©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Analysis of Myosin Heavy Chain Kinase A from Dictyostelium
EVIDENCE FOR A HIGHLY DIVERGENT PROTEIN KINASE DOMAIN, AN AMINO-TERMINAL COILED-COIL DOMAIN, AND A DOMAIN HOMOLOGOUS TO THE beta-SUBUNIT OF HETEROTRIMERIC G PROTEINS (*)

(Received for publication, August 10, 1994; and in revised form, October 28, 1994)

Lidia M. Futey (1) Quintus G. Medley (2) Graham P. Côté (2) Thomas T. Egelhoff (1)(§)

From the  (1)Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, Ohio 44106 and the (2)Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report here the cloning and characterization of the gene encoding the 130-kDa myosin heavy chain kinase (MHCK A) from the amoeba Dictyostelium. Previous studies have shown that purified MHCK A phosphorylates threonines in the carboxyl-terminal tail portion of the Dictyostelium myosin II heavy chain and that phosphorylation of these sites is critical in regulating the assembly and disassembly of myosin II filaments in vitro and in vivo. Biochemical analysis of MHCK A, together with analysis of the primary sequence, suggests that the amino-terminal 500 amino acids form an alpha-helical coiled-coil domain and that residues from position 860 to the carboxyl terminus (residue 1146) form a domain with significant similarity to the beta-subunit of heterotrimeric G proteins. No part of the MHCK A sequence displays significant similarity to the catalytic domain of conventional eukaryotic protein kinases. However, both native and recombinant MHCK A displayed autophosphorylation activity following renaturation from SDS gels, and MHCK A expressed in Escherichia coli phosphorylated purified Dictyostelium myosin, confirming that MHCK A is a bona fide protein kinase. Cross-linking studies demonstrated that native MHCK A is a multimer, consistent with the presence of an amino-terminal coiled-coil domain. Southern blot analysis indicates that MHCK A is encoded by a single gene that has no detectable introns.


INTRODUCTION

Conventional myosin (myosin II) has been implicated as having important roles in a wide array of cellular contractile events. In Dictyostelium, genetic and cellular analyses have demonstrated that myosin II is essential for cytokinesis, multicellular morphogenesis, capping of cell-surface receptors, and efficient amoeboid locomotion(1, 2, 3, 4, 5) . Although myosin II seems to play similar roles in a variety of cell types, the in vivo mechanisms regulating myosin II assembly and localization during these processes are not well understood in most systems. In the Dictyostelium system, strong evidence now indicates that myosin II heavy chain phosphorylation has a critical role in the control of myosin assembly and localization within cells.

Purified myosin II from Dictyostelium can be phosphorylated by endogenous myosin heavy chain (MHC) (^1)kinases on target sites near the carboxyl-terminal portion of the myosin tail. Two distinct threonine-specific Dictyostelium MHC kinases (MHCKs) have been purified to homogeneity(6, 7) , and phosphorylation of purified MHC by either of these enzymes is capable of driving the disassembly of bipolar myosin filaments at physiological salt concentrations. One of these enzymes (MHCK A) has a molecular mass of 130 kDa on SDS gels and is expressed both in growth phase cells and in starved cells that have entered the Dictyostelium developmental pathway(8) . In vitro target sites for this enzyme have been mapped to threonine residues 1823, 1833, and 2029 of Dictyostelium MHC (9, 10) .

The second Dictyostelium MHCK has a molecular mass of 84 kDa and is expressed only during development(11) . This enzyme has been recently cloned and, based on sequence homology, seems to be a member of the protein kinase C family(6, 11) . Phosphorylation of Dictyostelium myosin II by the 84-kDa MHCK promotes filament disassembly in vitro(6) , although it is not clear whether the target sites for the 84-kDa MHCK are identical to those for MHCK A. Dictyostelium also seems to contain an MHC kinase that phosphorylates serine residues and may contain additional threonine-specific MHC kinases, but these activities have not been purified(12, 13) .

Exposure of Dictyostelium cells to chemoattractants or to agents that stimulate receptor capping results in a transient recruitment of myosin II from a soluble pool into the cytoskeleton, suggesting that regulated assembly and disassembly of myosin filaments into the cytoskeleton are important for these processes(14, 15, 16, 17) . MHC phosphorylation on threonine residues has been observed in vivo concomitant with the transient relocalization of myosin II to the cytoskeleton, suggesting that MHC kinases may play a role in redirecting myosin II back to the soluble pool after chemoattractant-stimulated recruitment(15, 18, 19) . In previous work, the target sites for MHCK A (threonine residues 1823, 1833, and 2029 on MHC) were mutated either to alanine, to inhibit phosphorylation, or to aspartic acid, to mimic phosphorylation(14) . Elimination of these MHCK A target sites resulted in gross overassembly of myosin into the cytoskeleton and caused an array of partial defects in myosin-related contractile processes. Conversion of these MHCK A target sites to aspartic acid rendered the myosin incapable of assembling functionally into the cytoskeleton. The phenotypes of the site-directed MHC mutants provide strong support for the idea that MHCK A has a central role in the control of myosin localization in vivo.

