(Received for publication, September 8, 1994)
From the
There are two vertebrate nonmuscle myosin heavy chain (MHC)
genes that encode two separate isoforms of the heavy chain, MHC-A and
MHC-B. Recent work has identified additional, alternatively spliced
isoforms of MHC-B cDNA with inserted sequences of 30 nucleotides
(chicken and human) or 48 nucleotides (Xenopus) at a site
corresponding to the ATP binding region in the MHC protein (Takahashi,
M., Kawamoto, S., and Adelstein, R. S.(1992) J. Biol. Chem. 267, 17864-17871) and Bhatia-Dey, N., Adelstein, R. S., and
Dawid, I. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,
2856-2859). The deduced amino acid sequence of these inserts
contains a consensus sequence for phosphorylation by
cyclin-p34 (cdc2) kinase. In cultured Xenopus XTC cells, we have identified two inserted MHC-B isoforms and a
noninserted MHC-A isoform by immunoblotting of cell extracts. When
myosin was immunoprecipitated from XTC cells and phosphorylated in
vitro with cdc2 kinase, the kinase catalyzed the phosphorylation
of both inserted MHC-B isoforms but not MHC-A. Isoelectric focusing of
tryptic peptides generated from MHC-B phosphorylated with cdc2 kinase
revealed one major phosphopeptide that was purified by reverse-phase
high performance liquid chromatography and sequenced. The
phosphorylated residue was Ser-214, the cdc2 kinase consensus site
within the insert near the ATP binding region. The same site was
phosphorylated in intact XTC cells during log phase of growth and in
cell-free lysates of Xenopus eggs stabilized in second meiotic
metaphase but not interphase. Moreover, Ser-214 phosphorylation was
detected during maturation of Xenopus oocytes when the cdc2
kinase-containing maturation-promoting factor was activated, but not in
G
interphase-arrested oocytes. These results demonstrate
that MHC-B phosphorylation is tightly regulated by cdc2 kinase during
meiotic cell cycles. Furthermore, MHC-A and MHC-B isoforms are
differentially phosphorylated at these stages, suggesting that they may
serve different functions in these cells.
Myosin is a superfamily of proteins which includes a number of
molecular motors that can move relative to actin filaments and can
generate force in a Mg-ATP-dependent
manner(1, 2) . Conventional myosin (myosin II) plays
both a structural and an enzymatic role in such diverse cellular
processes as muscle contraction (3) , cell
division(4) , cell locomotion(5) , and intracellular
movements(4, 6, 7) . The myosin II molecule
consists of a dimer of two heavy chains of approximately 200 kDa
noncovalently associated with two pairs of light chains of
approximately 20 and 17 kDa. The myosin heavy chains (MHCs) (
)form two globular amino-terminal heads followed by
-helical coiled-coil tails. The heads contain an actin-activated
ATPase activity, and the tails are involved in filament formation. The
myosin heads can be divided by three protease-sensitive regions into
peptides of 25, 50, and 20 kDa (Fig. 1). The ATP binding region
is near the 25-50 kDa junction, and the actin binding region is
near the 50-20 kDa junction.
Figure 1: Inserted sequences near the ATP binding region of nonmuscle myosin heavy chain B. Above are the amino acid sequences around and including the insert in MHC-B. The top two lines show the sequence of the noninserted and inserted forms, respectively, of the MHC-B protein in chickens as deduced from brain cDNA(17) . The insert in MHC-B in chickens consists of 10 amino acids beginning at amino acid 212. The bottom line shows that in Xenopus, this same region of the MHC-B, beginning at amino acid 212, contains a 16-amino acid insert. Unlike chickens, in Xenopus there is no noninserted MHC-B(19) . The underlined amino acids denote the consensus sequence for phosphorylation by cdc2 kinase. The S with the asterisk represents the potential phosphorylation site for this kinase. The arrowhead in the bottom diagram points to the approximate location of the insert in MHC-B, which is near the ATP binding region.
