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Identification of myosin II kinase from sea urchin eggs as protein kinase CK2
Gene 275 (2001) 141–148 www.elsevier.com/locate/gene
Identification of myosin II kinase from sea urchin eggs as protein kinase CK2 Shigeru Komaba, Hajime Hamao, Maki Murata-Hori 1, Hiroshi Hosoya* Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan Received 31 January 2000; received in revised form 6 June 2001; accepted 18 July 2001 Received by T. Sekiya
Abstract Here we purified and identified a myosin II kinase from sea urchin eggs. The activity of this myosin II kinase in the egg extract was not significantly affected by Ca 21/calmodulin (CaM). Using sequential column chromatographies, we purified the myosin II kinase from the egg extract as a complex composed of 36- (p36) and 28-kDa (p28) proteins. Partial amino acid sequences of these two components were highly coincident with those of the a and b subunits of protein kinase CK2 (formerly casein kinase II) in sea urchin eggs, respectively. To confirm that the purified myosin II kinase was CK2, we obtained a cDNA which encodes p36 from a cDNA library of sea urchin eggs. The amino acid sequence derived from the obtained cDNA showed over 70% homology to CK2 from various eukaryotes. Furthermore, recombinant p36, as well as the purified myosin II kinase, phosphorylated MRLC. One dimensional phosphopeptide mapping revealed that the phosphorylation site(s) of MRLC by both recombinant p36 and the purified myosin II kinase was identical. These clearly showed that the Ca 21/CaMindependent myosin II kinase activity in sea urchin eggs was identical to CK2. q 2001 Elsevier Science B.V. All rights reserved. Keywords: CK2; Myosin II regulatory light chain; Phosphorylation; Sea urchin egg; Myosin light chain kinase
1. Introduction Phosphorylation of the myosin II regulatory light chain (MRLC) is believed to be important for modulating the interaction between filamentous myosin II and actin filaments that generates force for the contraction of smooth muscle and non-muscle cells (Sellers and Adelstein, 1987). It is well known that the actin-activated MgATPase activity of myosin II increases upon phosphorylation of MRLC by Ca 21/calmodulin (CaM)-dependent myosin light chain kinase (MLCK), at least in vitro (Kamm and Stull, 1985; Ikebe and Hartshorne, 1985). Phosphorylation sites of MRLC by MLCK are Ser19 and Thr18 (MLCK sites) and the phosphorylation at both sites results in higher MgATPase activity of myosin II than that of single site phposphorylated myosin II (Ikebe, 1989). Recently, it was reported that several Ca 21/CaM-independent kinases, which Abbreviations: MAPKAPK-2, MAP kinase activated protein kinase-2; MLCK, myosin light chain kinase; MRLC, myosin regulatory light chain; PKC, protein kinase C; CK2, casein kinase 2; PMSF, phenyl methyl sulfonyl fluoride * Corresponding author. Tel.: 181-824-24-7443; fax: 181-824-24-0734. E-mail address: [email protected] (H. Hosoya). 1 Present address: Department of Physiology, University of Massachusetts Medical School, 377 Plantation ST., Worcester, MA 01605, USA.
might have important roles in cell division, such as RSK-2 (Suizu et al., 2000), MAPKAPK-2 (Komatsu et al., 1997) and AIM-1 (one of Aurora/Ipl1 kinase family) (Murata-Hori et al., 2000), Rho-kinase (Amano et al., 1996), p21-activated protein kinase (Chew et al., 1998) phosphorylate MRLC at Ser19. Moreover, phosphorylated MRLC at Ser19 was localized along the contractile ring in dividing non-muscle cells (Matsumura et al., 1998; Murata-Hori et al., 1998). These results suggest that the phosphorylation of MRLC at Ser 19 may be involved in the regulation of cytokinesis. We also found that ZIP kinase from non-muscle cells could more efficiently phosphorylate MRLC at both Ser19 and Thr18 than MLCK (Murata-Hori et al., 1999), suggesting that phosphorylation of MRLC at both Ser19 and Thr18 might occur in non-muscle cells. On the other hand, phosphorylation sites of MRLC at Ser1/Ser2 and Thr9 by PKC (PKC sites) down-regulated MgATPase activity of myosin II phosphorylated by MLCK (Nishikawa et al., 1984; Ikebe et al., 1987). Satterwhite et al showed that p34 cdc2 kinase also phosphorylated the same sites of MRLC as did PKC (Satterwhite et al., 1992). The cumulative evidence suggests that changes in the phosphorylation state of MRLC may play an important role in the cytokinesis signaling pathway.
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00626-6
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Recently, the activity of kinases capable of phosphorylating MRLC was analyzed throughout the first cell cycle of sea urchin eggs (Totsukawa et al., 1996). Total kinase activity in vitro did not fluctuate during the cell cycle but the activity of the kinase(s) responsible for phosphorylation of MRLC at PKC sites showed a significant increase at metaphase. Using a selective inhibitor of p34 cdc2 kinase, this myosin II kinase activity for PKC sites was recognized as a p34cdc2 kinase. In contrast, MRLC phosphorylation at MLCK sites showed no significant changes during the first cell cycle. As this myosin II kinase activity for MLCK sites was observed both in the absence and presence of Ca 21/ CaM, it is supposed to be different from the Ca 21/CaM dependent MLCK. Thus, it is important to elucidate which kinase(s) participate in temporal and spatial regulation of phosphorylation of MRLC during cell division. Here, we tried to purify and identify the kinase that phosphorylates MRLC from sea urchin egg extract. As the result of this approach, a myosin II kinase was purified from sea urchin eggs and identified as a multifunctional serine/threonine kinase, protein kinase CK2. CK2 might have important roles in cell cycle progression throughout phosphorylation of MRLC during the early development of sea urchin eggs.