Biochemical studies on purified MHCK A have shown that the kinase requires autophosphorylation for activity, but have been unable to identify physiologically relevant molecules that may serve to regulate MHCK A activity in vivo(7, 20) . To gain further insights into the mechanisms regulating myosin II phosphorylation and localization in vivo, we have isolated and characterized the gene for the 130-kDa MHCK A. The primary sequence reported here demonstrates, surprisingly, that MHCK A does not display any significant homology to the catalytic domains of conventional eukaryotic protein kinases, yet biochemical analysis of the recombinant MHCK A protein verifies the presence of intrinsic protein kinase activity. In addition, MHCK A appears to contain a coiled-coil domain that may function in oligomerization of the kinase and a carboxyl-terminal domain containing WD sequence repeats similar to beta-subunits of heterotrimeric G proteins.


MATERIALS AND METHODS

Monoclonal Antibodies

Monoclonal antibodies were prepared by standard techniques following immunization of mice with native MHCK A, purified as described(7) . Twenty-four hybridomas secreting antibodies against MHCK A were detected by enzyme-linked immunosorbent assay and subcloned. The antibody designated A1 was purified using protein A-Sepharose from the ascites fluid of mice injected with the corresponding hybridoma line. The other three monoclonal antibodies used in this study (A16, A21, and A22) were derived from hybridoma supernatants.

For immunoblot analysis, washed Dictyostelium cells were boiled directly in SDS sample buffer and subjected to SDS-PAGE. Samples were either stained directly with Coomassie Blue or electroblotted to Immobilon-P (Millipore Corp.) and probed with monoclonal antibody. Signal was detected using a horseradish peroxidase-linked goat anti-mouse antibody and a chemiluminescence assay (Amersham Corp.).

Cloning of the MHCK A Gene and Sequence Analysis

The MHCK A gene was cloned from a gt11 cDNA expression library made from Dictyostelium RNA (CLONETECH). Standard protocols were used for library plating, screening, and all phage manipulations(21) . The library was probed with a pool of two monoclonal antibodies (A16 and A21). Positive plaques were observed at 1/100 the frequency of plaques positive for a myosin heavy chain antiserum that was used as a control. The overall frequency of MHCK A-positive phage in the library was 1/10,000. Eight clones were picked for further characterization. Three clones were observed to react with only one of the four antibodies (either A1 or A16), one clone reacted with two antibodies (A1 + A22), one clone reacted with three of the antibodies (A1 + A16 + A22), and three of the isolated clones reacted with all four monoclonal antibodies. The cDNA inserts of the clones that reacted with three or less antibodies were determined to be 1.5 kb or less in size, while the inserts from clones reacting with all four antibodies were 2 kb or larger. These reactivity patterns suggested that each of the four antibodies recognized distinct epitopes and that the smaller inserts observed correspond to partial cDNA products. Inserts from two clones (3.1 and 1.1) that reacted with all four monoclonal antibodies were subcloned into plasmid vectors. Restriction enzyme analysis and subsequent sequence analysis indicated partial overlap between these inserts. The 3.1 insert spanned from nucleotides 21 to 2025 (as numbered in Fig. 2), and the 1.1 insert spanned from nucleotides 246 to 3465.


Figure 2: Sequence of the MHCK A gene. Nucleotide numbering is shown to the left, and amino acid numbering to right. Candidate residues for the nucleotide-binding site (GXGXXG at positions 778-783), invariant lysine (either position 795 or 808), and invariant glutamate (position 834) are doubleunderlined. Highly conserved residues of the carboxyl-terminal G-like repeat are boldfaceunderlined. Underlineditalic residues correspond to five regions that match peptide sequence derived from direct sequencing of purified MHCK A tryptic fragments, confirming the identity of the cloned gene.



