(Received for publication, November 14, 1996, and in revised form, December 23, 1996)
From the Department of Biochemistry, Queen's University, Kingston,
Ontario K7L 3N6 and the Department of Physiology and
Biophysics, Case Western Reserve University,
Cleveland, Ohio 44106
Myosin heavy chain kinase A (MHCK A) in Dictyostelium was identified as a biochemical activity that phosphorylates threonine residues in the myosin II tail domain and regulates myosin filament assembly. The catalytic domain of MHCK A has now been mapped through the functional characterization of a series of MHCK A truncation mutants expressed in Escherichia coli. A recombinant protein comprising the central nonrepetitive domain of MHCK A (residues 552-841) was isolated in a soluble form and shown to phosphorylate Dictyostelium myosin II, myelin basic protein, and a synthetic peptide substrate. The functionally mapped catalytic domain of MHCK A shows no detectable sequence similarity to known classes of eukaryotic protein kinases but shares substantial sequence similarity with a transcribed Caenorhabditis elegans gene and with the mammalian elongation factor-2 kinase (calcium/calmodulin-dependent protein kinase III). We suggest that MHCK A represents the prototype for a novel, widely occurring protein kinase family.
Assembly of Dictyostelium myosin-II into the
cytoskeleton in vivo is regulated by myosin heavy chain
phosphorylation (1, 2). The biochemically identified 130-kDa myosin
heavy chain kinase (MHCK A)1 has a
demonstrated role in regulating myosin II assembly both in
vitro (3) and in vivo (4). A surprising feature of the primary sequence of MHCK A is that it displays no detectable similarity to the conserved catalytic domains found in conventional eukaryotic protein kinases (5). MHCK A also displays no detectable similarity to
"histidine protein kinases" (6) or related proteins such as
-ketoacid dehydrogenase kinase (7, 8). MHCK A consists of an
amino-terminal domain with probable coiled-coil structure, a central
nonrepetitive domain, and a carboxyl-terminal domain consisting of
seven WD repeats (9). The central nonrepetitive domain of MHCK A does
contain a GXGXXG motif similar to that present in
the nucleotide-binding site of conventional protein kinases, but this
motif is located only about 75 residues distant from the start of the
first WD repeat. In conventional protein kinases the
GXGXXG motif is located in the first of the 12 distinct kinase subdomains and is followed by about 250 residues of
conserved sequence that comprises the rest of the catalytic domain
(10).
In this paper we define the location and extent of the novel MHCK A protein kinase catalytic domain by expressing in Escherichia coli and functionally characterizing a series of MHCK A truncation mutants. The results show that neither the MHCK A coiled-coil domain nor the WD repeat domain are required for kinase activity and identify the catalytic activity as residing solely in the 40-kDa MHCK A central domain. Although the central domain of MHCK A exhibits no homology with conventional protein kinases, it does share significant sequence similarity with the rat eukaryotic elongation factor-2 (eEF-2) kinase and with a possible eEF-2 kinase homolog from Caenorhabditis elegans. The results indicate that the novel kinase catalytic domain of MHCK A is evolutionarily conserved and thus may represent the prototype for a novel family of protein kinases.
A plasmid that
expresses "full-length" MHCK A (residues 8-1132) in E. coli with a hexahistidine (His6) tag at the carboxyl terminus was
constructed using the pET21d vector and has been described (5). The
first seven codons of the MHCK A coding region are absent in this
construct, replaced by four codons from the pET21 vector. At the COOH
terminus the last 14 codons of MHCK A are removed at the fusion site to
the vector His6 tag. Subsequent constructs were generated using
standard methods to create the set of truncations presented in Fig. 1.
Each recombinant protein contains several polylinker-derived amino
acids at the NH2 terminus and a His6 tag at the COOH
terminus. Both wild type and 3X ALA Dictyostelium myosin II
were purified as described in Ref. 11, with the addition of a final
Sephacryl S300 chromatography step to remove residual contaminating
proteins.
Expression and Purification of Truncated Forms of MHCK A
Transformed E. coli strain BL21 was grown at either
37 °C (for purification of proteins from inclusion bodies) or
24 °C (for isolation of soluble protein). Induction, lysis, and
purification on nickel chelation chromatography resin was performed
according to standard protocols provided with the His-Bind resin
(Novagen). For purification of insoluble protein from inclusion bodies,
8 M urea was included during binding and elution steps.
Recombinant proteins were eluted with 0.1 M NaCl, 0.2 M imidazole, 5 mM Tris, pH 8.0, dialyzed
overnight against 20% glycerol, 20 mM NaCl, 1 mM dithiothreitol, 20 mM Hepes, pH 7.0, and
stored at 80 °C. Proteins stored in this manner retained activity
for several months.