Both the heavy chain and the
light chain subunits of myosin exist as isoforms. Our interest has been
in the structure and function of the heavy chain isoforms of smooth
muscle and nonmuscle myosin. There is one smooth muscle MHC gene and at
least two pairs of alternatively spliced products of the smooth muscle
MHC mRNA. One pair of isoforms is generated from alternative splicing
in the 3` end of the mRNA, resulting in MHC proteins with carboxyl
termini that differ in length and sequence(8, 9) . We (10) and others (11, 12, 13) found
that the smooth muscle MHC mRNA is also alternatively spliced at the 5`
end. Intestinal, but not vascular, MHC mRNA contains an insert of 21
nucleotides encoding 7 amino acids. This insert occurs beginning at
amino acid 212, which is near the 25-50 kDa junction in the
primary sequence of the heavy chain. In the three-dimensional structure
of the heavy chain(14) , this insert is located near the ATP
binding pocket in a region that was not resolved in the crystal
structure, suggesting that this region may be flexible. This insert
most likely affects the ATP binding pocket because we found that the
presence of the insert correlates with a higher velocity of movement of
actin filaments in an in vitro motility assay and a higher
actin-activated Mg-ATPase activity(10) .
In contrast to a single gene encoding smooth muscle MHC isoforms,
there are at least two genes encoding nonmuscle MHC isoforms. The
products of these two nonmuscle MHC genes are referred to as MHC-A and
MHC-B(15, 16) . The recent cDNA cloning of chicken
brain nonmuscle MHC-B provided evidence for multiple inserted forms of
neuronal MHC-B(17) . One of these brain cDNA isoforms contained
a 30-nucleotide insert encoding 10 amino acids in a region
corresponding exactly to the region of the insert in the smooth muscle
MHC isoform (see Fig. 1, arrowhead). However, in
nonmuscle MHC-B the insert is a different size and has an amino acid
sequence different from that of the smooth muscle insert. One
interesting feature of the nonmuscle insert, which is not true of the
smooth muscle insert, is that it contains a putative phosphorylation
site for p34 (cdc2) kinase: a serine followed by a
proline and a basic residue(18) . A similar insert of 48
nucleotides, encoding 16 amino acids (see Fig. 1), also
containing a consensus sequence site for phosphorylation by cdc2
kinase, was found in Xenopus nonmuscle MHC-B(19) .
cdc2 kinase is a cell cycle-regulated kinase that catalyzes the entry of cells into meiosis and mitosis(20, 21, 22) . The kinase is part of a protein complex called maturation-promoting factor (MPF). MPF consists of cdc2 kinase, which is the catalytic subunit(23, 24, 25) , and a regulatory subunit called cyclin(26, 27) . MPF activity cycles, being active during meiotic and mitotic metaphase but inactive during interphase, due partly to the repetitive synthesis and degradation of cyclin(28, 29, 30) . Transitions from interphase to meiosis or mitosis in eukaryotic cells entail a wide variety of changes in cell structure (30, 31, 32, 33) . In addition, entry of cells into meiosis and mitosis is accompanied by a dramatic increase in the level of phosphorylation of many proteins involved in both regulatory and structural aspects of mitosis and meiosis. There is substantial evidence that phosphorylation by cdc2 kinase of certain cytoskeletal structural proteins such as the nuclear lamins(34) , vimentins(35) , and caldesmon (36, 37) plays an important role in the induction of mitosis and/or meiosis. Recently it was also shown that the regulatory light chain of Xenopus nonmuscle myosin is phosphorylated by cdc2 kinase during metaphase, but not interphase, in Xenopus egg lysates(38) .
The aim of the present study was to determine if nonmuscle MHC-B is a physiological substrate for cdc2 kinase. We used cultured Xenopus XTC cells as well as egg lysates and intact oocytes to examine the meiotic and mitotic metaphase-specific phosphorylation of MHC-B. We found that the major MHC isoform in Xenopus is MHC-A, which does not contain a cdc2 kinase phosphorylation site near the ATP binding region. Phosphorylation of this isoform was not different in metaphase and interphase cells. In contrast, MHC-B containing an insert with a cdc2 kinase consensus sequence phosphorylation site near the ATP binding region was a minor myosin isoform in Xenopus. We found that cdc2 kinase phosphorylates MHC-B in vitro at a single site, Ser-214, which is within the insert near the ATP binding pocket. This same site was phosphorylated in intact XTC cells during exponential growth as well as in metaphase, but not interphase, Xenopus egg lysates and intact oocytes, suggesting that phosphorylation of MHC-B may play a role in mitosis and meiosis.