DNA sequence analysis was performed by constructing nested deletions of each of these inserts from both ends using the Erase-a-Base system (Promega) and sequencing with the Sequenase system (U. S. Biochemical Corp.). All portions of the gene were sequenced at least once on both strands of the gene. Analysis of the compiled sequence indicated a complete 3`-end, but did not reveal an upstream start codon. Following Southern blot analysis, a plasmid library was constructed in ClaI-digested pBR328 from a size-selected portion of a ClaI digest of genomic Ax2 DNA. Colony screens were performed (21) with a 5`-probe derived from the 3.1 cDNA insert, allowing the isolation of a 1.5-kb ClaI genomic fragment that overlapped the cloned cDNA and contained upstream flanking sequences. Sequence analysis of this clone revealed a putative ATG start codon 21 bases beyond the 5`-end of the 3.1 clone. Upstream of this in-phase ATG codon, the sequence becomes extremely AT-rich (83%), a feature diagnostic for noncoding regions in the Dictyostelium genome.

Sequence analysis was performed with the Wisconsin GCG package running on a VAX computer. Data base searches with the BLAST program were performed via Internet at the National Center for Biotechnology Information at the National Institutes of Health. Computer analysis of alpha-helical coiled-coil potential was performed using an algorithm devised by Lupas et al. ((23) ; see also (22) ).

Southern Blot Analysis

Agarose gel electrophoresis, blotting to nitrocellulose, and hybridization were performed using standard conditions(21) . Final filter washes were performed in 1 times SSC + 0.1% SDS at 65 °C for 90 min. Genomic DNA was prepared as described previously(24) . Approximately 1 µg of genomic DNA was loaded per gel lane. Probes were made using a random primer method following the manufacturer's protocol (Boehringer Mannheim). Probe DNA fragments were isolated as restriction fragments from the cDNA. Probe A corresponds to nucleotides 122-1493 of the sequence as numbered in Fig. 2, probe B corresponds to nucleotides 1493-2025, and probe C corresponds to nucleotides 2765-3465.

Cross-linking Analysis

Purified native MHCK A (7) at 10 µg/ml in 5 mM TES, 0.1 M KCl, 6% glycerol, and 0.5 mM dithiothreitol, pH 7.5, was autophosphorylated for 30 min at 25 °C by the addition of 0.1 mM [-P]ATP (0.2 Ci/mmol). Cross-linking was carried out in 0.5 M KCl by the addition of 0.5 mM bis(sulfosuccinimidyl)suberate (Pierce) for 5 min at 25 °C.

Autophosphorylation Analysis

For renaturation of MHCK A purified from Dictyostelium, MHCK A (0.2 µg) was subjected to SDS-PAGE and renatured following incubation in 6 M guanidine HCl as described(25) . The gel was then incubated for 1 h at 25 °C with 3 ml of 10 mM TES, pH 7.5, 2 mM MgCl(2), 1 mM dithiothreitol, and 50 µM [-P]ATP (0.5 Ci/mmol). Radioactivity was removed by washing in 5% trichloroacetic acid, 1% sodium pyrophosphate. The gel was then stained with Coomassie Blue, dried, and exposed to x-ray film. For analysis of recombinant protein, the MHCK A gene was expressed in Escherichia coli strain BL21 from the vector pET21d (Novagen). Following induction with isopropyl-1-thio-beta-D-galactopyranoside, cells were washed and resuspended in 5 mM imidazole, 0.5 M NaCl, and 40 mM Tris, pH 7.9, and then lysed by sonication. An inclusion body fraction was isolated by centrifugation for 15 min at top speed in a microcentrifuge. Either total bacterial protein lysates or inclusion body pellets were suspended in SDS sample buffer, heated to 100 °C, and subjected to SDS-PAGE (10% gel). For all samples, an amount of material equivalent to 0.1 A units of original culture was loaded per lane. The ``full-length'' MHCK A plasmid construct expresses a fusion protein in which the first seven codons of MHCK A are replaced by four codons from the vector. The truncated MHCK A construct expresses a fusion protein with the same 4 amino-terminal vector amino acids fused to residues 634-1132 of MHCK A, followed by the polyhistidine tag from the pET21d vector. The last 14 codons of the MHCK open reading frame (residues 1133-1146) are absent in both constructs, with amino acid 1132 of MHCK A fused in phase with the polyhistidine tag of pET21d. SDS-PAGE, transfer of proteins to nitrocellulose, and denaturation/renaturation were done as described (26) . Phosphorylation assays were performed by incubating the filter for 30 min in 30 mM Tris, pH 7.5, 10 mM MgCl(2), 2 mM MnCl(2), and 0.08 µM [-P]ATP (3000 Ci/mmol; Amersham Corp.) at 23 °C. Following the phosphorylation step, the filter was washed in Tris-buffered saline; then with 7 M guanidine, 50 mM Tris, pH 7.5, 2 mM EDTA, and 0.25% milk for 2 h; and then with 2 N HCl for 2 h, followed by a brief Tris-buffered saline rinse before being exposed to film.