Soluble T-4 and T-5 recovered from E. coli grown at 24 °C were dialyzed overnight against 20 mM NaCl, 1 mM dithiothreitol, and 20 mM Tris, pH 7.5, and dephosphorylated by addition of 2 mM MgCl2 and 10 µg/ml CIAP. After 1 h at room temperature samples were chromatographed over the His-Bind resin as described above or loaded onto a Mono Q column (Pharmacia Biotech Inc.) equilibrated in 20 mM NaCl, 1 mM dithiothreitol, 20 mM Tris, pH 7.5. T-4 and T-5 were eluted from the Mono Q column with an NaCl gradient to 1 M. Both procedures separated T-4 and T-5 from CIAP.
Phosphorylation AssaysIn some cases recombinant proteins
were assayed for the ability to autophosphorylate following SDS-PAGE
and electrophoretic transfer to nitrocellulose filters (5). Briefly,
filters were subjected to a denaturation/renaturation regime and then
incubated in buffer containing 10 mM MgCl2, 2 mM MnCl2, 50 nM
[-32P]ATP (1000 Ci/mmol), and 30 mM Tris,
pH 7.5. The filters were then washed and exposed to x-ray film as
described (5). Soluble proteins were assayed for the ability to
autophosphorylate and phosphorylate exogenous substrates in a buffer
containing 2 mM MgCl2, 1 mM
dithiothreitol, 0.1 mM [
-32P]ATP (0.1 Ci/mmol), and 10 mM Hepes, pH 7.0, unless noted otherwise. Incorporation of phosphate into peptide and protein substrates was
quantitated using P81 phosphocellulose paper as described (12) or by
excising the protein bands of interest following SDS-PAGE and Coomassie
Blue staining and counting the bands in liquid scintillation fluid
using a Beckman LS 7500 Scintillation Counter. Peptide phosphorylation
assays were performed using MH-3 peptide (RKKFGEAEKTKAKEFL) described
previously (12). Wild type and 3X ALA myosin phosphorylation was
performed in 10 mM Hepes, 0.2 mM
MgCl2, 2 mM ATP, 1 mM DTT, pH 7.4, containing 0.3 µg/µl myosin and either 6 ng/µl MHCK A or 20 ng/µl T-4. Reactions were incubated 10 min at 22 °C and then
subjected to SDS-PAGE and autoradiography and phosphorimaging for
quantitation.
A schematic representation of the MHCK A constructs used in this study is shown in Fig. 1A. All the recombinant proteins were designed to contain a COOH-terminal His6 tag to facilitate purification. The recombinant proteins were expressed at high levels when E. coli were induced at 37 °C, but in all cases they were aggregated into inclusion bodies and were recovered quantitatively in the pellet fraction following cell lysis and centrifugation. The pellets were solubilized in buffer containing 8 M urea, and the recombinant proteins were purified by chromatography over an immobilized metal ion affinity column (His-Bind resin). Coomassie Blue-stained SDS gels showed that the recombinant proteins were recovered as single bands with the expected molecular mass, with the exception of N-MHCK, T-2, and T-6, which were recovered as two or more bands (Fig. 1B). The largest forms of N-MHCK and T-6 had apparent molecular masses equivalent to those expected for the full-length proteins (75 and 35 kDa, respectively), whereas T-2, predicted to have a molecular mass of 64 kDa, was isolated as three fragments of 58, 50, and 35 kDa. In all cases the smaller fragments likely result from proteolysis at the amino terminus, because the ability of the recombinant proteins to interact with the His-Bind resin demonstrates the presence of the COOH-terminal His6 tag.