2. Material and methods 2.1. Material Light chains from myosin II (MLCs) and myosin light chain kinase (MLCK) were prepared from chicken gizzard as described elsewhere (Murata-Hori et al., 1999). Protein kinase C (PKC) purified from rabbit brain was kindly provided by Drs K. Mizuno and S. Ohno (Yokohama City University). CaM from bovine brain, TPCK treated trypsin (Type XIII), calyculin A and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO). Leupeptin and pepstatin A were from the Peptide Institute (Osaka, Japan). Endoproteinase Glu-C from Staphylococcus aureus V8 was purchased from Boehringer Mannheim (Mannheim, Germany). An antibody raised against the peptide for the consensus amino acid sequence of the CK2 peptide was kindly provided by Drs Moriyama and I. Yahara (Tokyo Metropolitan Institute of Medical Science, Tokyo). 2.2. Preparation of sea urchin egg extracts Unfertilized eggs from sea urchin, Hemicentrotus pulcherrimus, were collected by centrifugation at 1700 £ g for 2 min and homogenized with an equal volume of buffer H (100 mM Pipes (pH 6.8), 10% (v/v) glycerol, 5 mM MgCl2, 10 mM EGTA, 5 mM PMSF, and 10 mg/ml leupeptin). The egg homogenates were centrifuged at 20 000 £ g for 30 min and the supernatant was removed. Then, the pellet was homogenized with an equal volume of buffer H
and stored at 2808C until use. All procedures were carried out at 48C. 2.3. Purification of myosin II kinase from sea urchin egg extracts Sea urchin egg extracts were obtained under high salt condition as described by Ishidate and Mabuchi (1989) with a slight modification. Briefly, the homogenate stocked at 2808C was resuspended in an equal volume of buffer H containing 1 M NaCl, swirled for 2 h and then centrifuged at 20 000 £ g for 30 min. The supernatant was applied to a gel filtration column (5 £ 89 cm) of Sephacryl S-300 (Amersham Pharmacia Biotech) equilibrated with buffer A (20 mM Tris–HCl, pH 8.0, 2 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml pepstatin A, and 0.1 mM DTT) containing 200 mM NaCl. Each fraction was assayed for myosin II kinase activity as described below. Fractions containing myosin II kinase activity were collected and applied to a column (2:5 £ 17 cm) of hydroxylapatite (Bio-Rad HTP) equilibrated with buffer B (20 mM potassium phosphate (pH 8.0), 2 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml pepstatin A, and 0.1 mM DTT). The column was washed with buffer B and retained proteins were eluted with 300 ml of buffer B in a linear gradient of 20–500 mM potassium phosphate. All of the fractions obtained were assayed for myosin II kinase activity, respectively. Fractions of myosin II kinase activity were collected, dialyzed against buffer A containing 20 mM NaCl and applied to a Resource Q anion exchange column (6 ml; Amersham Pharmacia Biotech) equilibrated buffer A containing 20 mM NaCl. The column was washed with buffer A containing 20 mM NaCl and retained proteins were eluted with 120 ml of buffer A in a linear gradient of 20–500 mM NaCl using FPLC system (Amersham Pharmacia Biotech). Each fraction was assayed for myosin II kinase activity. Fractions containing myosin II kinase activity eluted at about 400 mM NaCl were collected, diluted with an equal volume of buffer A and applied to a Resource Q anion exchange column (6 ml) equilibrated with a buffer A containing 200 mM NaCl again. The column was washed with buffer A containing 200 mM NaCl and retained proteins were eluted with 120 ml of buffer A in a linear gradient of 200–500 mM NaCl using FPLC system. Each fraction was assayed for myosin II kinase activity. Fractions containing myosin II kinase activity were pooled and stored at 2808C until use. All procedures were carried out at 48C. 2.4. Myosin II kinase assay An aliquot from each fraction (10 ml) was added to a reaction mixture containing 120 mM Tris–HCl (pH 8.0), 4 mM EGTA, 8 mM MgCl2, 0.2 mM calyculin A, 100 mg/ml leupeptin, 2.5 mM PMSF, and 0.4 mM [g- 32P] ATP with MLCs (0.06 mg/ml), incubated for 15 min at room temperature and subjected to SDS-PAGE. The radiolabeled bands of
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MRLC were visualized by the Bio Imaging Analyzer BAS 2000 (Fuji). 2.5. Construction of sea urchin embryo cDNA library Total RNA was extracted from embryos of sea urchin, Hemicentrotus pulcherrimus, at the hatched blastula stage by the hot phenol method (Feramisco et al., 1982). Messenger RNA was extracted from exponentially growing cells using oligo(dT)-latex (Roche). The cDNA library was synthesized in the same manner as described elsewhere (Murata-Hori et al., 1999). 2.6. cDNA cloning and sequencing The cDNA fragment encoding a subunit of CK2 was amplified from HeLa cell cDNA library (Murata-Hori et al., 1999) with the first set of oligonucleotide primers (5 0 ACGAGTCACATGTGGTGGAA-3 0 and 5 0 -GCCAGCATACAACCCAAACT-3 0 ) using PCR. The sea urchin cDNA library (1:25 £ 105 pfu) was screened with the PCR product as a probe using ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech). Positive phages were isolated and the inserted cDNAs were subcloned into pBluescript SK(2) according to the manufacturer’s instruction. DNA sequencing was carried out as described previously (Murata-Hori et al., 1999). 2.7. Preparation of recombinant proteins The glutathione S-transferase (GST) fused recombinant proteins were prepared, expressed and purified in the same manner as described previously (Murata-Hori et al., 1999). The purified GST fused p36 proteins were digested with Factor Xa (New England Biolabs) and then Factor Xa was removed by treatment with p-aminobenzamidine beads (Amersham Pharmacia Biotech). The recombinant p36 protein obtained was used for the assay of myosin II kinase activity. 2.8. Phosphorylation of MRLC or intact myosin II by the purified myosin II kinase or recombinant p36 Phosphorylation of MLCs (0.06 mg/ml) or intact myosin II (0.32 mg/ml) by the purified myosin II kinase or recombinant p36 was carried out in 60 mM Tris–HCl (pH 8.0), 260 mM NaCl, 4 mM MgCl2, 2 mM EGTA, 1 mM CaCl2, 0.1 mM calyculin A, 50 mg/ml leupeptin, 1.25 mM PMSF and 0.18 mM [g- 32P]ATP. 2.9. Phosphorylation of MRLC by other kinases Phosphorylation of MRLC with MLCK was carried out in a buffer containing 22 mM Tris–HCl (pH 7.2), 110 mM NaCl, 2 mM MgCl2, 1.1 mM EGTA, 2 mM CaCl2, 25 mg/ ml CaM, 0.1 mM calyculin A, 50 mg/ml leupeptin, 1.25 mM PMSF and 0.18 mM [g- 32P] ATP. Phosphorylation of MRLC with PKC was carried out in 50 mM Tris–HCl (pH
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8.0), 10 mM NaCl, 2 mM MgCl2, 0.1 mM EGTA, 1 mM CaCl2, 50 ng/ml l-a-phosphatidyl-l-serine, 10 ng/ml 12-Otetradecanoyl-phorbol 13-acetate (TPA), 0.1 mM calyculin A, 50 mg/ml leupeptin, 1.25 mM PMSF and 0.18 mM [g- 32P] ATP. 2.10. Other procedures Amino acid sequencing analysis was described previously (Okubo et al., 1999). Phosphopeptide mapping and phosphoamino acid analyses were described elsewhere (Totsukawa et al., 1996; Murata-Hori et al., 2000). SDSPAGE gels were stained with a silver stain kit (Wako Pure Chemical Industries).
