(Received for publication, August 28, 1995; and in revised form, November 6, 1995)
From the
We have expressed two truncated isoforms of chicken nonmuscle
myosin II-B using the baculovirus expression system. One of the
expressed heavy meromyosins (HMM) consists of two 150-kDa
myosin heavy chains (MHCs), comprising amino acids 1-1231 as well
as two pairs of 20-kDa and 17-kDa myosin light chains (MLCs) in a 1:1:1
molar ratio. The second HMM
was identical except that it
contained an insert of 10 amino acids (PESPKPVKHQ) at the
25-50-kDa domain boundary in the subfragment-1 region of the MHC.
These 10 amino acids include a consensus sequence (SPK) for
proline-directed kinases. Expressed HMMs were soluble at low ionic
strength and bound to rabbit skeletal muscle actin in an ATP-dependent
manner. These properties afforded a rapid purification of milligram
quantities of expressed protein. Both isoforms were capable of moving
actin filaments in an in vitro motility assay and manifested a
greater than 20-fold activation of actin-activated MgATPase activity
following phosphorylation of the 20-kDa MLC. HMM
with the
10-amino acid insert was phosphorylated by Cdc2, Cdk5, and
mitogen-activated protein kinase in vitro to 0.3-0.4 mol
of PO
/mol of MHC. The site phosphorylated in the MHC was
identified as the serine residue present in the 10-amino acid insert
and its presence was confirmed in bovine brain MHCs. Characterization
of the baculovirus expressed noninserted and inserted MHC isoforms with
respect to actin-activated MgATPase activity and ability to translocate
actin filaments in an in vitro motility assay produced the
following average values following MLC phosphorylation: noninserted
HMM
, V
= 0.28
s
, K
= 12.7
µM; translocation rate = 0.077 µm/s; inserted
HMM
, V
= 0.37
s
, K
= 15.1
µM; translocation rate = 0.092 µm/s.
Myosin II is a hexamer composed of two heavy chains of
approximately 200 kDa and two pairs of light chains of 20 and 17 kDa.
It is a member of an expanding family of myosin motor proteins (1) and can be subdivided into a number of different isoforms
present in striated muscle, smooth muscle, and nonmuscle cells. Our
recent interest has focused on vertebrate cytoplasmic myosin II, which
is present in both muscle and nonmuscle cells. To date, two separate
genes, located on two different human
chromosomes(2, 3) , have been shown to encode
vertebrate nonmuscle myosin heavy chains (MHCs), ()which we
refer to as MHC II-A (22q11.2) and MHC II-B (17p13). Since it is now
clear that some of these two isoforms are present in distinct locations
in a single cell (4, 5) , there is reason to believe
that each isoform may have a specific function, in addition to possibly
overlapping functions. Moreover, previous studies have shown that these
two isoforms are expressed in a tissue-dependent manner with brain and
testes being particularly enriched for MHC II-B and spleen and
intestines containing mostly MHC II-A, while human platelets and rat
basophil leukemic cells contain only MHC
II-A(6, 7, 8) .
MHC subfragment-1 (S-1) can be proteolytically cleaved at two sites, one located about 25 kDa and the second about 75 kDa from the amino-terminal end, giving rise to 25-, 50-, and 20-kDa tryptic fragments(9) . These two proteolytically susceptible sites correspond to regions of the molecule that were not resolved in the three-dimensional crystallographic structure of chicken skeletal myosin S-1 and probably are present as disordered surface loops(10) . The region at the junction of the 25/50-kDa fragments is termed loop 1 and is near the ATP binding domain. The region at the junction of the 50/20-kDa kDa fragments is termed loop 2 and is near an actin binding domain(10, 11, 12) . Recently, we and others have demonstrated that these regions serve as sites for alternative splicing of mRNA to produce isoforms of nonmuscle MHC II-B (13, 14, 15) and smooth muscle MHCs (16, 17, 18) .
mRNA encoding nonmuscle
MHC-B has been shown to generate two different insertions in loop 1,
referred to as MHC II-B1, and one in loop 2, referred to as MHC II-B2. ()The insert in loop1 starts just after amino acid 211, and
consists of either 10 or 16 amino acids (see Fig. 1)(14) . Sequence of human genomic DNA from this
area revealed the presence of two exons, one encoding 10 amino acids
and the second encoding 6 amino acids, consistent with the idea that
these sequences are generated by alternative splicing of pre-mRNA and
that the 16-amino acid insert differs from the 10-amino acid insert in
that it is encoded by both exons instead of just one (19) (Fig. 1). The 10-amino acid inserted isoform has
been shown to be highly expressed in mammalian cerebral cortex and
retina, whereas mRNA encoding the 16-amino acid inserted isoform has
only been detected in human retinoblastoma cell lines, following
treatment with butyrate(14) .