MHC Phosphorylation

E. coli cells (BL21) expressing either the truncated MHCK A (residues 634-1132) or the full-length MHCK A construct were grown at 22 °C for 7 h in LB medium containing 50 µg/ml ampicillin and 1 mM isopropyl-1-thio-beta-D-galactopyranoside. Cells were harvested by centrifugation; washed once in 10 mM Tris, pH 7.5, 1 mM EDTA; and then resuspended in 50 mM Tris, pH 7.5, 50 mM NaCl, 50 mM EDTA, 2 mM dithiothreitol, 10 µg/ml pepstatin, and 10 µg/ml phenylmethylsulfonyl fluoride. Cells were then lysed by sonication, and a pellet was obtained by centrifugation in a microcentrifuge. The pellet was solubilized in 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, and 1.5% Sarkosyl(27) ; centrifuged to remove insoluble material; and dialyzed overnight at 4 °C against 10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. Phosphorylation reactions contained 5 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM MgCl(2), 5 µM [-P]ATP (50 Ci/mmol), and 0.4 mg/ml Dictyostelium myosin(7) . Reactions were conducted at room temperature for 15 min, and samples were then subjected to SDS-PAGE and autoradiography.


RESULTS

Isolation of MHCK A cDNA and Sequence Analysis

The monoclonal antibodies generated against MHCK A all reacted strongly with both the unphosphorylated and autophosphorylated forms of the purified MHCK, which have SDS-PAGE mobilities of 130 and 140 kDa, respectively(28) . These antibodies specifically detect bands of 130 and 140 kDa when used to probe total Dictyostelium protein extracts (Fig. 1), which presumably represent the unphosphorylated and autophosphorylated forms of MHCK A in vivo.


Figure 1: Immunoblot of a crude Dictyostelium cell extract probed with monoclonal antibody A1. Lane1, Coomassie Blue-stained protein sample of total Dictyostelium proteins; lane2, corresponding Western blot. The faint band (140 kDa) detected slightly above the major MHCK A band (130 kDa) is believed to represent the active autophosphorylated form of MHCK A. Monoclonal antibodies A16, A21, and A22 used to isolate the MHCK A gene all produced very similar staining patterns (data not shown).



Four of the monoclonal antibodies were used to isolate the MHCK A gene from a Dictyostelium gt11 cDNA expression library as described under ``Materials and Methods.'' The complete sequence contains a single open reading frame of 1146 codons, encoding a protein with a predicted size of 128.9 kDa (Fig. 2). This closely matches the observed size of native MHCK A (130 kDa by SDS-PAGE).

Amino acid sequence analysis of the MHCK A protein isolated from Dictyostelium indicated that the amino terminus of native MHCK A was blocked. Sequencing was therefore performed on purified tryptic peptide fragments of native MHCK A to confirm the identity of the cDNA clone. Five independent peptide sequences were obtained, and all were found to match the predicted translation product of the MHCK A gene (underlineditalic residues in Fig. 2).

The MHCK A sequence was used to search the data base of known sequences using the BLAST algorithm. Weak but significant homologies were found in the amino-terminal portion of MHCK A (residues 100-500) to a variety of alpha-helical coiled-coil proteins, including myosin II tail domains, tropomyosin, paramyosins, and intermediate filament proteins. The carboxyl-terminal portion of MHCK A (residues 880-1146) displayed strong similarity to members of the ``WD repeat'' family of proteins (also known as ``WD40'' or ``-transducin-like proteins'')(29, 30) . Both coiled coils and WD motifs have repetitive character, so the MHCK A sequence was analyzed with the Dotplot program of the Wisconsin GCG package to identify regions with repetitive character (Fig. 3). This analysis revealed an amino-terminal region (residues 100-500) with weak repetitive character that corresponds to the portion of MHCK A bearing similarity to known coiled-coil proteins. The central portion of the MHCK A protein displayed no significant repetitive character (residues 500-880), while the carboxyl-terminal portion (residues 880-1146) displayed a distinct 7-fold repeat of the WD or beta-transducin-like repeat. We have tentatively designated each of these segments of MHCK A as distinct domains (Fig. 3, bottom), and analysis of each domain is discussed below.


