethanol, and 2 mM KPO
, pH 7.0. The
column was washed extensively with equilibration buffer and eluted with
a 200-ml linear KPO
gradient (0.002-0.3 M,
pH 7.0) in the equilibration buffer (Fig. 2). The peak of
activity eluting at 50 mM KPO
was pooled, dialyzed
overnight against 1 liter of 5% sucrose, 40 mM KCl, 1
mM dithiothreitol, 20 mM Tris, pH 7.8, and loaded
onto an FPLC Mono Q HR 5/5 column (Pharmacia LKB Biotech Inc.)
equilibrated with the same buffer. After extensive washing with the
equilibration buffer the column was eluted with a 35-ml linear KCl
gradient (0.04-0.2 M) (Fig. 3). The most active
fractions, eluting at 0.13 M KCl, were immediately pooled and
stored in liquid nitrogen in 50-µl aliquots.
Figure 1:
Phosphocellulose chromatography of the
initial high speed supernatant. A high speed supernatant obtained from
200 g wet weight packed Dictyostelium was chromatographed over
a phosphocellulose P-11 column as described under ``Experimental
Procedures.'' The flow rate was 80 ml/h and fractions of 13 ml
were collected. The presence of ATP in the column buffer required that
the elution of protein be monitored using the Bradford assay
(). Salt concentration was determined by conductivity
measurements (
). Aliquots of 2 µl from every second fraction
were assayed for the ability to stimulate the actin-activated MgATPase
activity of MyoD (
) as described under ``Experimental
Procedures.'' The basal actin-activated MgATPase of the MyoD used
in these experiments was 0.15 µmol/min/mg. In the absence of added
MyoD, none of the column fractions displayed significant
actin-activated MgATPase activity. Fractions that activated MyoD
activity eluted at a KCl concentration of 0.2 M and were
pooled as indicated by the solid
bar.
Figure 2:
Chromatography of the phosphocellulose
pool over hydroxylapatite. The pooled material from the
phosphocellulose column was chromatographed over a 2.5 4-cm
hydroxylapatite HT column as described under ``Experimental
Procedures.'' The flow rate was 20 ml/h and fractions of 2 ml were
collected. The absorbance at 280 nm (
) and the
conductivity (
) were monitored. Aliquots of 2 µl were assayed
for the ability to activate the actin-activated MgATPase activity of
MyoD (
) as described in the legend to Fig. 1. Active fractions
eluted at a KPO
concentration of 50 mM and were
pooled as indicated by the solid
bar.
Figure 3:
Chromatography of the hydroxylapatite pool
over Mono Q. The pooled material from the hydroxylapatite column was
chromatographed over a Mono Q HR 5/5 as described under
``Experimental Procedures.'' The flow rate was 20 ml/h and
fractions of 0.5 ml were collected. The absorbance at 280 nm
() and the conductivity (
) were monitored.
Aliquots of 1 µl taken from 10
diluted fractions were
assayed for the ability to activate the actin-activated MgATPase
activity of MyoD (
) as described in the legend to Fig. 1. Active
fractions eluted at a KCl concentration of 0.13 M and were
pooled as indicated by the solid
bar.
Actin-activated MgATPase Assays
The MgATPase
activity of MyoD was assayed in a buffer containing 5 mM
MgCl, 1 mM [
-
P]ATP (2
Ci/mol), 1 mM dithiothreitol, and 10 mM Tes, pH 7.5.
Unless stated otherwise, actin-activated MgATPase assays were performed
in the same buffer with 10 µM F-actin. Assays were carried
out for 20 min at 25 °C and ATPase activity was measured by the
release of
P
from the
[
-
P]ATP
(33) . Assays designed to
measure the ability of fractions to stimulate the actin-activated
MgATPase activity of MyoD were performed as follows. First, the
fractions were dialyzed against 20 mM KCl, 1 mM
dithiothreitol, and 20 mM Tes, pH 7.5, by spotting 10-µl
samples of the fractions onto a 0.025-micron type VS filter (Millipore)
floating on the dialysis solution. After 1 h an aliquot of the dialyzed
sample was removed and added to a tube containing 10 µl of 0.25
mM ATP, 2 mM MgCl
, 1 mM
dithiothreitol, 10 nM okadaic acid, 0.1 mg/ml bovine serum
albumin, and 10 mM Tes, pH 7.0. In most cases a preincubation
for 20 min at 25 °C was performed and then a 10-µl sample of
MyoD (0.5-1 µM) was added and incubation allowed to
proceed for another 20 min at 25 °C. At the end of this time 180
µl of MgATPase buffer containing actin was added and the MgATPase
rate determined as described above. The volume of the column fractions
to be assayed was chosen so that the most active fraction always
produced less than the maximum possible MyoD actin-activated MgATPase
(
3 µmol/min/mg). Specific activities were determined by
varying the length of time (from 1 to 20 min) that MyoD was incubated
with the fractions. A linear relationship between the time of
incubation and the percent enhancement of the MyoD actin-activated
MgATPase activity was observed so long as MyoD was stimulated to less
than 50% of its maximal activity. This result indicates that addition
of the actin-containing MgATPase assay buffer essentially terminated
further activation of MyoD. Activities determined by this assay are
presented in units, with 1 unit being arbitrarily defined as the
activity required to cause a 100% increase in the basal actin-activated
MgATPase rate of MyoD.