Extracts were prepared by homogenizing oocytes in 200 µl of buffer (50 mM Tris, pH 7.5, 25% glycerol, 50 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mM vanadate, 10 µg/ml leupeptin, 2 µg/ml pepstatin) on ice. The extracts were cleared of yolk and cortical debris by two centrifugations in a microcentrifuge for 15 min each at 4 °C, and frozen at -80 °C. Before immunoprecipitation (as described above), the extracts were thawed and diluted to 500 µl with extraction buffer (see above).
Figure 2: Identification of inserted forms of nonmuscle myosin heavy chain B in Xenopus XTC cells. Panel A, Coomassie Blue staining of XTC cell extracts and immunoprecipitated XTC cell myosin electrophoresed in SDS-5% polyacrylamide gels. Panel B, immunoblot of XTC cell extracts electrophoresed as described for panel A and transferred to Immobilon. The left lane shows the reactivity with an antibody specific for MHC-A, the middle lane shows the reactivity with MHC-B-specific antibodies, and the right lane shows the reactivity of the extract to antibodies generated against the Xenopus 16-amino acid inserted sequence in MHC-B.
Figure 3:
Phosphorylation of XTC cell myosin heavy
chains in vitro by cyclin-p34 kinase and
analysis of tryptic phosphopeptides. Myosin was immunoprecipitated from
XTC cells with MHC-B carboxyl-terminal-specific antibodies and
phosphorylated in vitro with purified cdc2 kinase. Panel
A, lanes 1 and 2 show the immunoprecipitates
stained with Coomassie Blue. The antibodies precipitate some of the
MHC-A and all of the MHC-B (MHC-B1 and MHC-B2) from the extracts. Panel B, lanes 3 and 4, show the
corresponding autoradiogram. The phosphorylated MHC-B1 and MHC-B2 bands
were cut separately from the gel using the Coomassie stain as a guide,
digested with trypsin, and approximately 1,000 cpm of each tryptic
digest was focused in an IEF gel as described under ``Materials
and Methods.'' Panel C shows the autoradiogram of the IEF
gel.
To determine whether the same sites were phosphorylated in MHC-B1 and MHC-B2 by cdc2 kinase, these MHC bands were cut separately from Coomassie Blue-stained 5% polyacrylamide gels, digested with trypsin, and approximately equal cpm of the tryptic digests were analyzed by IEF. Fig. 3C is an autoradiogram of the focused tryptic phosphopeptides and shows that there is only one major phosphopeptide that is the same in MHC-B1 and MHC-B2. A tryptic phosphopeptide focusing to the same position was seen following tryptic digestion of MHC-B1 and MHC-B2 phosphorylated with Xenopus cdc2 kinase (gift of Dr. James Maller, University of Colorado) and sea star oocyte cdc2 kinase (Upstate Biotechnology Inc., Lake Placid, NY; results not shown).
Figure 4:
Identification of the cdc2 kinase
phosphorylation site in MHC-B. Panel A, MHC-B peptides that
eluted from a C18 reverse phase HPLC column in a single radioactive
peak at an acetonitrile concentration of 20% (see ``Materials and
Methods'') were injected onto the same C18 column. The column was
developed with an acetonitrile gradient of 10-30% in 0.1%
trifluoroacetic acid over 60 min at 1.0 ml/min (panel A). The arrow points to the single radioactive peak that was collected
and used for amino acid sequencing. Radioactivity was determined by
Cerenkov counting. Peptides were detected by A.
The y axis denotes the percent of full scale where full scale
is 100 for CH
CN, 400 for cpm and 32 milliAbsorbance units
for A
. Panel B, the peptide purified as
shown in panel A was sequenced as described under
``Materials and Methods.'' The sequence obtained is underlined and is shown between the two arrows marking the predicted tryptic cleavage sites. The serine with the asterisk (Ser-214) marks the phosphorylated amino acid that
was determined by phosphoamino acid analysis. The purified tryptic
phosphopeptide was subjected to acid hydrolysis followed by thin layer
electrophoresis as described under ``Materials and Methods.''
