©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of a Dictyostelium Protein Kinase Required for Actin Activation of the MgATPase Activity of Dictyostelium Myosin ID (*)

Sheu-Fen Lee , Graham P. Côté (§)

From the (1) Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/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.


INTRODUCTION

Myosins comprise a superfamily of motor proteins characterized by a conserved amino-terminal 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) .

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,() 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.

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.


EXPERIMENTAL PROCEDURES

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.

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.


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.

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, -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).

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 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 KEDTA 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) .

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 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.

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 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 KEDTA 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

Starting material was 200 g of wet packed Dictyostelium.


  
Table: Kand Vvalues of the 110-kDa protein kinase for synthetic peptide substrates

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 Kand 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.).



FOOTNOTES

*
This research was supported by a grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Queen's University, Kingston, Ont. K7L 3N6, Canada. Tel.: 613-545-2998; Fax: 613-545-2497; E-mail: coteg@qucdn.queensu.ca.

The abbreviations used are: MyoA-E, Dictyostelium myosins IA-IE; Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

S. F. Lee and G. P. Côté, unpublished observations.


ACKNOWLEDGEMENTS

We thank Quintus G. Medley for helpful discussions.


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