Figure 1: Amino acid sequences in loop 1 of myosin heavy chain II-B. The 10-amino acid (A) and 16-amino acid (B) inserts present at loop 1 are shown in bold type for three species. C, the major sites of tryptic cleavage of chicken MHC II-B1 in this region are indicated by large arrows. A possible minor tryptic cleavage site is also marked. The phosphorylatable serine in the chicken sequence is underlined.
Expression of MHC II-B1 in
nonmuscle cells appears to vary in both a species- and tissue-dependent
manner. In Xenopus, an almost identical insert of 16 amino
acids in MHC II-B, starting after amino acid 211, is present in all
cells examined to date(20) . Unlike avian and mammalian cells,
MHC II-B (lacking the insert) is not expressed in Xenopus. In
contrast to its ubiquitous expression in Xenopus cells,
expression of MHC II-B1 (with the 10-amino acid insert) in avian and
mammalian cells is almost always confined to neuronal tissue and
neuronal cell lines, where it is accompanied by expression of the
noninserted isoform. Of note, the constitutively expressed Xenopus MHC II-B1 isoform has been shown to be phosphorylated by cyclin
p34 (Cdc2) kinase both in vitro and in situ within the inserted region(21) .
To date, there has been no careful characterization of the differences between myosin containing MHC II-B1 and MHC II-B. This has largely been due to the inability to obtain sufficient quantities of pure myosin II-B1 from avian and mammalian cells. In this paper, we focus on the differences between nonmuscle MHC II-B and MHC II-B1, the isoform containing the 10-amino acid insert. We used the baculovirus system to express a heavy meromyosin (HMM)-like product of these two alternatively spliced isoforms along with both the 20-kDa and 17-kDa myosin light chains. We then characterized each isoform with respect to its ATPase activity and ability to propel actin filaments in an in vitro motility assay. The MHC II-B1, but not the MHC II-B isoform, contains a putative phosphorylation site for proline-directed kinases, which we demonstrate can be phosphorylated by cyclin-dependent and mitogen-activated protein (MAP) kinases. The site phosphorylated is within the 10-amino acid insert. We also show that MHC purified from bovine brains contains this site and can also be phsophorylated.
For construction of the transfer vector containing the 30-nucleotide insert between T633 and G634, the MHC II-B cDNA encoding the HMM fragment was removed from pBlueBac using BamHI and subcloned into pBluescript. Clone S4, which is derived from the same chicken brain library and contains the 30-nucleotide insert flanked by NsiI sites(13) , was restricted and the resulting 539-base pair fragment was ligated into the same site in pBluescript containing the truncated clone MHC II-B(13) . The cDNA encoding the MHC II-B1 HMM-like fragment was then ligated into pBlueBac II at the NheI site.
cDNAs
encoding bovine nonmuscle MLC(22) (a gift of Dr.
David Hathaway, Bristol Myers Squibb Co., Princeton, NJ) and chicken
nonmuscle MLC
(23) (a gift of Dr. C. Chandra Kumar,
Schering-Plough Corp., Bloomfield, NJ) were cloned into the pAcUW51
transfer vector using polymerase chain reaction-derived clones. cDNA
encoding MLC
was cloned into the BglII site,
placing it under control of the p10 promoter, and cDNA encoding
MLC
was cloned into the BamHI site, under control
of the polyhedrin promoter. Orientation and sequence of both light
chains were verified using the dideoxy chain termination
method(24) .
The partially thawed pellet was
extracted (5-8 ml/ml of packed cells) with 0.6 M NaCl,
25 mM Tris HCl (pH 7.5), 50 mM NaP
O
, 5 mM EGTA, 5
mM EDTA, 10 mM ATP, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 5 mM dithiothreitol,
5 µg/liter leupeptin, 3 mM NaN
, and 1% Nonidet
P-40 following homogenization in a ground glass homogenizer. Extraction
at 4° with stirring continued for 1 h followed by sedimentation at
47,000
g for 10 min. This step effectively separates
soluble from insoluble MHCs. The supernatant is sedimented at 300,000
g for 1 h and the resulting supernatant made 5 mM ATP, 10 mM MgCl
and subjected to
(NH
)
SO
fractionation. The
40-60% fraction was solubilized in 10 ml of 0.5 M NaCl,
10 mM MOPS (pH 7.0), 0.1 mM EGTA, 3 mM NaN
, 0.1 mM PMSF, and 5 mM dithiothreitol (Buffer A) and dialyzed in 50 volumes of Buffer A
overnight with one change.