Figure 3: Dotplot of the MHCK A protein versus itself and tentative domain assignments. The Compare and Dotplot programs of the Wisconsin GCG package were run with a window of 20 and stringency of 10. Weak repetitive character characteristic of coiled coils is apparent (residues 100-500), and the 7-fold WD repeat of the carboxyl-terminal region (residues 880-1146) is also apparent.



Southern Blot Analysis

Southern blot analysis was performed with DNA probes corresponding to each of the three segments of the gene to establish a restriction site map of the genomic locus and to determine whether the MHCK A gene is single copy or a member of a homologous gene family. Fig. 4A presents the schematic domain assignment of the MHCK A open reading frame aligned with a DNA map indicating the position of the internal HindIII restriction site at nucleotide 1493 and the positions of each of the probes used for Southern blot analysis. Fig. 4B presents a map of the MHCK A locus derived from Southern blot and cDNA sequence analyses indicating the positions of HindIII (H) and XbaI (X) sites. Probes A, B, and C all hybridized to the same genomic DNA XbaI fragment of 8 kb (Fig. 4C, lanes2). In a HindIII digest (lanes1), probe A hybridized to a 5`-HindIII fragment of 11 kb, and probes B and C each hybridized to a 3`-HindIII fragment of 9 kb. In an XbaI + HindIII double digest (lanes3), probe A reacted with a 4-kb 5`-fragment, and probes B and C both reacted with a 4.5-kb 3`-fragment. These digests, as well as several other single and double digest combinations (data not shown), indicate that the MHCK A cDNA corresponds to a single gene that has no detectable introns (small introns of less than 80 base pairs might not be detected in this analysis).


Figure 4: Southern blot analysis of the MHCK A locus. A, schematic alignment of predicted MHCK A domains with the cDNA for the gene, indicating the position of the internal HindIII restriction site and the positions of probe fragments used for Southern blotting. B, diagram of the MHCK A locus derived from Southern blot analysis and cDNA sequence analysis. The position and orientation of the MHCK A coding region are indicated by the box and arrow, respectively. HindIII (H) and XbaI (X) restriction sites are indicated. Fineticks represent 1 kb. C, autoradiogram from Southern blot analysis. Genomic DNA samples of the cell line Ax2 were digested with restriction enzymes as follows: lanes1, HindIII; lanes2, XbaI; lanes3, HindIII and XbaI.



Evidence for a Coiled-coil Domain and Oligomerization

The MHCK A sequence was analyzed for coiled-coil character using a computer algorithm devised by Lupas et al.(23) that accurately recognizes known coiled coils in proteins such as myosin as well as short coiled-coil segments such as the ``leucine zippers.'' The algorithm predicted that large stretches of amino acids within the amino-terminal 500 residues of MHCK A (corresponding to the region displaying weak repetitive character and weak similarity to known coiled-coil proteins) have an almost 100% chance of forming a coiled-coil structure (Fig. 5a).


Figure 5: alpha-Helical coiled-coil structure of MHCK A. a, percent probability of primary sequence forming coiled-coil structure as predicted by the algorithm of Lupas et al.(23) . b, chemical cross-linking of Dictyostelium myosin and MHCK A. Shown are the results from SDS-PAGE of myosin (lanes1 and 2) and MHCK A (lanes3 and 4) before (lanes1 and 3) and after (lanes2 and 4) cross-linking with bis(sulfosuccinimidyl)suberate. Myosin was visualized by staining with Coomassie Blue, and P-labeled autophosphorylated MHCK A was detected by autoradiography.



Proteins with coiled-coil domains are classically found to be assembled in their native state into dimers, trimers, or tetramers. Chemical cross-linking studies were therefore performed with purified MHCK A to assess whether native MHCK A has an oligomeric structure. MHCK A autophosphorylated in the presence of [P-]ATP was used for these experiments to allow cross-linked products to be visualized with high sensitivity by autoradiography. Parallel cross-linking of Dictyostelium myosin II was performed as a control. Prior to cross-linking, MHC migrated on SDS gels with a mobility corresponding to that of a monomer (240 kDa), while after cross-linking, it electrophoresed with a much lower mobility, presumably corresponding to the molecular mass of an MHC dimer (Fig. 5b, lanes1 and 2). A dramatic shift in mobility was also observed for MHCK A following cross-linking. Prior to cross-linking, MHCK A migrated at a molecular mass corresponding to 130 kDa (lane3), but after cross-linking, MHCK migrated with a much lower mobility (lane4), suggesting the formation of either a trimer or tetramer. These results are consistent with the formation of a coiled-coil domain by the amino-terminal portion of MHCK A. This prediction is also in agreement with earlier observations that showed that the 130-kDa MHCK A elutes from gel filtration columns with an apparent molecular mass of >700 kDa(7) .