Protein Kinase and Phosphoamino Acid Assays
Assays
were carried out by addition of substrate to an equal volume of 2
mM MgCl, 2 mM dithiothreitol, 20
mM Tes, pH 7.0, and 0.5 mM
[
-
P]ATP (500 Ci/mol) followed by addition
of the 110-kDa protein kinase to initiate the reaction. Kinase and
substrate concentrations for each experiment are provided in the figure
and table legends. When substrate concentrations were varied, the final
ionic strength was kept constant by addition of the appropriate buffer.
Autophosphorylation of the 110-kDa protein kinase was performed by
diluting the kinase into 2 volumes of 0.4 mM
[
-
P]ATP (500 Ci/mol), 3 mM
MgCl
, 1.5 mM dithiothreitol, 0.15 mg/ml bovine
serum albumin, and 15 mM Tes, pH 7.0. All reactions were
performed at 25 °C. Protein phosphorylation or autophosphorylation
activities were determined by removing aliquots of 10-20 µl
from the assays at time intervals and immediately adding them to a
one-fifth volume of boiling hot SDS sample buffer (5% SDS, 30% sucrose,
2.5%
-mercaptoethanol). Samples were subjected to SDS-PAGE, the
gel stained with Coomassie Blue and the appropriate protein band
excised and counted in liquid scintillation fluid in a scintillation
counter. Kinase activity assays were carried out such that less than
0.1 mol of P
was incorporated per mole of protein. Under
these conditions the incorporation of
P into substrate was
linear with time and proportional to the amount of kinase. Phosphate
incorporation into the synthetic peptides was determined by spotting
10-µl aliquots of the assay mixture onto squares of P-81
phosphocellulose paper (Whatman) that were then washed in 0.1%
phosphoric acid as described
(34) . Phosphoamino acid analysis
was performed essentially as described
(35) .
Renaturation Following SDS-PAGE
The 110-kDa
protein was subjected to SDS-PAGE and renatured in the polyacrylamide
gel following incubation in 6 M guanidine HCl as described
(36). The gel was then equilibrated in 2 mM MgCl,
1 mM dithiothreitol, 10 mM Tes, pH 7.5, and incubated
for 1 h at 25 °C in 3 ml of the same buffer containing 0.1
mM [
-
P]ATP (200 Ci/mol).
Radioactivity was removed by washing the gel extensively in 5%
trichloroacetic acid, 1% sodium pyrophosphate. The gel was then stained
with Coomassie Blue, dried, and subjected to autoradiography.
Miscellaneous Methods
Protein concentrations were
determined by the colorimetric assay of Bradford
(37) . In some
cases the concentrations of MyoD and the 110-kDa protein kinase were
determined following SDS-PAGE. The gels were stained with Coomassie
Blue, scanned at 596 nm using a laser densitometer (LKB 2202 Ultro),
and the areas of the peaks calculated. For both methods bovine serum
albumin was used as the standard. For autoradiography dried gels and
thin layer sheets were exposed at -80 °C to x-ray Hyperfilm
(Amersham Corp.) with an intensifying screen (Du Pont, Cronex Lightning
Plus).
RESULTS
Purification of a 110-kDa Protein That Stimulates the
Actin-activated MgATPase Activity of MyoD
As isolated, MyoD
displays an actin-activated MgATPase activity of less than 0.2
µmol/min/mg, which is considerably below the 2-8
µmol/min/mg actin-activated MgATPase rates reported for other
Acanthamoeba and Dictyostelium myosin I isozymes (23,
26, 27, 30, 38). Initial experiments in which MyoD was incubated with
crude Dictyostelium homogenates were unsuccessful in detecting
any stimulation of the MyoD actin-activated MgATPase activity. The
crude homogenates exhibited a high background level of actin-activated
MgATPase activity (perhaps resulting from the presence of myosin II)
that tended to obscure any activity contributed by the added MyoD.