Previous studies have shown that bacterially expressed full-length MHCK
A is able to autophosphorylate after being solubilized from inclusion
bodies, subjected to SDS-PAGE, transferred to nitrocellulose, and
incubated with Mg2+, Mn2+, and
[-32P]ATP (5). When subjected to the same procedure
neither N-MHCK nor C-MHCK, which represent, respectively, the
NH2- and COOH-terminal halves of MHCK A, displayed the
ability to incorporate 32P (Fig. 1C). As a
result, attention was focused on the possibility that an intact central
domain might be essential for kinase catalytic activity. A series of
recombinant proteins (T-1 through T-6) were constructed in which
progressively larger NH2- and COOH-terminal portions of
MHCK A were deleted. As judged by the nitrocellulose renaturation
assay, T-1, T-2, T-3, and T-4 all retained the ability to
autophosphorylate strongly (Fig. 1C). The smallest of these proteins, T-4, corresponds closely in extent to the central
nonrepetitive domain of MHCK A and is completely lacking both the
NH2-terminal
-helical coiled-coil domain and the
COOH-terminal WD repeat domain. Removal of 53 residues from the
NH2 terminus of T-4 yielded a 35-kDa protein, T-5, with a
reduced capacity to autophosphorylate (Fig. 1, B and
C). The removal of 58 residues from the COOH terminus of T-4
yielded a 35-kDa protein, T-6, that displayed no ability to
autophosphorylate (Fig. 1, B and C).
The reduced autophosphorylation activity observed for T-5 and T-6 in
the nitrocellulose renaturation assays could potentially result from
the loss of intrinsic protein kinase activity, the elimination of key
autophosphorylation sites, or an inability to renature on the
nitrocellulose. Attempts were therefore made to isolate T-4, T-5, and
T-6 in a soluble form so that their protein kinase activity could be
analyzed in more detail. It was found that induction of expression in
E. coli at 24 °C rather than 37 °C resulted in the
recovery of T-4, T-5, and T-6 in the supernatant fraction of cell
lysates, albeit with lower total yield. Chromatography on His-bind
resin provided nearly homogenous purification (Fig. 2A).
When incubated with Mg2+ and [-32P]ATP,
the soluble T-4 autophosphorylated to a significantly greater extent
than soluble T-5, whereas no autophosphorylation of soluble T-6 was
detected (Fig. 2B). The soluble form of the T-4 construct
migrated with a higher apparent mass on SDS-PAGE than did T-4 isolated
from inclusion bodies. This difference could be attributed to in
vivo autophosphorylation of the soluble material in E. coli, because treatment of the soluble T-4 with CIAP shifted its
mobility to the same position as the T-4 isolated from inclusion
bodies. In contrast, soluble T-5 migrated with the same mobility on
SDS-PAGE as T-5 from inclusion bodies and exhibited no mobility shift
when treated with CIAP. Subsequent in vitro
autophosphorylation of the CIAP-treated soluble T-4 protein resulted in
an upwards mobility shift (data not shown) and the incorporation of 4 mol Pi/mole protein. This behavior is consistent with the
shift in mobility displayed by MHCK A upon autophosphorylation (12).
CIAP-treated T-5 protein incorporated only 1 mol Pi/mol protein when incubated with MgATP, confirming that several
autophosphorylation sites are located between residues 499 and 551.
Native MHCK A and the recombinant soluble T-4 protein were assayed for their ability to phosphorylate wild type Dictyostelium myosin II and a mutant myosin II in which the major MHCK A phosphorylation sites in the tail (threonines 1823, 1833, and 2029) have been replaced with alanines (3X ALA myosin; Ref. 2). The 3X ALA myosin was phosphorylated to only about 10% of the level of wild type myosin by both T-4 and MHCK A (Fig. 2, C and D), indicating that T-4 retains the major determinants of substrate specificity.
A more detailed analysis of the kinase activity of T-4 and T-5 was
performed using the synthetic peptide substrate MH-3, which is based on
the sequence surrounding the Thr-2029 MHCK A phosphorylation site in
Dictyostelium myosin II. The dephosphorylated (CIAP-treated) forms of T-4 and T-5 phosphorylated MH-3 (Fig. 3,
A and B, squares). Preincubation of
the dephosphorylated T-4 and T-5 with MgATP to allow for
autophosphorylation increased the activity of T-4 (Fig. 3A,
circles) but did not alter the activity of T-5 (Fig.
3B, circles). Although the activation by
autophosphorylation observed with T-4 (~3-fold) is small compared
with that observed with intact MHCK A (~50-fold), the results provide
evidence that residues 499-551, which link the coiled-coil domain of
MHCK A to the central domain, may function as an autoinhibitory
sequence that can be regulated by autophosphorylation.