The inset in panel A shows the autoradiogram of the
phosphoamino acids from MHC-B (left lane) and the positions of
the phosphoamino acid standards (right
lane).
Figure 5: Analysis of tryptic phosphopeptides of MHC-B phosphorylated in logarithmically growing XTC cells and in metaphase and interphase Xenopus egg lysates.Panel A, autoradiogram, and panel B, PhosphorImage of IEF gels containing tryptic phosphopeptides of MHC-B phosphorylated in vitro with purified cdc2 kinase (panel A, left lane), in actively dividing XTC cells (panel A, right lane), in metaphase Xenopus egg lysates (panel B, left lane) and in interphase egg lysates (panel B, right lane). Ser-214 points to the peptide containing phosphate at Ser-214 as determined by comigration with one shown to be phosphorylated at Ser-214 by amino acid sequencing.
Because cdc2 kinase regulates the entry of cells into meiosis and
mitosis, we sought to determine whether MHC-B is specifically
phosphorylated at these stages of the cell cycle by cdc2 kinase. To
study the metaphase-specific phosphorylation of MHC-B by cdc2 kinase,
we used lysates of Xenopus eggs. Xenopus cytoplasmic
lysates that were stabilized in either metaphase or interphase were
labeled with [-P
]ATP, and the MHC-A and
MHC-B isoforms were immunoprecipitated separately with specific
carboxyl-terminal peptide antibodies. Autoradiograms of
immunoprecipitates of MHC-B electrophoresed in polyacrylamide gels
showed a prominent 200-kDa phosphoprotein from both metaphase and
interphase lysates (results not shown). In contrast, the MHC-A
immunoprecipitates did not appear to be phosphorylated in either the
metaphase or interphase lysates. The radiolabeled MHC-B bands were
digested with trypsin, and the tryptic phosphopeptides were separated
by IEF (Fig. 5B). A peptide that comigrates with the
peptide phosphorylated at Ser-214 in MHC-B phosphorylated in vitro with cdc2 kinase, is present in MHC-B from metaphase lysates, but
not interphase lysates, suggesting that this site is phosphorylated by
cdc2 kinase specifically during metaphase of the cell cycle. The
additional phosphopeptides seen in both the mitotic and interphase
lysates have not yet been identified, but it appears that
phosphorylation of MHC-B at sites other than Ser-214 may also differ in
metaphase and interphase.
Figure 6: Phosphorylation of Xenopus oocyte myosin heavy chains during meiosis. Panel A, autoradiogram of an SDS-4% polyacrylamide gel (PAGE) showing MHC-B and MHC-A immunoprecipitated from oocytes that were not treated(-) or were treated (+) with progesterone. Panel B, PhosphorImage of an IEF gel showing phosphopeptides from tryptic digests of the MHC-A and MHC-B bands from the gel in panel A. Ser-214 points to a tryptic phosphopeptide that comigrates with one phosphorylated on this site by cdc2 kinase in vitro.
MHC-B and MHC-A tryptic digests were analyzed by one-dimensional IEF (Fig. 6B). The first lane shows two major phosphopeptides and one minor phosphopeptide from MHC-B of untreated interphase oocytes. The same phosphopeptides are generated from MHC-B of progesterone-treated metaphase oocytes (second lane), but, in addition, a marked increase in the phosphopeptide containing Ser-214 was observed. No differences are detectable in the phosphopeptides of MHC-A from oocytes incubated with or without progesterone (Fig. 6B, third and fourth lanes). This experiment was repeated, with similar results, using four different preparations of untreated and progesterone-treated oocytes. Therefore, maturation of Xenopus oocytes leads to the specific phosphorylation of MHC-B but not MHC-A. This phosphorylation at Ser-214, a site that is located within an inserted sequence of the heavy chain near the ATP binding region, is also the precise site of cdc2 kinase phosphorylation in vitro, in intact log phase XTC cells, and in metaphase-arrested egg lysates.
