(Received for publication, August 10, 1994; and in revised form, October 28, 1994)
From the
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
-helical coiled-coil domain and that residues from
position
860 to the carboxyl terminus (residue 1146) form a domain
with significant similarity to the
-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.
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) ()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
-subunits of heterotrimeric G proteins.
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.).
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 -helical coiled-coil
potential was performed using an algorithm devised by Lupas et al. ((23) ; see also (22) ).
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
-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
-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.
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.
Figure 5:
-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) .
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) .
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`).
Figure 7:
Homology of the MHCK A carboxyl-terminal
repeat to the -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
-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.
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
-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
- 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.
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].