Analysis of the Central Nonrepetitive Domain

The central portion of MHCK A (residues 500-880) is nonrepetitive in character and seems to be the most likely portion of the protein to contain the kinase catalytic functions. Surprisingly, computer data base searches using the BLAST algorithm failed to detect any homology between this region and members of the conserved family of eukaryotic protein kinases(31, 32) . The large majority of known eukaryotic protein kinases belong to this conserved family and display blocks of similarity to each other in 11 different subregions(33) . In direct examination by eye, a possible nucleotide-binding motif (GXGXXG) was observed at position 778 (subregion I of the conventional protein kinase family; (31) ), and candidate residues for an invariant lysine (subregion II) are present at position 795 or 808. A candidate for a conserved glutamate (subregion III) is present at position 834. Beyond this limited similarity, however, no significant similarity to known kinase domains could be identified by eye, by analysis with the BLAST data base search algorithm, or by direct line-up using Bestfit or Dotplot analysis against known protein kinases.

Another MHCK A feature suggesting significant divergence from (or unrelatedness to) the conventional family of eukaryotic protein kinases is the position of the identified GXGXXG motif relative to the G-like domain. The G-like domain of MHCK A begins at approximately residue 880, only 100 residues past the GXGXXG sequence. This contrasts with the large majority of currently characterized eukaryotic protein kinase catalytic domains, which all contain at least 200 or more amino acids of conserved sequence with functional importance on the carboxyl-terminal side of the GXGXXG nucleotide-binding site(31, 33) .

Intrinsic Protein Kinase Activity of MHCK A

Although native MHCK A from Dictyostelium has been extensively characterized biochemically(7, 20) , additional tests were performed to ensure that the MHCK A gene does encode a protein with bona fide kinase activity. To rule out the possibility that a minor contaminant might be responsible for the protein kinase activity in preparations of MHCK A, protein kinase reactions were carried out after gel electrophoresis of purified MHCK A, removal of SDS, and renaturation of the polypeptide in the gel(25) . Incorporation of P into the band corresponding to the 130-kDa MHCK A was observed, indicating that protein kinase activity is an intrinsic property of this protein (Fig. 6A). Autophosphorylation of a lower molecular mass protein was also detected. This protein reacted with monoclonal antibodies to MHCK A (data not shown) and presumably represents a minor degradation product that renatures more efficiently than full-length MHCK A.


Figure 6: Protein kinase activity of native and recombinant MHCK A proteins. A, MHCK A purified from Dictyostelium was subjected to SDS-PAGE, renatured on the gel as described under ``Materials and Methods,'' incubated with [-P]ATP, and then stained with Coomassie Blue. The Coomassie Blue-stained gel (lane1) and the corresponding autoradiogram (lane1`) are shown. B, shown is the autophosphorylation of recombinant MHCK A expressed in E. coli. Lanes 1-4 show the Coomassie Blue-stained profile of E. coli extracts following SDS-PAGE, and lanes1`-4` show the autoradiogram from an identical gel in which the proteins were transferred to nitrocellulose, renatured, and assayed for the ability to autophosphorylate as described under ``Materials and Methods.'' Lanes1 and 1`, total extract of cells expressing a truncated MHCK A fusion protein (expresses residues 634-1132 of MHCK A); lanes2 and 2`, total extract of cells expressing the full-length MHCK A protein; lanes3 and 3`, inclusion body pellet from cells expressing the truncated MHCK A protein; lanes4 and 4`, inclusion body pellet from cells expressing full-length MHCK A. Both the truncated and full-length MHCK A proteins are major proteins visible in the stained profile (molecular masses of 58 and 130 kDa, respectively). Proteolytic products of both proteins are also present in the E. coli extracts, visible by Coomassie Blue staining and by Western blot analysis with MHCK A-specific antibodies (data not shown). C, shown is the phosphorylation of Dictyostelium MHC by recombinant MHCK A. Lanes1 and 2 show a Coomassie Blue-stained gel of phosphorylation reactions, and lanes1` and 2` show a corresponding autoradiogram. Lanes1 and 1`, MHC phosphorylation reaction containing E. coli extract expressing the truncated MHCK A protein; lanes 2 and 2`, MHC phosphorylation reaction performed with E. coli extract expressing the full-length MHCK A construct. The MHC band is apparent just above the 170-kDa marker.



