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Volume 270, Number 3, Issue of January 20, 1995 pp. 1395-1401
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Xenopus Nonmuscle Myosin Heavy Chain Isoform Is Phosphorylated by Cyclin-p34 Kinase during Meiosis (*)

(Received for publication, September 8, 1994)

Christine A. Kelley (§), ,  Froma Oberman (1), (¶),  Joel K. Yisraeli (1), (**),  Robert S. Adelstein

From the Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Anatomy and Embryology, The Hebrew University-Hadassah Medical School, Jerusalem 91010, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2) 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.

INTRODUCTION

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) (^1)form two globular amino-terminal heads followed by alpha-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.

MATERIALS AND METHODS

Cell Culture

Xenopus XTC cells were grown at 25 °C in Leibovitz L-15 medium diluted to 61% and supplemented with fetal bovine serum to 10% as described previously(39) .

Antibody Production and Affinity Purification

Antibodies specific for nonmuscle MHC-B were generated against a synthetic peptide (SSSRSGRRQLHI) corresponding to a chicken MHC-B carboxyl-terminal sequence unique to this isoform(17) . Antibodies specific for the Xenopus inserted region were made against a synthetic peptide (TESPKAIKHQSGSLLY) corresponding to the deduced amino acid sequence of the insert(19) . The MHC-A carboxyl-terminal antiserum was generated as described previously (17) against the peptide GKAEAGDAKATE. Conjugation of the peptides to keyhole limpet hemocyanin, rabbit immunization, and affinity purification of the antisera were performed as described previously(40) .

Extraction of Cells

Total cell extracts were prepared for immunoprecipitation by solubilization of whole cells in a nondenaturing, Nonidet P-40 high salt extraction buffer at pH 7.5 containing protease inhibitors as described previously(41) .

Immunoblotting

Cell extracts subjected to electrophoresis in SDS-5% polyacrylamide gels were transferred to Immobilon-P (Millipore Corp., Bedford, MA) and immunostained with antiserum or affinity-purified antibodies as described(40) .

Immunoprecipitation and SDS-Polyacrylamide Gel Electrophoresis

Cell extracts were incubated overnight at 4 °C with anti-MHC-B or anti-MHC-A carboxyl-terminal antibodies. For studies on phosphorylation in intact cells, the myosin-antibody complexes were precipitated with Pansorbin (Calbiochem), washed several times with extraction buffer, and then boiled in SDS sample diluter(42) . For in vitro phosphorylation of immunoprecipitated myosin, the precipitates were washed several times with phosphorylation buffer (described below) before incubating with kinase and ATP. Immunoprecipitates were subjected to electrophoresis in either 4 or 5% polyacrylamide gels in the presence of SDS according to the method of Laemmli(42) . The incorporation of P was visualized with a PhosphorImager (Molecular Dynamics Incorporated, Sunnyvale, CA) or by autoradiography.

In Vitro Phosphorylation and Determination of Stoichiometry

Immunoprecipitated XTC cell myosin was incubated at 30 °C for 1 h in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl(2), 0.2 mM EGTA, 1 mM dithiothreitol (phosphorylation buffer), 500 nM peptide inhibitor to protein kinase A (Sigma), 500 µM ATP (10 µCi of [-P]ATP, 30 Ci/mmol, DuPont NEN) and human HeLa cell cdc2 kinase (kindly provided by Dr. Fumio Matsumura, Rutgers University) in a total volume of 100 µl. Reactions were terminated by the addition of SDS sample dilutor and boiled. Samples were electrophoresed in SDS-5% polyacrylamide gels, and the stoichiometry of MHC-B phosphorylation was determined by extraction of the P-phosphorylated MHCs from the gels using Solvable (DuPont NEN) followed by liquid scintillation counting. The total P content was then calculated from the radiospecific activity of [-P]ATP. The protein concentration of the two P-phosphorylated MHC-B isoforms was determined by densitometric scanning of the gels compared with a standard curve of gizzard smooth muscle MHCs.

Phosphorylation in Intact XTC Cells

XTC cells were labeled metabolically with P(i) (0.2 mCi/ml, DuPont NEN) in phosphate-free L-15 medium for 4 h. The labeling medium was removed, and cell extracts were prepared for immunoprecipitation as described above.

Phosphopeptide Mapping

The MHC-B subunits phosphorylated in intact cells or in vitro with cdc2 kinase were separated by 4% or 5% polyacrylamide gel electrophoresis in the presence of SDS. The heavy chain isoforms were excised from the gels and digested with trypsin as described previously(6) . The tryptic peptides were separated by one-dimensional isoelectric focusing (IEF) electrophoresis, pH 2.5-8.0, as described(43) . The gels were dried, and the phosphopeptides were detected with a PhosphorImager or by autoradiography.