Further studies with the synthetic peptide MH-3 showed that the activity of T-4 was dependent on Mn2+ or Mg2+ and was not supported by Ca2+ (Fig. 3C). T-4 activity was strongly inhibited at higher ionic strengths, with 80% loss of activity at 100 mM KCl. T-4 displayed a Km for ATP of ~50 µM and was able to utilize GTP as a substrate only poorly (rate less than 1% that of ATP). T-4 and T-5 displayed Km values for MH-3 that were about 4-5-fold higher than native MHCK A and Vmax values that were about 2-4-fold lower (Table I). Histone III-S (a lysine-rich fraction) and histone VIII-S (arginine-rich fraction) were poor substrates for T-4, but myelin basic protein was as good a substrate as MH-3 (Table I). T-6 displayed no activity when assayed under various conditions with MH-3 or myelin basic protein, and we conclude that this construct is inactive.
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It is noteworthy that the Vmax of native MHCK A with the peptide substrate MH3 (2.2 µmol/mg·min) is roughly similar to those of members of the conventional family of protein kinases such as cAMP-dependent protein kinase, phosphorylase kinase, and chicken gizzard myosin light chain kinase, which range from 10 to 15 µmol/mg·min (12, 13). In contrast, most reported "unconventional" protein kinases either have not been characterized kinetically or display extremely low activity. For example, a publication reporting that topoisomerase I has protein kinase activity indicated a specific activity of 3 nmol/mg·min (14), approximately 1000-fold less active than MHCK A or conventional protein kinases.
The results presented here map the biochemical protein kinase activity
of MHCK A as residing between residues 552 and 841 and further define a
probable autoinhibitory peptide sequence containing multiple
autophosphorylation sites just proximal to this region (residues
~500-551). Although the primary sequence of the MHCK A kinase
catalytic domain displays no detectable similarity to conventional
protein kinases, there is substantial similarity throughout this entire
segment of MHCK A to two published sequences. The first is a sequence
in GenBankTM, which was deposited by the C. elegans genome
project (genomic, accession number U10414[GenBank]; cDNA, accession number
D27775[GenBank]). The second is the recently published sequence of eEF-2 kinase
(15), also known as calcium/calmodulin-dependent protein
kinase III. The published sequence of the eEF-2 kinase is highly
similar to the C. elegans open reading frame (ORF)
throughout its entire length, suggesting that the C. elegans
ORF may encode an eEF-2 kinase, but the sequence similarity of these
proteins to MHCK A is limited to the region that we have mapped as
constituting the MHCK A catalytic domain (Fig. 4). The
catalytic domain of MHCK A displays 41 and 42% identity with the
C. elegans ORF and the rat eEF-2 kinase, respectively.
In the conventional eukaryotic protein kinase superfamily the conserved GXGXXG nucleotide binding loop constitutes the first of 12 conserved subdomains and is positioned at the extreme amino-terminal portion of the primary sequence (16). It is interesting that MHCK A, eEF-2 kinase, and the C. elegans ORF all display a highly conserved GXGXXG motif but that this motif lies at the extreme carboxyl-terminal portion of the mapped MHCK A catalytic domain (residues 778-783 of MHCK A; Fig. 4). If this motif retains its function as a nucleotide binding loop, it seems likely that the structural fold of the MHCK A catalytic domain will bear little resemblance to that of conventional protein kinases. In their analysis of the eEF-2 kinase sequence, Redpath and colleagues have proposed that the catalytic domain of the eEF-2 kinase extends to the carboxyl-terminal side of the conserved GXGXXG motif as is typical of conventional protein kinases (15). However, the authors comment on the surprisingly low degree of conservation of this region of the eEF-2 kinase with other serine/threonine protein kinases. For example, the highly conserved catalytic site sequence DXKXXN (residues 166-171 of protein kinase A) is missing in the eEF-2 kinase. We suggest instead that the eEF-2 kinase shares the novel kinase catalytic domain found in MHCK A, so that in fact the eEF-2 kinase catalytic domain lies to the amino-terminal side of the GXGXXG motif and is represented by the region that shares 42% identity with the catalytic domain of MHCK A. Functional mapping of the eEF2 catalytic domain will be needed to resolve this issue. It will also be important to identify the key residues involved in nucleotide binding and catalysis within the MHCK A kinase catalytic domain. The residues that show complete conservation between the Dictyostelium, C. elegans, and mammalian kinases (Fig. 4) are obvious candidates for further investigation.
The detailed mapping of the catalytic domain of MHCK A presented here, together with the demonstration of highly related primary sequences in both C. elegans and mammalian eEF-2 kinase, suggests the presence of a novel and widespread family of eukaryotic protein kinases that is unrelated to the previously characterized conventional protein kinase superfamily.