Figure 2:
Purification of HMM and
HMM
from baculovirus-infected Sf9 cells. A Coomassie
Blue-stained SDS 8-16% gradient polyacrylamide gel is shown for
HMM
(no insert) and HMM
(containing the
10-amino acid insert). Lanes 1 and 4 show the
polypeptide pattern of the Sf9 cell extract prior to sedimentation. A
total of 2 µl and 3 µl out of 50 ml were electrophoresed for
HMM
and HMM
, respectively. Lanes 2 and 5 show the 40-60% ammonium sulfate fraction. Sample size
was 3 µl out of 15 ml. Lanes 3 and 6 show the
purified HMM
and HMM
following addition of ATP
and sedimentation to release actin (see ``Experimental
Procedures'' for details). Sample size was 2 µl out of 0.9
ml.
Purification of HMM without the insert
(HMM) and with the insert (HMM
) is shown in Fig. 2. The only difference between these two isoforms is the
presence of the 10 amino acids in loop 1 starting after residue 211 in
the MHC. Lanes 1 and 4 show the pattern of
polypeptide staining following the initial extraction of Sf9 cells.
Examination of both the low speed and high speed (300,000
g) pellets revealed that most of the MHC
was
soluble under these conditions (data not shown). Lanes 2 and 5 show the polypeptide pattern following fractionation of the
extract supernatant with 40-60% ammonium sulfate. Lanes 3 and 6 show the purified HMM
following
release from F-actin by MgATP. Virtually all of the HMM
bound to actin in the absence of ATP and was released into the
supernatant in the presence of ATP (data not shown). Thus, most of the
expressed HMM heavy chains combined with light chains was soluble and
bound to actin in an ATP-dependent manner. Although both the extract
and 40-60% ammonium sulfate fraction show overexpression of
MLC
compared to MHC
, scanning of the
purified HMM
gave a molar ratio of 1:1:1 for the
MHC
and two MLCs. Using the procedure outlined above and
detailed under ``Experimental Procedures,'' we are able to
purify between 0.4 and 2 mg of purified HMM from 10
infected cells (650 ml of 1.5
10
cells/ml).
Figure 3:
Movement
of actin filaments by HMM and HMM
. Histogram of
the mean velocity of actin filament translocation by different
preparations of HMM
(open bars) and HMM
(cross-hatched bars). Paired bars represent preparations
of the isoforms purified and assayed on the same day. Conditions are as
described under ``Experimental Procedures.'' Data are shown
as the mean velocity with error bars indicating the standard
deviation.
Figure 4:
In vitro phosphorylation of
HMM and HMM
using Cdk5 kinase. Purified
HMM
and HMM
was incubated with
(+K) and without (-K) Cdk5 kinase, or
kinase alone was incubated without HMM (kinase; see ``Experimental
Procedures''). The panel on the left is an SDS
8-16% gradient polyacrylamide gel of the samples following
incubation with the phosphorylation mixture, and the corresponding
autoradiogram of the gel is on the right. Only the MHC
containing the 10-amino acid insert is phosphorylated (autoradiogram, lane 2). The 20-kDa MLC (MLC
) is also
phosphorylated to a small extent (lanes 1 and 2). The M
of standard proteins is shown on the left.
In order to identify the site(s) phosphorylated by Cdk5 kinase, both
the phosphorylated MHC and MLC
bands were
excised from the gel, digested with trypsin, and subjected to gel
isoelectric focusing. Fig. 5shows that the major phosphopeptide
generated by trypsin comigrates with a synthetic phosphopeptide with
the amino acid sequence: DHNIPPESPKPVK. This peptide represents the
amino acid sequence of the predicted tryptic peptide from the inserted
MHC sequence, assuming that trypsin would not cleave the KP peptide
bond (Fig. 1). Comigration of the MHC tryptic phosphopeptide
with the phosphorylated standard peptide is consistent with serine 214,
the only serine (or threonine) present in the inserted sequence being
the phosphorylated residue. The minor peptide seen near the bottom of
the gel is most likely due to partial cleavage of the RKD sequence at
the amino-terminal end of the expected tryptic peptide to yield
KDHNIPPESPKPVK, in addition to the expected peptide (see Fig. 1).