However, following chromatography over a phosphocellulose P-11 column
fractions that were capable of stimulating the actin-activated MgATPase
activity of MyoD were readily detected (Fig. 1). Control assays
demonstrated that the column fractions alone displayed negligible
actin-activated MgATPase activity and that the observed activity was
dependent on the addition of both MyoD and actin. The most active
column fractions were capable of stimulating the actin-activated
MgATPase of MyoD greater than 10-fold, to a rate approaching 2.5
µmol/min/mg. It should be noted that in order to obtain this high
degree of activation it was found necessary to preincubate the column
fractions with MgATP prior to the addition of MyoD.
Figure 4:
SDS
gel analysis of fractions obtained during purification. Samples were
electrophoresed on an 8% SDS-polyacrylamide gel and stained with
Coomassie Blue. The samples and the amount of proteins loaded in each
lane are: a, initial homogenate, 20 µg; b, high
speed supernatant, 20 µg; c, phosphocellulose pool, 20
µg; d, hydroxylapatite pool, 10 µg; and e,
Mono Q pool, 1 µg. The molecular mass standards in kDa are
indicated to the left of the gel and were, from top to bottom: skeletal muscle myosin heavy chain,
-galactosidase, phosphorylase b, bovine serum albumin,
and ovalbumin.
The specific
activities of the fractions obtained following each column
chromatography step were determined by performing time courses of the
stimulation of MyoD actin-activated MgATPase activity as described
under ``Experimental Procedures.'' The specific activity of
the phosphocellulose pool was determined to be 11.5 units/min/mg,
giving a total activity at this stage of the purification of 2150
units/min (). Chromatography over hydroxylapatite resulted
in an 50-fold elevation in specific activity and a 2-fold rise in
total activity. The last step in the purification yielded a 1350-fold
increase in specific activity, despite the fact that the purity of the
110-kDa protein increased less than 10-fold. The total activity at this
step jumped to 72,000 units/min. It is apparent that some factor that
acts to prevent stimulation of the actin-activated MgATPase activity of
MyoD is removed as the purification procedure proceeds.
-globulin, and thyroglobulin). This value is consistent with the
native 110-kDa protein being a globular, monomeric protein. Assays of
the column demonstrated that the 110-kDa protein co-eluted exactly with
those fractions that stimulated the actin-activated MgATPase activity
of MyoD (data not shown).
Protein Kinase Activity of the 110-kDa Protein
The
110-kDa protein was found to incorporate P when incubated
in a buffer containing Mg
and
[
-
P]ATP while no phosphorylation occurred
in the absence of Mg
(Fig. 5A).
Further studies showed that a 2 mM Mg
concentration was optimal and that phosphorylation of the 110-kDa
protein could be supported by 1 mM Mn
but
not by Ca
(data not shown). The incorporation of
phosphate caused no detectable change in the mobility of the 110-kDa
protein on SDS gels (Fig. 5A). The 110-kDa protein was
still capable of incorporating
P after being subjected to
SDS-PAGE and renaturation in the gel (Fig. 5B). Since
this result strongly suggests that the 110-kDa protein contains
intrinsic protein kinase and autophosphorylating activity, it will
henceforth be referred to as the 110-kDa protein kinase.
Figure 5:
Autophosphorylation of the 110-kDa protein
kinase. A, the 110-kDa protein (final concentration 0.23
µM) was incubated as described under ``Experimental
Procedures'' but with no MgCl (lanes a and
c) or with a final concentration of 2 mM
MgCl
(lanes b and d). At 40 min
aliquots of 20 µl were boiled in SDS sample buffer and subjected to
SDS-PAGE. Lanes a and b show the Coomassie
Blue-stained gel and lanes c and d, the corresponding
autoradiogram. Note that the band at 66 kDa represents bovine serum
albumin. B, the 110-kDa protein (2 µg) was subjected to
SDS-PAGE, renatured in the gel, and incubated with
[
-
P]ATP as described under
``Experimental Procedures.'' An autoradiogram of the dried
gel is shown and demonstrates that the 110-kDa protein is
phosphorylated. C, time course of the autophosphorylation of
the 110-kDa protein kinase. The 110-kDa protein kinase was incubated as
described in A with 2 mM MgCl
. At the
indicated times aliquots of 20 µl were boiled in SDS sample buffer
and
P incorporation into the 110-kDa band was measured
following SDS-PAGE as described under ``Experimental
Procedures.'' The inset shows an autoradiogram of the
phosphoamino acid analysis of maximally phosphorylated 110-kDa protein.