A single eukaryotic cell may possess a myriad of myosin isoforms. Two important questions are: how are these various actin-based motors regulated in vivo, and what is the significance of multiple myosin isoforms. The results of the present study demonstrate that Xenopus nonmuscle MHC-B, but not MHC-A, is a physiological substrate for cdc2 kinase. The nonmuscle MHC-B isoform is phosphorylated on a single serine residue, Ser-214, which is near the ATP binding region on the MHC. This site is phosphorylated in vitro by purified cdc2 kinase as well as in intact Xenopus XTC cells during log phase of growth. To determine if the phosphorylation in intact cells was metaphase-specific, we initially tried to synchronize XTC cells to obtain pure mitotic and interphase cell populations. However, we were unable to synchronize these cells using methods that successfully synchronized rat embryo fibroblasts (REF-4A cells). Therefore, to study metaphase-specific phosphorylation, we turned to lysates of Xenopus eggs stabilized in either metaphase or interphase. These lysates are capable of carrying out many of the events of the cell cycle in vitro, such as nuclear envelope breakdown and reformation, as well as membrane vesicle fusion. We found no phosphorylation at Ser-214 in extracts from eggs that are stabilized in the interphase following meiosis II (the equivalent of the interphase following fertilization) but marked Ser-214 phosphorylation in lysates of eggs stabilized in metaphase of meiosis II.
We then examined the phosphorylation of Ser-214 in MHC-B
in intact oocytes undergoing meiosis. In stage VI oocytes that were
arrested in the G interphase preceding meiosis I, there was
little or no phosphorylation of MHC-B at Ser-214. In contrast, when
stage VI oocytes were treated with progesterone to stimulate their
progression through meiosis and their arrest in metaphase of meiosis
II, MHC-B became phosphorylated at Ser-214. cdc2 kinase activation
induces both meiosis and mitosis whereas its inactivation triggers the
onset of anaphase and progression into interphase. Thus, MHC-B
phosphorylation and dephosphorylation at Ser-214 correlate with the
activation of cdc2 kinase in meiotic metaphase and its inactivation in
interphase, respectively. It is of interest to note that in addition to
the Ser-Pro sequence in the inserted region of MHC-B, there are three
other Ser-Pro or Thr-Pro sequences in this heavy chain isoform. These
other cdc2 kinase consensus sequence sites are not phosphorylated,
suggesting that phosphorylation at Ser-214 is specific and most likely
important.
Progesterone induction of maturation of Xenopus oocytes results in the synchronous activation of cdc2 kinase and mitogen-activated protein (MAP) kinase(47, 48, 49, 50) . The minimum consensus sequence for phosphorylation by MAP kinase, similar to cdc2 kinase, is Ser-Pro or Thr-Pro. Therefore, Ser-214 might also be phosphorylated by MAP kinase. Attempts to phosphorylate Xenopus MHC-B in vitro using purified Xenopus MAP kinase (gift of Dr. James Maller, University of Colorado) or sea star MAP kinase (Upstate Biotechnology, Inc., Lake Placid, NY) were unsuccessful (data not shown), suggesting that the meiotic-specific phosphorylation of MHC-B on Ser-214 in vivo is most likely catalyzed by cdc2 kinase.
In Xenopus XTC cells, oocytes, and eggs we found one species of MHC-A and two inserted MHC-Bs that migrate slightly differently in low percentage polyacrylamide gels. Although we are not certain why there are two MHC-Bs in Xenopus, it may be that these MHCs are the products of duplicated genes. In Xenopus a number of genes are represented by two copies with generally less than 10% sequence divergence(51) . Therefore, it is possible that the two MHC-B bands represent the products of two very similar duplicated genes. There appears to be only one MHC-A in Xenopus based on our polyacrylamide gels. It is possible that the MHC-A gene is not present in a duplicated form or that we are unable to resolve the two MHC-A bands in our gel electrophoresis systems.
The phosphopeptide maps of MHC-B and MHC-A from metaphase and interphase oocytes revealed the presence of two phosphopeptides in addition to the phosphopeptide containing Ser-214. These other phosphopeptides have not yet been identified, although previous studies have shown that both MHC-A and MHC-B contain amino acids that can be phosphorylated by protein kinase C (6, 52) and casein kinase II(53) . It is important to note that the increased phosphorylation of Ser-214 was the only reproducible change in MHC-B phosphorylation we observed between metaphase and interphase in four separate experiments. For example, phosphorylation of the additional two peptides in MHC-B did not always decrease in metaphase compared with interphase oocytes, as shown in Fig. 6B.