Figure 5:
Analysis of myosin heavy chain and myosin
light chain tryptic phosphopeptides. An autoradiogram of an isoelectric
focusing gel is shown. Lanes 1 and 2 are different
loadings of the tryptic digest of the heavy chain of HMM phosphorylated by Cdk5 kinase from the samples electrophoresed in Fig. 4. Lane 3 shows the focusing of a synthetic
phosphopeptide, DHNIPPESPKPVK, that was phosphorylated using the same
kinase. This peptide corresponds to the sequence of amino acids
207-219 in HMM
(see Fig. 1). Lane 4 shows the tryptic phosphopeptides of the MLC from the same sample.
The identification of the two light chain phosphopeptides as Ser-1 and
Ser-1` was based on standard phosphopeptides not shown (44) and
is due to partial cleavage by trypsin. The identification of the MHC
peptide as containing Ser-214 is based on the sequence of MHC
II-B1(13) . The peptide in lanes 1 and 2,
migrating nearer to the negative pole, is most likely due to partial
cleavage of the Arg-Lys sequence at the amino terminus of the tryptic
peptide (see Fig. 1).
We studied the ability of two other
proline-dependent kinases to catalyze phosphorylation of
MHC. Both MAP kinase (data not shown) and Cdc2 kinase
were capable of phosphorylating the heavy chain of HMM
, but
not HMM
. It was also of interest to see if tissue-purified
bovine brain myosin, which had previously been shown to contain both
MHC II-B and MHC II-B1(14) , could be phosphorylated. Fig. 6shows the result of an in vitro phosphorylation
assay using bovine brain myosin as well as HMM
as
substrate. Panel A is a Coomassie Blue-stained SDS-10%
polyacrylamide gel, and panel B is the corresponding
autoradiogram. The figure shows that purified bovine brain MHC II-B1 is
a substrate for Cdc2 kinase, and panel C shows that the site
phosphorylated is identical to that found for HMM
. In
addition to proline-directed kinases, we assayed a number of other
kinases to see if they could catalyze phosphorylation of
HMM
. Neither cAMP-dependent protein kinase, CaM kinase II,
nor protein kinase C could catalyze phosphorylation of the heavy chain
of this isoform (data not shown).
Figure 6:
In vitro phosphorylation of
bovine brain myosin and HMM by Cdc2 kinase. Purified bovine
brain myosin and HMM
were phosphorylated using Cdc2 kinase. A, Coomassie Blue-stained SDS-12.5% polyacrylamide gel showing
the incubation mixture of bovine brain myosin in the absence of kinase (-K), kinase alone, bovine brain myosin + Cdc2
kinase (+K), HMM
- kinase (-K), and HMM
+ kinase (+K). B, autoradiogram of A showing
phosphorylation of MHC from brain myosin and HMM
. There is
also autophosphorylation of Cdc2 kinase and phosphorylation of the
MLCs. C, isoelectric focusing gel. The bovine brain MHC and
HMM
MHC from the gel in panel A were excised from
the gel and digested with trypsin. The tryptic peptides were analyzed
by an isoelectric focusing gel, and the major phosphopeptide was
identified as containing Ser-214 based on its comigration with
standards (see Fig. 5).
The pre-mRNA encoding MHC II-B, but not MHC II-A, is subject to alternative splicing to produce a number of different isoforms that are only expressed in avian and mammalian neuronal cells. The function of the isoforms produced by this splicing is not known and they appear to be confined to cells that are part of the central nervous system. In chicken brain, the B1 isoform, detected by quantitative polymerase chain reaction, is already present by embryonic day 4 (the first day analyzed) and it reaches a peak on embryonic day 10(14) . Using S-1 nuclease analysis, Takahashi et al.(13) were only able to detect small amounts of mRNA encoding MHC II-B1 in the adult chicken brain. On the other hand, human cerebral cortex and retina have been shown to be highly enriched for mRNA encoding MHC II-B1 and have been shown to express the 10-amino acid inserted peptide(14) .