3. Results 3.1. Purification of myosin II kinase from sea urchin egg extract Phosphorylation activity for MRLC (myosin II kinase activity) contained in the egg extract was just slightly higher in the absence of Ca 21/CaM than in the presence of Ca 21/ CaM (Fig. 1A, inset). We therefore tried to purify this Ca 21/ CaM-independent myosin II kinase from the egg extract. Fig. 1 showed the sequential purification steps of myosin II kinase from sea urchin eggs using Sephacryl S-300 gel filtration (Fig. 1A), hydroxylapatite (Fig. 1B) and the double Resource Q column chromatographies (Fig. 1C,D). At each purification step, only one strong peak of MRLC kinase activity was detected. The peak of myosin II kinase activity in each step was analyzed by SDS-PAGE (Fig. 2A). Finally, we found that the myosin II kinase was composed of two components, the molecular mass of which were 36 kDa (p36) and 28 kDa (p28), respectively (Fig. 2B). 3.2. Identification of the purified myosin II kinase in egg extracts The N-terminal amino acid sequences of several fragments from V8 protease digests of each component were determined as shown in Fig. 3. The p36 and p28 were highly homologous to a and b subunits of CK2 from various organisms, respectively (Fig. 3A,B). This strongly suggests that the sea urchin egg Ca 21/CaM-independent myosin II kinase was CK2. To assess this possibility, we screened a cDNA library from sea urchin eggs using the human CK2 cDNA as a probe and obtained a cDNA encoded p36 of myosin II kinase. The determined nucleotide and the deduced amino acid sequences of this cDNA clone were shown in Fig. 4A, respectively. The decided nucleotide sequence was 2667 bp long and had a single open reading frame, which started at the position of 144 and ended at that of 1323. The determined amino acid sequences in Fig. 3 were contained in the
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Fig. 1. Sequential purification steps of myosin II kinase from sea urchin egg extracts. (A) A Sephacryl S-300 gel filtration chromatography of the egg extracts of sea urchin. Fractions from 22 to 46 were collected for the next purification step. Inset; Original myosin II kinase activity in the egg extract was just slightly higher in the absence of Ca 21/CaM than in the presence of Ca 21/CaM. (B) Hydroxylapatite column chromatography of the Sephacryl S-300 fraction. The Sephacryl S-300 fractions containing myosin II kinase activity were applied to a hydroxylapatite column. Proteins were eluted with a 20–500 mM linear potassium phosphate gradient. Each fraction was assayed for myosin II kinase. Fractions from 92 to 99 were pooled for the next purification step. (C) First Resource Q anion exchange column chromatography of the hydroxylapatite fractions. The dialyzed hydroxylapatite fractions were applied to a Resource Q anion exchange column. Proteins were eluted with a 20–500 mM linear NaCl gradient using FPLC system. Two fractions (71 and 72) containing myosin II kinase activity were pooled for the next purification step. (D) Second Resource Q anion exchange column re-chromatography of first Resource Q fraction. The first Resource Q fraction diluted with buffer A was applied to a Resource Q anion exchange column. Proteins were eluted with a 200–500 mM linear NaCl gradient using a FPLC system. myosin II kinase activity was mainly observed in fractions 51–53. W, absorbance of 280 nm; X, myosin II kinase activity.
deduced amino acids from this cDNA sequence (Fig. 4A, underlines). We further compared the deduced amino acid sequence of the a subunit of sea urchin CK2 to those of various eukaryotes (Fig. 4B). p36 has over 70% homology to CK2 from human (NP_001886), chicken (P21868), zebrafish (CAA68229), Xenopus (P28020) and Drosophila (P08181). Furthermore, an antibody raised against the peptide for the consensus amino acids of CK2 recognized both the purified myosin II kinase and the recombinant p36 (data not shown). These allowed us to determine that p36 was CK2 of sea urchin eggs. 3.3. Characterization of myosin II kinase from sea urchin eggs In order to know that the p36 protein has myosin II kinase activity, the obtained cDNA was expressed as a GST fusion
protein (GST-p36). GST-p36 was digested by Factor Xa and the p36 obtained was used as a recombinant p36 in an assay for myosin II kinase activity. As shown in Fig. 5, recombinant p36 phosphorylated isolated MRLC, but not MRLC of intact myosin II, with the same activity as the purified myosin II kinase from sea urchin eggs. Interestingly, both recombinant p36 and the purified myosin II kinase have the ability to phosphorylate myosin II heavy chains (see Fig. 5, lanes 4 and 8). A previous report showed that CK2 from bovine brain mainly phosphorylates the heavy chain of myosin II in intact myosin II (Murakami et al., 1984). This coincides well with our results. We further determined the phosphorylation site of MRLC by recombinant p36 and the purified myosin II kinase using phosphopeptide mapping analysis. As shown in Fig. 6, the mapping pattern of phosphorylated MRLC by the purified myosin II kinase (lane 3) was identical to that by both recombinant p36 (lane 4) and human glioblastoma CK2
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4. Discussion 4.1. Presence of the myosin II kinase activity in insoluble fraction of eggs
Fig. 2. Purification processes of the myosin II kinase from sea urchin egg extraction on a SDS-PAGE. (A) The original egg extract (lane 1), Sephacryl S-300 fraction (lane 2), hydroxylapatite fraction (lane 3), first Resource Q fraction (lane 4), second Resource Q fraction (lane 5). Each fraction was electrophoresed on a SDS-PAGE gel and stained with CBB. (B) Silver staining of the second Resource Q fraction loaded on a SDS-PAGE gel. Two bands corresponding to the molecular mass of 36 and 28 kDa were detected.