Purification of Tryptic Phosphopeptides

Immunoprecipitated XTC cell MHC-B was phosphorylated in vitro by cdc2 kinase as described above. The heavy chains were separated on SDS-5% polyacrylamide gels and digested with trypsin as described for the phosphopeptide mapping experiments. The tryptic digests were lyophilized and dissolved in 0.1% trifluoroacetic acid in water and injected onto a C18 reverse phase high performance liquid chromatography (HPLC) column (Vydac, The Nest Group, Southborough, MA). Peptides were eluted with a linear gradient of 10-60% (v/v) acetonitrile, 0.1% trifluoroacetic acid over 60 min at 1.0 ml/min. A single radioactive peak that eluted in an acetonitrile concentration of approximately 20% was lyophilized, injected onto the same C18 column, but developed with an acetonitrile gradient of 10-30%. A single radioactive peak was obtained and used for amino acid sequencing.

Peptide Microsequencing

The amino acid sequence was kindly determined by Dr. Wilson Burgess, American Red Cross, Rockville, MD. Multiple rounds of Edman degradation were performed using an Applied Biosystems model 477 gas phase protein sequenator. Phenylthiohydantoin derivatives were identified with an on-line model 120A HPLC system.

Phosphoamino Acid Analysis

The P-labeled phosphopeptide from the IEF gel was hydrolyzed in 6 N HCl for 3 h at 106 °C. The acid-hydrolyzed peptides were subjected to electrophoresis at 1,000 V for 3 h in acetic acid/formic acid/water, 78:25:897, pH 1.9. Identification of radioactive phosphoamino acids was determined by autoradiography, and their migration was compared with that of standards of phosphotyrosine, phosphothreonine, and phosphoserine stained with ninhydrin.

Preparation and Phosphorylation of Xenopus Egg Lysates

Xenopus egg metaphase and interphase lysates were prepared from unfertilized (metaphase II) eggs as described previously(44, 45) . Extracts were incubated with 100 µCi of [-P]ATP (6,000 Ci/mmol, DuPont NEN) for 40 min at 25 °C. The lysates were then diluted with the cell extraction buffer described above and immunoprecipitated with antibodies to the carboxyl terminus of MHC-B and MHC-A also as described above.

Oocyte Labeling and Maturation

Xenopus female frogs were anesthetized by immersion in a solution of 0.04% benzocaine, and stage VI oocytes were manually isolated in 1 times modified Barth's saline-Hepes(46) . Oocytes (approximately 100 oocytes/100 µl) were radioactively labeled overnight at 20 °C in 1 times modified Barth's saline-Hepes containing 10 mCi/ml P(i) (370 mBq, Amersham Corp.) in the presence or absence of 10 µM water-soluble progesterone (Sigma). Oocytes were washed twice with 1 times modified Barth's saline-Hepes to remove unincorporated label, and matured oocytes were identified on the basis of germinal vesicle breakdown.

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).

RESULTS

Identification of Isoforms of the Nonmuscle MHC in Xenopus

The first goal of our studies was to determine whether the inserted form of MHC-B is a substrate for cdc2 kinase in vitro. We began these studies using the cultured Xenopus cell line, XTC. First, it was necessary to identify the inserted form of MHC-B at the protein level. Fig. 2A shows a Coomassie Blue-stained 5% polyacrylamide gel. The left lane is a total XTC cell extract that shows one major protein band around 200 kDa, which is the molecular mass of the MHC. When this extract is incubated with antibodies specific for the carboxyl terminus of MHC-B, the major band at 200 kDa is immunoprecipitated as well as two slower migrating minor bands at around 200 kDa (Fig. 2A, right lane). We identified these bands as MHCs by immunoblotting of XTC cell extracts as shown in Fig. 2B. The first lane shows the immunoreactivity of the extract with antibodies specific for an amino acid sequence in the carboxyl terminus of chicken MHC-A. The antibodies recognize the major, faster migrating MHC, but not the slower migrating, minor bands. The second lane shows that peptide antibodies generated against an amino acid sequence in the carboxyl terminus of chicken MHC-B recognize the two slower migrating, minor forms of myosin, MHC-B1 and MHC-B2, but not the major MHC isoform, MHC-A. Both of the MHC-B isoforms were also immunoreactive with peptide antibodies generated against the Xenopus insert amino acid sequence as shown in the last lane. Thus, in these Xenopus cells, there are two inserted forms of MHC-B as well as a noninserted, MHC-A isoform (see ``Discussion''). The identification of the major MHC isoform as MHC-A has been verified further by cDNA cloning and sequencing. (^2)We believe that MHC-A is nonspecifically immunoprecipitated with the MHC-B-specific antibodies through its interaction with MHC-B because when immunoprecipitations of cell extracts were performed in the presence of ATP, no MHC-A was immunoprecipitated along with MHC-B (data not shown).

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.