Electrophoresis on cellulose sheets was carried out at pH 1.9
(migrating from bottom to top) and then at pH 3.5
(migrating from left to right). The position of
phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine
(P-Tyr) standards are indicated. D, the specific activity of
autophosphorylation of the 110-kDa protein kinase was determined in
assays carried out as described in C, above, except that the
concentration of the 110-kDa protein was varied from 0.04 to 0.4
µM. Aliquots of 20 µl were taken from the reactions at
2 and 4 min and
P incorporation into the 110-kDa band was
measured following SDS-PAGE. In all reactions phosphate incorporation
into the 110-kDa band was linear over this time
period.
A time
course of the autophosphorylation reaction indicated that a maximum of
1 mol of phosphate could be incorporated per mole of the 110-kDa
protein kinase (Fig. 5C). Phosphoamino acid analysis of
the maximally autophosphorylated 110-kDa protein kinase revealed only
phosphoserine (Fig. 5C, inset). The rate at
which the 110-kDa protein kinase autophosphorylated was highly
dependent on its concentration and ionic strength. A 10-fold increase
in the concentration of the 110-kDa protein kinase resulted in a
20-fold increase in the specific activity of autophosphorylation
(Fig. 5D), while activity measured at 200 mM
KCl was only 10% of that obtained at 20 mM KCl (data not
shown).
Phosphorylation of MyoD Heavy Chain by the 110-kDa
Protein Kinase
SDS-PAGE analysis demonstrated that the purified
MyoD consisted of a 125-kDa heavy chain and a 16-kDa light chain
(Fig. 6A, lane a). Incubation of MyoD with the
110-kDa protein kinase in the presence of Mg and
[
-
P]ATP resulted in the incorporation of
P into the MyoD heavy chain but not the light chain
(Fig. 6A, lane b). No phosphorylation of MyoD
was observed if Mg
or the 110-kDa protein kinase was
omitted (data not shown). The time course of MyoD phosphorylation was
initially linear when assays were performed using autophosphorylated
110-kDa protein kinase, but displayed a distinct initial lag phase when
the unphosphorylated 110-kDa protein kinase was used (Fig. 6B).
It is likely that some portion of the 110-kDa protein kinase is
autophosphorylated during the initial lag phase, yielding the
subsequent increased rate of MyoD phosphorylation. If only the initial
rates (measured over the first 40 s) are compared, the
autophosphorylated form of the 110-kDa protein kinase displays a
40-fold greater rate of MyoD phosphorylation than the unphosphorylated
form. A maximum of only about 0.5 mol of P
per mole of MyoD
heavy chain was incorporated by the 110-kDa protein kinase
(Fig. 6B). This stoichiometry was obtained in several
experiments, and was not exceeded even if additional aliquots of
autophosphorylated 110-kDa protein kinase were added to the
phosphorylation reaction (Fig. 6B). Maximally
phosphorylated MyoD contained only phosphoserine
(Fig. 6B, inset) and displayed a triphasic
MgATPase curve as a function of actin concentration
(Fig. 6C).
for MyoD. Increasing
the KCl concentration to 50 mM inhibited the rate of MyoD
phosphorylation by 50%, while at 150 mM KCl no MyoD heavy
chain phosphorylation was detected (data not shown).
Substrate Specificity of the 110-kDa Protein
Kinase
Synthetic peptides corresponding to the sequence
surrounding the heavy chain phosphorylation site of Acanthamoeba myosin IC (PC9)
(39) and the homologous regions of MyoB and
MyoD were poor substrates for the 110-kDa protein kinase. These
peptides displayed V values considerably lower
than the rate measured with MyoD as a substrate, even though, as
discussed above, MyoD was employed at a concentration that was likely
well below its K
(). The
110-kDa protein kinase-phosphorylated histone fraction 2A (12.5
µM concentration) with a rate of 0.15 µmol/min/mg but
did not phosphorylate purified Dictyostelium myosin II or
MyoB. The MyoB used in these experiments displayed good
K
EDTA activity (5 µmol/min/mg), indicating that
the active site was intact, and a low actin-activated MgATPase activity
of 0.2 µmol/min/mg, suggesting that it was not already activated by
phosphorylation.