(lane 5) but not that by chicken gizzard MLCK (lane 1) or rabbit brain PKC (lane 2). Taken together, the purified myosin II kinase was identified to be CK2 of sea urchin egg and it phosphorylated the different sites of MRLC from those phosphorylated by MLCK or PKC.
It is known that CK2 tends to aggregate at low salt concentration (Glover, 1986). Thus, CK2 activity might not be contained in the soluble cytoplasmic fraction of sea urchin eggs prepared at a low salt concentration. Our previous study showed that the soluble cytoplasmic fraction of sea urchin eggs did not exhibits CK2 activity (Totsukawa et al., 1996). In this study, the activity was actually purified from the solubilized fraction of insoluble materials of sea urchin eggs as a myosin II kinase activity. It has been also reported that CK2 activity is detected in the myosin preparations obtained from chicken brain, suggesting that the kinase is likely to be enriched in the cytoskeletal structures such as myosin filaments (Matsumura et al., 1983). Although CK2 might associate with myosin filaments in insoluble fractions of sea urchin eggs, the precise relationship between CK2 and cytoskeletal structures including myosin filaments still remains obscure. 4.2. Physiological roles of the myosin II kinase in sea urchin egg Recently, it has been reported that CK2 must be implicated in hatching and blastula/gastrula transition during early development of the sea urchin (Delalande et al.,
Fig. 3. Comparison of partial amino acid sequences of p36 or p28 among CK2 from various organisms. The N-terminus sequences of two peptides from p36 (A) and one of p28 (B) were compared with a and b subunit of CK2 from human (NP_001886), mouse (U17112), chicken (P21868), Xenopus (P28020), Drosophila (P08181) and C. elegans (J05274), respectively. The numbers in the parentheses were accession numbers in PIR and GenBank databases. X represents undetermined amino acids and identities of amino acids are shown by boxes. The starting and ending amino acid position in each subunit of CK2 are indicated at the left and the right, respectively.
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Fig. 4. Determined nucleotides and the deduced amino acid sequence of sea urchin CK2. (A) Sequence of sea urchin CK2 a subunit. The nucleotide sequence is shown, together with the deduced amino acid sequence of the encoded protein. Underlined amino acids indicate matches with the determined protein sequence as shown in Fig. 3A. (B) Amino acid sequence alignment among p36 and a subunit of CK2 from various eukaryotes. Deduced amino acid sequence of p36 was compared with those from human (NP_001886), chicken (P21868), Xenopus (P28020), zebrafish (CAA68229) and Drosophila (P08181), which were obtained from PIR and GenBank databases (the numbers in parentheses indicated the accession numbers) using a software (GENETYX). Dots indicate identical amino acid. The nucleotide sequence data of cDNA encoded p36 will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession number AB024599.
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Fig. 5. Phosphorylation of the isolated MRLC and MRLC of intact myosin II by the purified myosin II kinase and the recombinant p36. The MLCs (0.06 mg/ml; lanes 1, 2, 5 and 6) or intact myosin II (0.32 mg/ml; lanes 3, 4, 7 and 8) were incubated with (lanes 2, 4, 6 and 8) or without (lanes 1, 3, 5 and 7) purified myosin II kinase (lanes 1–4) or the recombinant p36 (lanes 5–8) for 30 min under the conditions described in Section 1. Each sample was analyzed by SDS-PAGE followed by autoradiography. HC indicates myosin II heavy chains.
1999). Especially, as it was demonstrated that specific prevention of hatching is not sufficient to affect development at the level of the blastula to gastrula transition, the targets of CK2 are not restricted to the production and/or activation of the hatching enzyme. In the stage of blastula/ gastrula transition, cells dynamically change their shape and relocate in embryos, suggesting occurrence of dynamic reassembly of cytoskeletal structures inside those cells. As increasing numbers of cytoskeletal proteins, including spectrin, troponin T and smooth muscle and non-muscle myosins, have been reported to be phosphorylated by CK2 (Tuazon and Traugh, 1991), phosphorylation of these proteins by CK2 are supposed to be involved in regulating blastula/gastrula transition. Several reports suggest that CK2 is required for progression through the cell division cycle. Marshak and Russo (1994) reported that, in HeLa cells, high CK2 activity is necessary for a normal G1 phase but that progression through the S phase requires inhibition of CK2. A likely target of CK2 during G1 is p34 cdc2, which has been shown to be a substrate for CK2 at Ser 39 in HeLa cells (Litchfield et al., 1991; Russo et al., 1992). CK2 phosphorylation at Ser 39 inhibits kinase activities of p34 cdc2 through prevention of cyclin binding to p34 cdc2 during G1 phase and may contribute to maintaining the cell in G1. p34 cdc2 has also been known to phosphorylate MRLC at the sites phorphorylatable by PKC and to inhibit actin-activated ATPase activities of myosin II (Satterwhite et al., 1992). Thus, CK2 may play important roles in the maintenance of myosin ATPase activities in G1.
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Fig. 6. Identification of the phosphorylation site of MRLC by the purified myosin II kinase or the recombinant p36. One-dimensional tryptic peptide mapping of MRLC phosphorylated by MLCK (lane 1), PKC (lane 2), the purified myosin II kinase (lane 3), the recombinant p36 (lane 4), and human glioblastoma CK2 (lane 5), respectively. MRLC phosphorylated by each kinase was digested with trypsin and then processed on a cellulose plate for electrophoresis. ‘ori’ indicates the origin.