Phosphorylation of XTC Cell MHCs in Vitro by cdc2 Kinase

We next examined whether the inserted MHC-B1 and MHC-B2 isoforms could be phosphorylated by cdc2 kinase in vitro. Because these MHC-B isoforms are relatively minor compared with MHC-A and, therefore, not easily purified, we used immunoprecipitated myosin for our in vitro phosphorylation studies. Myosin was immunoprecipitated from XTC cells using the MHC-B carboxyl-terminal antibodies and phosphorylated in vitro with cdc2 kinase purified from mitotic HeLa cells. Fig. 3A shows a Coomassie Blue-stained 5% polyacrylamide gel of immunoprecipitated myosin incubated without (lane 1), or with (lane 2), cdc2 kinase. The corresponding autoradiogram (Fig. 3B) shows that in the presence of cdc2 kinase, the two inserted MHC-B isoforms, MHC-B1 and MHC-B2, are phosphorylated, but the noninserted MHC-A isoform is not phosphorylated (lane 4). Autoradiography does not resolve the two MHC-B isoforms as well as Coomassie staining; therefore, confirmation that both are phosphorylated was determined by separately cutting the bands from Coomassie-stained gels for peptide mapping (see below). Using these in vitro conditions, the stoichiometry of total MHC-B phosphorylation is estimated to be at least 0.3 mol of phosphate/mol of MHC-B. When [-P]ATP, but no kinase, is added to the immunoprecipitated myosin, there is no phosphorylation of any of the MHCs (lane 3).

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).

Identification of the cdc2 Kinase-catalyzed Phosphorylation Site in MHC-B

To determine the site of phosphorylation, tryptic peptides of MHC-B phosphorylated by cdc2 kinase in vitro were separated by reverse phase HPLC. The arrow in Fig. 4A points to the single peak of radioactivity observed, which was used for amino acid sequencing. The sequence obtained is shown in Fig. 4B (underlined sequence). Phosphoamino acid analysis demonstrated the presence of serine phosphate, but not threonine phosphate, in the purified tryptic phosphopeptide (Fig. 4A, inset). These data localized the phosphorylation site to Ser-214, shown with the asterisk in Fig. 4B. This serine is within the inserted region of MHC-B, and it is followed by a proline and a basic residue, which is typical of cdc2 kinase phosphorylation sites.

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(3)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).


Cell Cycle-dependent Phosphorylation of MHC-B in Intact XTC Cells and Xenopus Egg Lysates

To determine whether Ser-214 in MHC-B can be phosphorylated in intact cells, XTC cells in log phase of growth were labeled with P(i). The MHC-B isoforms were then immunoprecipitated from cell extracts, digested with trypsin, and the tryptic phosphopeptides were analyzed by IEF. The MHC-B tryptic phosphopeptides obtained after in vitro phosphorylation by cdc2 kinase were compared with those obtained following MHC-B phosphorylation in intact log phase XTC cells (Fig. 5A). A common radiolabeled phosphopeptide was observed which contained the site phosphorylated by cdc2 kinase in vitro. These results suggest that Ser-214 of MHC-B is phosphorylated in intact cells by cdc2 kinase or a cdc2-like kinase.

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.

Meiosis-specific Phosphorylation of MHC-B by cdc2 Kinase

Xenopus oocytes were used to examine the meiosis-specific phosphorylation of MHC-B in vivo. Quiescent stage VI oocytes are arrested in G(2) interphase of meiosis I and contain an inactive cdc2 kinase. When these oocytes are stimulated with progesterone, cdc2 kinase is activated, and the oocytes resume the meiotic cell cycle before arresting in metaphase of meiosis II(22) . Control and progesterone-treated oocytes were metabolically labeled with P(i), and MHC-B and MHC-A were immunoprecipitated separately from extracts of the labeled oocytes. Fig. 6A shows that MHC-B is phosphorylated in both untreated and progesterone-treated oocytes (first two lanes). Significantly, a slight retardation is discernible in the electrophoretic mobility of MHC-B from progesterone-treated oocytes, suggesting an additional site of phosphorylation. MHC-A was also phosphorylated, but no differences in the phosphorylation or migration of MHC-A between untreated and progesterone-treated oocytes were observed (Fig. 6A, last two lanes).

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.

DISCUSSION

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(2) 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 (^3)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.

FOOTNOTES

*
Part of this work was supported by a grant from the United States-Israel Binational Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bldg. 10, Rm. 8N-202, 10 Center Dr. MSC 1762, Bethesda, MD 20892-1762. Tel.: 301-496-5639; Fax: 301-402-1542.

Recipient of a postdoctoral fellowship from the Israel Cancer Research Foundation.

**
Recipient of a research career development award from the Israel Cancer Research Foundation.

(^1)
The abbreviations used are: MHC, myosin heavy chain; cdc2 kinase, cyclin-p34 kinase; MPF, maturation-promoting factor; IEF, isoelectric focusing; HPLC, high performance liquid chromatography; MAP kinase, mitogen-activated protein kinase; LC, 20-kDa myosin light chain.

(^2)
M. A. Conti, unpublished result.

(^3)
C. A. Kelley and I. C. Baines, unpublished data.

ACKNOWLEDGEMENTS

We thank Drs. Fumio Matsumura and Shigeko Yamashiro for help in trying to synchronize XTC cells, Dr. Don Bottaro for help in the purification of the MHC-B phosphopeptide and for critical reading of the manuscript, Dr. Naina Bhatia-Dey for helpful information about Xenopus, Dr. Cheryl Sato for use of her frog facilities, Dr. Igor Dawid for providing XTC cells, Drs. Jim Sellers and Sachiyo Kawamoto for critical reading of the manuscript, and Cathy Magruder for help in preparing the manuscript.

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