Previously, Satterwhite et al.(38) reported the
phosphorylation of the regulatory light chain (LC) of Xenopus myosin by cdc2 kinase in metaphase, but not interphase
egg lysates. Ser-1 or Ser-2 and Thr-9 on LC
were
phosphorylated by cdc2 kinase. Yamakita et al.(54) found that Ser-1 and/or Ser-2 of LC
was also
phosphorylated in mitotic but not interphase REF-4A cells. These sites
are the same sites phosphorylated by protein kinase C and are known to
result in inhibition of the actin-activated ATPase activity of smooth
muscle and nonmuscle myosin by decreasing the affinity of myosin for
actin(55, 56, 57) . Satterwhite et
al. (38) speculated that inhibition of myosin ATPase
activity by cdc2 kinase-catalyzed phosphorylation at these sites during
prophase and metaphase might be involved in the regulation of the
timing of cytokinesis. In contrast to our results, they found that the
MHC band immunoprecipitated with an antibody to Xenopus myosin
II was not phosphorylated in either the metaphase or interphase
extracts(38) . One possible explanation for this difference is
that their antibody precipitated both MHC-A and MHC-B and, since MHC-B
is a minor isoform in these lysates, its phosphorylation may not have
been apparent. In addition, their SDS-polyacrylamide gel
electrophoresis system would not have resolved the MHC-A and MHC-B
isoforms as we have shown in the present report.
Together, our
results and those of Satterwhite et al.(38) suggest
that MHC-B may be regulated by both light chain and heavy chain
phosphorylation during meiosis, whereas MHC-A may be regulated by light
chain phosphorylation alone. It will be of interest to examine
separately the metaphase and interphase phosphorylation of the
LCs associated with MHC-A and MHC-B since the report by
Satterwhite et al.(38) only studied total LC
phosphorylation. Examination of the phosphorylation of the 20-kDa
light chains associated with the individual MHC isoforms during
metaphase and interphase should provide information about whether these
isoforms are differentially phosphorylated on the light chain, as well
as the heavy chain, as we have reported here.
The relatively small
amount of MHC-B relative to MHC-A in Xenopus cells poses a
number of problems (see Fig. 2A, left lane).
It has made purification and in vitro characterization of the
purified MHC-B isoform (in the absence of MHC-A) difficult. Although we
have been able to incorporate a significant amount of phosphate into
the unique cdc2 kinase site in vitro (see
``Results''), it has not been possible to determine the
stoichiometry of phosphorylation of the MHC-B isoform in intact cells.
On the other hand, recent studies by Maupin et al.(58) with mammalian cell lines as well as preliminary
experiments by us ()using Xenopus cells suggest
distinct localizations for the MHC-B and MHC-A isoforms. This latter
finding strongly supports a different function for the two isoforms and
is consistent with a distinct function for MHC-B during meiosis.
The identification of Xenopus MHC-B as a physiological substrate for cdc2 kinase during meiosis is important because it may explain some of the many structural changes that accompany the conversion of fully grown oocytes to fertilization-competent eggs. During maturation, the cortical actin network is reorganized; e.g. oocyte microvillae retract from intercalated follicle cells and the eggs become capable of undergoing cortical contraction in response to sperm penetration. This cortical contraction is a myosin-mediated contraction of the cortex toward the apex of the animal hemisphere of the egg and is believed to aid in moving the male pronucleus closer to the female pronucleus, thereby facilitating pronuclear fusion(31) . MHC-B is unphosphorylated on Ser-214 before entering meiosis, it becomes phosphorylated on Ser-214 by cdc2 kinase during meiosis, and is again dephosphorylated at this site in the interphase, which is equivalent to the fertilized egg. Thus, MHC-B phosphorylation by cdc2 kinase, at a site located near the ATP binding region, is correlated with the cortical reorganization that occurs in meiosis, and dephosphorylation at this site correlates with cortical contraction. Further experiments should elucidate the precise function of this modification during early Xenopus development.