5. Conclusion 1. We purified a Ca 21/CaM-independent myosin II kinase from sea urchin egg extract. 2. The purified myosin II kinase was identified as protein kinase CK2 from sea urchin eggs. 3. Expressed protein (P36) of sea urchin CK2 in E. coli phosphorylated the same site of MRLC as did the purified myosin II kinase.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H.H. and M.M.H., and the Hayashi Memorial Foundation for Female Natural Scientists to M.M.H. and the Sasakawa Scientific Research Grant from The Japan Science Society to S.K. References Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., Kaibuchi, K., 1996. Phosphorylation and activation of
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Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells. J. Cell Biol. 140, 119–129. Murakami, N., Matsumura, S., Kumon, A., 1984. Purification and identification of myosin heavy chain kinase from bovine brain. J. Biochem. 95, 651–660. Murata-Hori, M., Murai, N., Komatsu, S., Uji, Y., Hosoya, H., 1998. Concentration of singly phosphorylated myosin II regulatory light chain along the cleavage furrow of dividing HeLa cells. Biomed. Res. 19, 111–115. Murata-Hori, M., Suizu, F., Iwasaki, T., Kikuchi, A., Hosoya, H., 1999. ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells. FEBS Lett. 451, 81–84. Murata-Hori, M., Fumoto, K., Fukuta, Y., Iwasaki, T., Kukuchi, A., Tatsuka, M., Hosoya, H., 2000. Myosin II regulatory light chain as a novel substrate for AIM-1, an Aurora/Ipl1 related kinase from rat. J. Biochem. 128, 903–907. Nishikawa, M., Sellers, J.R., Adelstein, R.S., Hidaka, H., 1984. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. J. Biol. Chem. 259, 8808–8814. Okubo, M.-A., Chiba, S., Nishikata, T., Matsuno, A., Hosoya, H., 1999. Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells. Dev. Growth Differ. 41, 381–389. Russo, G.L., Vandenberg, M.T., Yu, I.J., Bae, Y.-S., Franza, B.R., Marshak, D.R., 1992. Casein kinase II phosphorylates p34 cdc2 kinase in G1 phase of the HeLa cell division cycle. J. Biol. Chem. 267, 20317– 20325. Satterwhite, L.L., Lohka, M.J., Wilson, K.L., Scherson, T.Y., Cisek, L.J., Cordon, J.L., Pollard, T.D., 1992. Phosphorylation of myosin-II regulatory light chain by cyclin-p34 cdc2; a mechanism for the timing of cytokinesis. J. Cell Biol. 118, 595–605. Sellers, J.R., Adelstein, R.S., 1987. In: Boyer, P.D., Krebs, E.G. (Eds.), The Enzymes, Vol. 18. Academic Press, Orlando, FL, pp. 381–418. Suizu, F., Ueda, K., Iwasaki, T., Murata-Hori, M., Hosoya, H., 2000. Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b. J. Biochem. 128, 435– 440. Totsukawa, G., Himi-Nakamura, E., Komatsu, S., Iwata, K., Tezuka, A., Sakai, H., Yazaki, K., Hosoya, H., 1996. Mitosis-specific phosphorylation of smooth muscle regulatory light chain of myosin II at Ser-1 and / or -2 and Thr-9 in sea urchin egg extract. Cell Struct. Funct. 21, 475– 482. Tuazon, P.T., Traugh, J.A., 1991. Casein kinase I and II-multipotential serine protein kinases: Structure, Function, and Regulation. Adv. Second Mess. Phosphoprotein Res. 23, 123–164.
Identification of myosin II kinase from sea urchin eggs as protein kinase CK2 Shigeru Komaba, Hajime Hamao, Maki Murata-Hori 1, Hiroshi Hosoya* Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan Received 31 January 2000; received in revised form 6 June 2001; accepted 18 July 2001 Received by T. Sekiya
Abstract Here we purified and identified a myosin II kinase from sea urchin eggs. The activity of this myosin II kinase in the egg extract was not significantly affected by Ca 21/calmodulin (CaM). Using sequential column chromatographies, we purified the myosin II kinase from the egg extract as a complex composed of 36- (p36) and 28-kDa (p28) proteins. Partial amino acid sequences of these two components were highly coincident with those of the a and b subunits of protein kinase CK2 (formerly casein kinase II) in sea urchin eggs, respectively. To confirm that the purified myosin II kinase was CK2, we obtained a cDNA which encodes p36 from a cDNA library of sea urchin eggs. The amino acid sequence derived from the obtained cDNA showed over 70% homology to CK2 from various eukaryotes. Furthermore, recombinant p36, as well as the purified myosin II kinase, phosphorylated MRLC. One dimensional phosphopeptide mapping revealed that the phosphorylation site(s) of MRLC by both recombinant p36 and the purified myosin II kinase was identical. These clearly showed that the Ca 21/CaMindependent myosin II kinase activity in sea urchin eggs was identical to CK2. q 2001 Elsevier Science B.V. All rights reserved. Keywords: CK2; Myosin II regulatory light chain; Phosphorylation; Sea urchin egg; Myosin light chain kinase
1. Introduction Phosphorylation of the myosin II regulatory light chain (MRLC) is believed to be important for modulating the interaction between filamentous myosin II and actin filaments that generates force for the contraction of smooth muscle and non-muscle cells (Sellers and Adelstein, 1987). It is well known that the actin-activated MgATPase activity of myosin II increases upon phosphorylation of MRLC by Ca 21/calmodulin (CaM)-dependent myosin light chain kinase (MLCK), at least in vitro (Kamm and Stull, 1985; Ikebe and Hartshorne, 1985). Phosphorylation sites of MRLC by MLCK are Ser19 and Thr18 (MLCK sites) and the phosphorylation at both sites results in higher MgATPase activity of myosin II than that of single site phposphorylated myosin II (Ikebe, 1989). Recently, it was reported that several Ca 21/CaM-independent kinases, which Abbreviations: MAPKAPK-2, MAP kinase activated protein kinase-2; MLCK, myosin light chain kinase; MRLC, myosin regulatory light chain; PKC, protein kinase C; CK2, casein kinase 2; PMSF, phenyl methyl sulfonyl fluoride * Corresponding author. Tel.: 181-824-24-7443; fax: 181-824-24-0734. E-mail address: [email protected] (H. Hosoya). 1 Present address: Department of Physiology, University of Massachusetts Medical School, 377 Plantation ST., Worcester, MA 01605, USA.