Kelley et al.(18) found that smooth muscle myosin containing an insert of seven amino acids (QGPSFSY) in the exact same place in loop 1 as that described for the nonmuscle MHC II-B1, translocates actin filaments 2.5 times faster than does smooth muscle myosin, which does not contain the inserted amino acids. Uyeda et al.(38) produced chimeric Dictyostelium myosins in which the amino acid sequence in the loop 2 region was exchanged for the homologous sequence from other types of myosins. These substitutions were found to modulate the actin-activated MgATPase activity of the chimeric myosins in a manner roughly proportional to the rate of the myosin from which the loop was derived, but did not have a similar effect on the rate of in vitro motility. Based on these two biochemical studies and the location of the two inserts in the crystal structure of myosin, Spudich suggested that the sequence in loop 1, which is near the ATP binding pocket, might have a profound effect on the translocation of actin filaments by myosin in the in vitro motility assay, whereas the sequence in loop 2 near an actin binding site may affect the actin-activated MgATPase activity(11) .
We found no major effect on either of these two activities when comparing side by side preparations of baculovirus expressed truncated MHC II-B isoforms that either contained or did not contain the inserted sequence in loop 1. Although both Fig. 3and Table 1show higher values for both the in vitro motility assay and the actin-activated MgATPase activity, these increases are, at best, modest. This suggests that the presence of this insert in neuronal cell myosin may have other functional consequences rather than to alter these two parameters of myosin activity or that this is a subtle modulatory mechanism.
The presence
of a consensus sequence for proline-directed kinases in the inserted
residues raised the possibility that this MHC might serve as a
substrate for a number of kinases, including Cdc2, Cdk5, and MAP
kinase. Previous work with Xenopus MHC II-B, which contains a
similar, although 6 amino acids longer, inserted region at loop 1, has
shown that the insert can be phosphorylated by Cdc2 kinase, but not MAP
kinase(21) . In this paper, we show that a number of
proline-directed kinases can phosphorylate the 10-amino acid insert in
the chicken nonmuscle MHC. This inability of Xenopus MHC-IIB
to be phosphorylated by MAP kinase may reflect the difference in
sequence (TESPK versus PESPK) between the species (see Fig. 1) and might also be related to the 6 extra amino acids
present in the Xenopus insert. We also found that Cdk5 kinase
could phosphorylate the MLC at the same site
phosphorylated by protein kinase C, in agreement with the observation
of Satterwhite et al.(39) using Cdc2 kinase. The
extent of MLC phosphorylation was considerably less than that of the
MHC with Cdk5, an enzyme that appears to be only active in neuronal
tissue(37) .
Despite multiple additions of kinase, only
30-40% of the MHC was phosphorylated. This did not appear to be
due to prior phosphorylation of the MHC since PO
-labeling of the Sf9 cells just prior to
harvesting showed no evidence for labeling of MHC
following SDS-polyacrylamide gel electrophoresis of a lysed cell
extract (data not shown). Failure to obtain more than 40%
phosphorylation of Ser-214 in vitro may mean that we have not
yet identified the relevant proline-directed kinase, the right
conditions for phosphorylation or both. In any case, the partial
phosphorylation of the MHC that we observed had no significant effect
on the actin-activated MgATPase activity.
What then could be the role of the 10-amino acid insert? Previous work has shown that splicing of the mRNA to introduce the insert is responsive to certain signal transduction pathways. For example, mRNA encoding MHC II-B can be induced to splice in the 30 nucleotides encoding MHC II-B1 by treating rat PC-12 cells with nerve growth factor, but not epithelial growth factor. The cells then cease to divide and initiate neurite outgrowth(14) . Since MHC II-B is the major isoform (perhaps the only isoform since the amount of MHC II-A in brain is small and may be expressed only in non neuronal cells) present in neuronal cells, it is conceivable that splicing in the insert acts as a localization mechanism, permitting the myosin II-B1 to be bound in a particular part of the cell. Phosphorylation of the myosin in the inserted sequence might then act to regulate this association. Studies to explore this possibility are presently under way.
Previous investigators have used the baculovirus expression system to express other HMMs, including smooth muscle HMM (40, 41, 42) and cardiac HMM(43) . Of particular interest have been studies using site-directed mutagenesis to understand the mechanism of smooth muscle myosin regulation(40) . To our knowledge, this is the first report on expression of a vertebrate nonmuscle myosin. The ability to express milligram quantities of an enzymatically active myosin fragment and to introduce discrete mutations is proving to be a powerful technique in understanding all forms of myosins. Our study has shown that the system is also a valuable technique for studying different functions of closely related isoforms of myosin that would prove extremely difficult to obtain in pure form.