As a further test of MHCK protein kinase A activity, the MHCK A gene was overexpressed in E. coli. Cultures were prepared from E. coli cells containing vector only, containing vector expressing the full-length MHCK A polypeptide, or containing vector with a truncated gene expressing MHCK A residues 634-1132. Protein products of both the full-length and truncated MHCK A aggregated into inclusion bodies. Low speed centrifugation of cell lysates was used to enrich for these inclusion bodies, and both total extracts and inclusion body fractions were then subjected to SDS-PAGE. Separated proteins were transferred to nitrocellulose and subjected to denaturation in guanidine hydrochloride and renaturation(26) . Subsequent incubation of the filter in kinase buffer containing [-P]ATP revealed the presence of P in the full-length MHCK A protein in the total extract (Fig. 6B, lanes2 and 2`) and in the inclusion body fraction (lanes4 and 4`), but a complete absence of P in the truncated protein both in total extracts (lanes1 and 1`) and in inclusion body fractions (lanes3 and 3`). It is noteworthy that proteolytic fragments of full-length MHCK A in the E. coli extract also autophosphorylate. The minimal domain necessary for catalytic activity and autophosphorylation seems to be smaller than the full-length 130-kDa protein.

In further analysis, we established conditions with which full-length recombinant MHCK A could be solubilized in functional form from inclusion bodies. This material phosphorylated Dictyostelium MHC efficiently in vitro (Fig. 6C, lanes2 and 2`), while the truncated recombinant protein prepared in the same manner had no MHC kinase activity (lanes1 and 1`).

The Carboxyl-terminal WD Domain

Dotplot analysis and data base searches revealed a 7-fold repeat motif in the carboxyl-terminal portion of the MHCK A protein (residues 880-1146) ( Fig. 3and 7a) that has significant similarity to the 7-fold repeat pattern of the beta-subunits of heterotrimeric G proteins. When aligned with the human G subunit, this region shows 24% identity (49% similarity) at the primary sequence level (Fig. 7b). In addition to overall homology to G subunits, the MHCK A G-like domain displays specific features that are conserved with many true G subunits, such as the presence of a recognizable ``D-X(5)-WD'' motif in all of the repeats, with this motif being less strongly conserved in the second repeat unit (34) .


Figure 7: Homology of the MHCK A carboxyl-terminal repeat to the beta-subunit of heterotrimeric G proteins. a, alignment of residues 860-1146 of MHCK A demonstrates a 7-fold repeat of 40 amino acids. Residues that are identical in at least four of the seven repeats are boxed. b, shown is an alignment of MHCK A residues 880-1146 with the human G subunit using the Bestfit program of the Wisconsin GCG package. The repeated D-X(5)-WD motif of each repeat is underlined. This motif is less conserved in the second repeat of both MHCK A and the human G subunit.




DISCUSSION

The characterization of the MHCK A gene and its protein product presented here provides important new insights into the structure of the MHCK A protein, but also poses a number of new questions. Substantial evidence from biochemical and in vivo analyses suggests that MHC phosphorylation regulates Dictyostelium myosin localization and assembly and that threonine residues at positions 1823, 1833, and 2029 of MHC are critical for this regulation (6, 14, 35) . As of this report, the primary sequence is now known for each of the two biochemically identified MHCKs that can drive Dictyostelium myosin filament disassembly, and these two enzymes appear completely unrelated to each other at the primary sequence level. The 84-kDa MHCK previously cloned by Ravid and Spudich (11) appears to be a member of the protein kinase C family based on sequence homology and has a catalytic domain with substantial identity to the catalytic domains of other eukaryotic protein kinases. The presence in Dictyostelium of two unrelated MHCKs, each of which seems capable of regulating myosin assembly, suggests that multiple signaling pathways may exist within these cells to allow control of myosin assembly and localization in response to different initial stimuli. Studies of mutants with elevated cGMP levels have suggested a role for cGMP in the regulation of myosin localization in Dictyostelium(19, 36) , but neither the 84-kDa protein kinase C-like MHCK nor MHCK A contains regions with any significant similarity to cyclic nucleotide-binding domains. Furthermore, neither of these MHCKs is activated in vitro by cyclic nucleotides (6, 7) . While these results make it unlikely that cGMP is involved directly in the regulation of the Dictyostelium MHCKs, cGMP may act indirectly by regulating phosphatases or upstream kinases.