might have important roles in cell division, such as RSK-2 (Suizu et al., 2000), MAPKAPK-2 (Komatsu et al., 1997) and AIM-1 (one of Aurora/Ipl1 kinase family) (Murata-Hori et al., 2000), Rho-kinase (Amano et al., 1996), p21-activated protein kinase (Chew et al., 1998) phosphorylate MRLC at Ser19. Moreover, phosphorylated MRLC at Ser19 was localized along the contractile ring in dividing non-muscle cells (Matsumura et al., 1998; Murata-Hori et al., 1998). These results suggest that the phosphorylation of MRLC at Ser 19 may be involved in the regulation of cytokinesis. We also found that ZIP kinase from non-muscle cells could more efficiently phosphorylate MRLC at both Ser19 and Thr18 than MLCK (Murata-Hori et al., 1999), suggesting that phosphorylation of MRLC at both Ser19 and Thr18 might occur in non-muscle cells. On the other hand, phosphorylation sites of MRLC at Ser1/Ser2 and Thr9 by PKC (PKC sites) down-regulated MgATPase activity of myosin II phosphorylated by MLCK (Nishikawa et al., 1984; Ikebe et al., 1987). Satterwhite et al showed that p34 cdc2 kinase also phosphorylated the same sites of MRLC as did PKC (Satterwhite et al., 1992). The cumulative evidence suggests that changes in the phosphorylation state of MRLC may play an important role in the cytokinesis signaling pathway.
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00626-6
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Recently, the activity of kinases capable of phosphorylating MRLC was analyzed throughout the first cell cycle of sea urchin eggs (Totsukawa et al., 1996). Total kinase activity in vitro did not fluctuate during the cell cycle but the activity of the kinase(s) responsible for phosphorylation of MRLC at PKC sites showed a significant increase at metaphase. Using a selective inhibitor of p34 cdc2 kinase, this myosin II kinase activity for PKC sites was recognized as a p34cdc2 kinase. In contrast, MRLC phosphorylation at MLCK sites showed no significant changes during the first cell cycle. As this myosin II kinase activity for MLCK sites was observed both in the absence and presence of Ca 21/ CaM, it is supposed to be different from the Ca 21/CaM dependent MLCK. Thus, it is important to elucidate which kinase(s) participate in temporal and spatial regulation of phosphorylation of MRLC during cell division. Here, we tried to purify and identify the kinase that phosphorylates MRLC from sea urchin egg extract. As the result of this approach, a myosin II kinase was purified from sea urchin eggs and identified as a multifunctional serine/threonine kinase, protein kinase CK2. CK2 might have important roles in cell cycle progression throughout phosphorylation of MRLC during the early development of sea urchin eggs.
2. Material and methods 2.1. Material Light chains from myosin II (MLCs) and myosin light chain kinase (MLCK) were prepared from chicken gizzard as described elsewhere (Murata-Hori et al., 1999). Protein kinase C (PKC) purified from rabbit brain was kindly provided by Drs K. Mizuno and S. Ohno (Yokohama City University). CaM from bovine brain, TPCK treated trypsin (Type XIII), calyculin A and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO). Leupeptin and pepstatin A were from the Peptide Institute (Osaka, Japan). Endoproteinase Glu-C from Staphylococcus aureus V8 was purchased from Boehringer Mannheim (Mannheim, Germany). An antibody raised against the peptide for the consensus amino acid sequence of the CK2 peptide was kindly provided by Drs Moriyama and I. Yahara (Tokyo Metropolitan Institute of Medical Science, Tokyo). 2.2. Preparation of sea urchin egg extracts Unfertilized eggs from sea urchin, Hemicentrotus pulcherrimus, were collected by centrifugation at 1700 £ g for 2 min and homogenized with an equal volume of buffer H (100 mM Pipes (pH 6.8), 10% (v/v) glycerol, 5 mM MgCl2, 10 mM EGTA, 5 mM PMSF, and 10 mg/ml leupeptin). The egg homogenates were centrifuged at 20 000 £ g for 30 min and the supernatant was removed. Then, the pellet was homogenized with an equal volume of buffer H
and stored at 2808C until use. All procedures were carried out at 48C. 2.3. Purification of myosin II kinase from sea urchin egg extracts Sea urchin egg extracts were obtained under high salt condition as described by Ishidate and Mabuchi (1989) with a slight modification. Briefly, the homogenate stocked at 2808C was resuspended in an equal volume of buffer H containing 1 M NaCl, swirled for 2 h and then centrifuged at 20 000 £ g for 30 min. The supernatant was applied to a gel filtration column (5 £ 89 cm) of Sephacryl S-300 (Amersham Pharmacia Biotech) equilibrated with buffer A (20 mM Tris–HCl, pH 8.0, 2 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml pepstatin A, and 0.1 mM DTT) containing 200 mM NaCl. Each fraction was assayed for myosin II kinase activity as described below. Fractions containing myosin II kinase activity were collected and applied to a column (2:5 £ 17 cm) of hydroxylapatite (Bio-Rad HTP) equilibrated with buffer B (20 mM potassium phosphate (pH 8.0), 2 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml pepstatin A, and 0.1 mM DTT). The column was washed with buffer B and retained proteins were eluted with 300 ml of buffer B in a linear gradient of 20–500 mM potassium phosphate. All of the fractions obtained were assayed for myosin II kinase activity, respectively. Fractions of myosin II kinase activity were collected, dialyzed against buffer A containing 20 mM NaCl and applied to a Resource Q anion exchange column (6 ml; Amersham Pharmacia Biotech) equilibrated buffer A containing 20 mM NaCl. The column was washed with buffer A containing 20 mM NaCl and retained proteins were eluted with 120 ml of buffer A in a linear gradient of 20–500 mM NaCl using FPLC system (Amersham Pharmacia Biotech). Each fraction was assayed for myosin II kinase activity. Fractions containing myosin II kinase activity eluted at about 400 mM NaCl were collected, diluted with an equal volume of buffer A and applied to a Resource Q anion exchange column (6 ml) equilibrated with a buffer A containing 200 mM NaCl again. The column was washed with buffer A containing 200 mM NaCl and retained proteins were eluted with 120 ml of buffer A in a linear gradient of 200–500 mM NaCl using FPLC system. Each fraction was assayed for myosin II kinase activity. Fractions containing myosin II kinase activity were pooled and stored at 2808C until use. All procedures were carried out at 48C. 2.4. Myosin II kinase assay An aliquot from each fraction (10 ml) was added to a reaction mixture containing 120 mM Tris–HCl (pH 8.0), 4 mM EGTA, 8 mM MgCl2, 0.2 mM calyculin A, 100 mg/ml leupeptin, 2.5 mM PMSF, and 0.4 mM [g- 32P] ATP with MLCs (0.06 mg/ml), incubated for 15 min at room temperature and subjected to SDS-PAGE. The radiolabeled bands of