DISCUSSION
The actin-activated MgATPase activity exhibited by myosins is
considered to be the physiologically important ATPase activity and to
be correlated with the ability of myosin to drive motile
events
(40) . We have previously reported that MyoD is purified
in a form that displays minimal actin-activated MgATPase
activity
(30) . By using an assay based on the ability of
fractions to stimulate the MyoD actin-activated MgATPase activity, we
have isolated a 110 kDa protein from Dictyostelium that
contains intrinsic protein kinase activity and incorporates phosphate
into the MyoD heavy chain. The characterization of the 110-kDa protein
as a MyoD heavy chain kinase confirms that heavy chain phosphorylation
plays a central role in activating the motile activities of
Dictyostelium myosin I isozymes. While the critical importance
of heavy chain phosphorylation in activating the actin-activated
MgATPase activities of the three myosin I isozymes from
Acanthamoeba has been well documented (23, 38), the only
previous direct experimental evidence showing that heavy chain
phosphorylation stimulates the activity of a Dictyostelium myosin I, MyoB, was obtained using a purified Acanthamoeba myosin I heavy chain kinase
(27) . More recent studies have
shown that MyoB can be purified from Dictyostelium extracts in
both an inactive and active form, which apparently represent different
phosphorylation states of the enzyme
(30) .
(
)
and ATP.
This requirement can now be explained by the need for the 110-kDa
protein kinase to be activated by autophosphorylation. The rate of
autophosphorylation was found to be strongly dependent on the
concentration of the 110-kDa protein kinase (Fig. 5D).
Mechanistically, autophosphorylation could occur through either an
intermolecular or intramolecular process. A plot of the log of the
initial reaction velocity versus the log of the enzyme
concentration (Van't Hoff plot) is expected to yield a straight
line with a slope equal to 1 for an intramolecular mechanism of
autophosphorylation or a straight line with a slope equal to 2 for an
intermolecular mechanism
(42, 43) . When the data in
Fig. 5D were replotted in this manner a straight line
with a slope of 2.3 was obtained, consistent with an intermolecular
process. The dependence of the rate of autophosphorylation on the
concentration of the 110-kDa protein kinase may partially explain the
increase in total MyoD activating activity that is observed throughout
the purification procedure and most notably following the final Mono Q
column step () since the kinase is concentrated as it is
purified. The apparent increase in activity might also reflect the
removal of protein phosphatases that reverse the autophosphorylation of
the 110-kDa protein kinase or the phosphorylation of MyoD.
values of 14
µmol/min/mg
(39) and 0.06 µmol/min/mg, respectively. One
interpretation of these results is that the 110-kDa protein kinase has
a stricter substrate specificity than the Acanthamoeba myosin
I kinase, whose major determinants for substrate recognition consist
only of a basic amino acid on the amino-terminal side of the
phosphorylation site and a tyrosine two residues away on the
carboxyl-terminal side
(39) . This interpretation is reinforced
by the somewhat surprising observation that the 110-kDa protein kinase
is unable to phosphorylate MyoB, which previously has been shown to be
phosphorylated and activated by the Acanthamoeba myosin I
kinase
(27) . The MyoB used in the present experiments was not
denatured since it displayed a high K
EDTA activity and
was unlikely to have been isolated in a predominantly phosphorylated
form, since it displayed a low actin-activated MgATPase activity. If
the 110-kDa protein kinase is specific for MyoD this would have
interesting implications for the in vivo regulation of myosin
I isozymes. Evidence from gene targetting experiments suggests that
there is functional overlap between the Dictyostelium myosin I
isozymes, but there are also indications that different myosin I
isoforms may play distinctive roles in cell movement, perhaps the best
example being the demonstration that only the Acanthamoeba myosin IC isozyme is involved in contractile vacuole
function
(45) . If different myosin I isozymes have unique
functions, it would seem reasonable that mechanisms must exist to
individually regulate each isozyme. It is important to note that MyoB
has been purified from Dictyostelium extracts in a form that
displays significant actin-activated MgATPase activity
(30) ,
implying that a heavy chain kinase capable of activating MyoB does
exist. It is conceivable that Dictyostelium contain a family
of myosin I heavy chain kinases, each able to activate a subset of the
myosin I isozymes.
Table:
Purification of Dictyostelium 110-kDa protein
kinase
Table: Kand
V
values of the 110-kDa protein kinase for
synthetic peptide substrates
and V
values
were obtained by fitting direct plots of the data to the equation, v
= V
/(1 +
K
/[peptide]) using a nonlinear
curve-fitting program based on the Marquardt-Levenberg algorithm
(included in the software SigmaPlot 4.1, Jandel Scientific, Corte
Madera, CA.).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.