The 84-kDa protein kinase C-like MHCK seems to be expressed only during the developmental phase of the Dictyostelium life cycle (11) , suggesting that it may have specific functions during chemotactic cell locomotion in response to extracellular cAMP or during multicellular development. In contrast, the presence of the 130-kDa MHCK A during both growth and development suggests that this enzyme could participate in myosin localization during an array of cellular contractile events, including cytokinesis and capping of cell-surface receptors as well as chemotactic cell locomotion. Gene targeting studies with the MHCK A gene should help elucidate the specific roles of this enzyme in these processes and should help establish the relative roles of MHCK A versus the 84-kDa protein kinase C-like MHCK in specific processes.

It is likely that the kinase catalytic functions of MHCK A lie in the central nonrepetitive portion of the protein. Although this region displays no significant similarity to any protein kinase sequences in the data base when searched with the BLAST algorithm, the biochemical data reported here clearly indicate that the recombinant MHCK A protein is capable of autophosphorylation and displays bona fide kinase activity directed against Dictyostelium MHC. A truncated protein (residues 634-1132) displayed no activity, indicating that sequences to the amino-terminal side of residue 634 are involved in catalytic activity. Further truncation analysis and site-directed mutagenesis will be required to identify the portions of MHCK A that participate in the kinase catalytic functions, substrate specificity, and autophosphorylation.

It should be noted that there are several examples in the literature where biochemical studies have identified enzymes that exhibit intrinsic protein kinase activity despite having little or no similarity to the conserved catalytic domain commonly found in eukaryotic protein kinases(37, 38, 39, 40, 41) . None of these enzymes, however, display any detectable similarity to MHCK A at the primary sequence level.

Our current hypothesis is that the amino-terminal coiled-coil domain and carboxyl-terminal WD domain of MHCK A serve either regulatory or structural roles. Cross-linking and gel filtration experiments indicate that native MHCK A is an oligomer of 130-kDa subunits, probably held together by interactions between coiled-coil domains. Native MHCK A would therefore contain multiple catalytic and WD domains in close proximity to each other. It can be speculated that the coiled-coil domain might also have additional roles, such as directing MHCK A to myosin filaments either via coassembly or via annealing of this domain onto previously assembled bipolar myosin filaments. It is also possible that this domain may serve as a pseudosubstrate or autoinhibitory domain, by analogy to the pseudosubstrate domains that have been observed in protein kinase C, myosin light chain kinases, and other protein kinases(42, 43, 44, 45) .

Although a growing number of proteins have been observed to contain varying numbers of WD repeats, Dictyostelium MHCK A appears to represent the first reported example of a domain of the same size as a conventional G subunit (each containing seven WD repeats) coupled to a protein kinase domain. Indeed, to our knowledge, this is the first reported example of a WD repeat protein that has demonstrated enzymatic activity of any sort(29, 30) . Although the role of the WD domain is presently unknown, it is interesting to note that the beta-adrenergic receptor kinase is targeted to membranes by a direct interaction with G subunits (46) and that one of the intracellular receptors for activated protein kinase C has recently been identified as a G homolog(47) . By analogy, the MHCK A G-like domain may be responsible for localizing the kinase to specific membrane fractions. The presence of this domain also opens up the possibility that MHCK A may interact directly with, and be regulated by, G protein alpha- or -subunits. Further molecular genetic and biochemical analyses will be needed to establish the role of the WD repeat domain in MHCK A function and to define the mechanisms that regulate the activity of MHCK A in vivo.

During the final revisions of this paper, a GenBank submission from the Caenorhabditis elegans genome project was made that contains an open reading frame with substantial homology to a short segment within the central nonrepetitive portion of MHCK A. The C. elegans open reading frame (F42A10.4; GenBank accession number U10414) contains a segment of 50 amino acids that is 75% identical to MHCK A residues 734-784 (including the GXGXXG motif noted in Fig. 2), surrounded by 100 residues with weaker conservation. The presence of this conserved segment may provide insights regarding the catalytic functions within the central domain of MHCK A and furthermore indicates that regions of the novel central domain present in MHCK A have been evolutionarily conserved in other organisms.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM50009 (to T. T. E.) and by the Medical Research Council of Canada (to G. P. C.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16856 [GenBank]and U17368[GenBank].

§
To whom correspondence should be addressed. Tel.: 216-368-6971.

(^1)
The abbreviations used are: MHC, myosin heavy chain; MHCK, MHC kinase; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s); TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Jim Spudich for generous support in the initial stages of this project and Tony Hunter for helpful comments on the manuscript. We also thank M. Carpenter and L. B. Smillie for amino acid sequence analysis, G. Cates for help with preparing monoclonal antibodies, and Richard Cheney for assistance in coiled-coil analysis.


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