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MRLC were visualized by the Bio Imaging Analyzer BAS 2000 (Fuji). 2.5. Construction of sea urchin embryo cDNA library Total RNA was extracted from embryos of sea urchin, Hemicentrotus pulcherrimus, at the hatched blastula stage by the hot phenol method (Feramisco et al., 1982). Messenger RNA was extracted from exponentially growing cells using oligo(dT)-latex (Roche). The cDNA library was synthesized in the same manner as described elsewhere (Murata-Hori et al., 1999). 2.6. cDNA cloning and sequencing The cDNA fragment encoding a subunit of CK2 was amplified from HeLa cell cDNA library (Murata-Hori et al., 1999) with the first set of oligonucleotide primers (5 0 ACGAGTCACATGTGGTGGAA-3 0 and 5 0 -GCCAGCATACAACCCAAACT-3 0 ) using PCR. The sea urchin cDNA library (1:25 £ 105 pfu) was screened with the PCR product as a probe using ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech). Positive phages were isolated and the inserted cDNAs were subcloned into pBluescript SK(2) according to the manufacturer’s instruction. DNA sequencing was carried out as described previously (Murata-Hori et al., 1999). 2.7. Preparation of recombinant proteins The glutathione S-transferase (GST) fused recombinant proteins were prepared, expressed and purified in the same manner as described previously (Murata-Hori et al., 1999). The purified GST fused p36 proteins were digested with Factor Xa (New England Biolabs) and then Factor Xa was removed by treatment with p-aminobenzamidine beads (Amersham Pharmacia Biotech). The recombinant p36 protein obtained was used for the assay of myosin II kinase activity. 2.8. Phosphorylation of MRLC or intact myosin II by the purified myosin II kinase or recombinant p36 Phosphorylation of MLCs (0.06 mg/ml) or intact myosin II (0.32 mg/ml) by the purified myosin II kinase or recombinant p36 was carried out in 60 mM Tris–HCl (pH 8.0), 260 mM NaCl, 4 mM MgCl2, 2 mM EGTA, 1 mM CaCl2, 0.1 mM calyculin A, 50 mg/ml leupeptin, 1.25 mM PMSF and 0.18 mM [g- 32P]ATP. 2.9. Phosphorylation of MRLC by other kinases Phosphorylation of MRLC with MLCK was carried out in a buffer containing 22 mM Tris–HCl (pH 7.2), 110 mM NaCl, 2 mM MgCl2, 1.1 mM EGTA, 2 mM CaCl2, 25 mg/ ml CaM, 0.1 mM calyculin A, 50 mg/ml leupeptin, 1.25 mM PMSF and 0.18 mM [g- 32P] ATP. Phosphorylation of MRLC with PKC was carried out in 50 mM Tris–HCl (pH
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8.0), 10 mM NaCl, 2 mM MgCl2, 0.1 mM EGTA, 1 mM CaCl2, 50 ng/ml l-a-phosphatidyl-l-serine, 10 ng/ml 12-Otetradecanoyl-phorbol 13-acetate (TPA), 0.1 mM calyculin A, 50 mg/ml leupeptin, 1.25 mM PMSF and 0.18 mM [g- 32P] ATP. 2.10. Other procedures Amino acid sequencing analysis was described previously (Okubo et al., 1999). Phosphopeptide mapping and phosphoamino acid analyses were described elsewhere (Totsukawa et al., 1996; Murata-Hori et al., 2000). SDSPAGE gels were stained with a silver stain kit (Wako Pure Chemical Industries).
3. Results 3.1. Purification of myosin II kinase from sea urchin egg extract Phosphorylation activity for MRLC (myosin II kinase activity) contained in the egg extract was just slightly higher in the absence of Ca 21/CaM than in the presence of Ca 21/ CaM (Fig. 1A, inset). We therefore tried to purify this Ca 21/ CaM-independent myosin II kinase from the egg extract. Fig. 1 showed the sequential purification steps of myosin II kinase from sea urchin eggs using Sephacryl S-300 gel filtration (Fig. 1A), hydroxylapatite (Fig. 1B) and the double Resource Q column chromatographies (Fig. 1C,D). At each purification step, only one strong peak of MRLC kinase activity was detected. The peak of myosin II kinase activity in each step was analyzed by SDS-PAGE (Fig. 2A). Finally, we found that the myosin II kinase was composed of two components, the molecular mass of which were 36 kDa (p36) and 28 kDa (p28), respectively (Fig. 2B). 3.2. Identification of the purified myosin II kinase in egg extracts The N-terminal amino acid sequences of several fragments from V8 protease digests of each component were determined as shown in Fig. 3. The p36 and p28 were highly homologous to a and b subunits of CK2 from various organisms, respectively (Fig. 3A,B). This strongly suggests that the sea urchin egg Ca 21/CaM-independent myosin II kinase was CK2. To assess this possibility, we screened a cDNA library from sea urchin eggs using the human CK2 cDNA as a probe and obtained a cDNA encoded p36 of myosin II kinase. The determined nucleotide and the deduced amino acid sequences of this cDNA clone were shown in Fig. 4A, respectively. The decided nucleotide sequence was 2667 bp long and had a single open reading frame, which started at the position of 144 and ended at that of 1323. The determined amino acid sequences in Fig. 3 were contained in the
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Fig. 1. Sequential purification steps of myosin II kinase from sea urchin egg extracts. (A) A Sephacryl S-300 gel filtration chromatography of the egg extracts of sea urchin. Fractions from 22 to 46 were collected for the next purification step. Inset; Original myosin II kinase activity in the egg extract was just slightly higher in the absence of Ca 21/CaM than in the presence of Ca 21/CaM. (B) Hydroxylapatite column chromatography of the Sephacryl S-300 fraction. The Sephacryl S-300 fractions containing myosin II kinase activity were applied to a hydroxylapatite column. Proteins were eluted with a 20–500 mM linear potassium phosphate gradient. Each fraction was assayed for myosin II kinase. Fractions from 92 to 99 were pooled for the next purification step. (C) First Resource Q anion exchange column chromatography of the hydroxylapatite fractions. The dialyzed hydroxylapatite fractions were applied to a Resource Q anion exchange column. Proteins were eluted with a 20–500 mM linear NaCl gradient using FPLC system. Two fractions (71 and 72) containing myosin II kinase activity were pooled for the next purification step. (D) Second Resource Q anion exchange column re-chromatography of first Resource Q fraction. The first Resource Q fraction diluted with buffer A was applied to a Resource Q anion exchange column. Proteins were eluted with a 200–500 mM linear NaCl gradient using a FPLC system. myosin II kinase activity was mainly observed in fractions 51–53. W, absorbance of 280 nm; X, myosin II kinase activity.
deduced amino acids from this cDNA sequence (Fig. 4A, underlines). We further compared the deduced amino acid sequence of the a subunit of sea urchin CK2 to those of various eukaryotes (Fig. 4B). p36 has over 70% homology to CK2 from human (NP_001886), chicken (P21868), zebrafish (CAA68229), Xenopus (P28020) and Drosophila (P08181). Furthermore, an antibody raised against the peptide for the consensus amino acids of CK2 recognized both the purified myosin II kinase and the recombinant p36 (data not shown). These allowed us to determine that p36 was CK2 of sea urchin eggs. 3.3. Characterization of myosin II kinase from sea urchin eggs In order to know that the p36 protein has myosin II kinase activity, the obtained cDNA was expressed as a GST fusion
protein (GST-p36). GST-p36 was digested by Factor Xa and the p36 obtained was used as a recombinant p36 in an assay for myosin II kinase activity. As shown in Fig. 5, recombinant p36 phosphorylated isolated MRLC, but not MRLC of intact myosin II, with the same activity as the purified myosin II kinase from sea urchin eggs. Interestingly, both recombinant p36 and the purified myosin II kinase have the ability to phosphorylate myosin II heavy chains (see Fig. 5, lanes 4 and 8). A previous report showed that CK2 from bovine brain mainly phosphorylates the heavy chain of myosin II in intact myosin II (Murakami et al., 1984). This coincides well with our results. We further determined the phosphorylation site of MRLC by recombinant p36 and the purified myosin II kinase using phosphopeptide mapping analysis. As shown in Fig. 6, the mapping pattern of phosphorylated MRLC by the purified myosin II kinase (lane 3) was identical to that by both recombinant p36 (lane 4) and human glioblastoma CK2
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4. Discussion 4.1. Presence of the myosin II kinase activity in insoluble fraction of eggs
Fig. 2. Purification processes of the myosin II kinase from sea urchin egg extraction on a SDS-PAGE. (A) The original egg extract (lane 1), Sephacryl S-300 fraction (lane 2), hydroxylapatite fraction (lane 3), first Resource Q fraction (lane 4), second Resource Q fraction (lane 5). Each fraction was electrophoresed on a SDS-PAGE gel and stained with CBB. (B) Silver staining of the second Resource Q fraction loaded on a SDS-PAGE gel. Two bands corresponding to the molecular mass of 36 and 28 kDa were detected.
(lane 5) but not that by chicken gizzard MLCK (lane 1) or rabbit brain PKC (lane 2). Taken together, the purified myosin II kinase was identified to be CK2 of sea urchin egg and it phosphorylated the different sites of MRLC from those phosphorylated by MLCK or PKC.
It is known that CK2 tends to aggregate at low salt concentration (Glover, 1986). Thus, CK2 activity might not be contained in the soluble cytoplasmic fraction of sea urchin eggs prepared at a low salt concentration. Our previous study showed that the soluble cytoplasmic fraction of sea urchin eggs did not exhibits CK2 activity (Totsukawa et al., 1996). In this study, the activity was actually purified from the solubilized fraction of insoluble materials of sea urchin eggs as a myosin II kinase activity. It has been also reported that CK2 activity is detected in the myosin preparations obtained from chicken brain, suggesting that the kinase is likely to be enriched in the cytoskeletal structures such as myosin filaments (Matsumura et al., 1983). Although CK2 might associate with myosin filaments in insoluble fractions of sea urchin eggs, the precise relationship between CK2 and cytoskeletal structures including myosin filaments still remains obscure. 4.2. Physiological roles of the myosin II kinase in sea urchin egg Recently, it has been reported that CK2 must be implicated in hatching and blastula/gastrula transition during early development of the sea urchin (Delalande et al.,
Fig. 3. Comparison of partial amino acid sequences of p36 or p28 among CK2 from various organisms. The N-terminus sequences of two peptides from p36 (A) and one of p28 (B) were compared with a and b subunit of CK2 from human (NP_001886), mouse (U17112), chicken (P21868), Xenopus (P28020), Drosophila (P08181) and C. elegans (J05274), respectively. The numbers in the parentheses were accession numbers in PIR and GenBank databases. X represents undetermined amino acids and identities of amino acids are shown by boxes. The starting and ending amino acid position in each subunit of CK2 are indicated at the left and the right, respectively.
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Fig. 4. Determined nucleotides and the deduced amino acid sequence of sea urchin CK2. (A) Sequence of sea urchin CK2 a subunit. The nucleotide sequence is shown, together with the deduced amino acid sequence of the encoded protein. Underlined amino acids indicate matches with the determined protein sequence as shown in Fig. 3A. (B) Amino acid sequence alignment among p36 and a subunit of CK2 from various eukaryotes. Deduced amino acid sequence of p36 was compared with those from human (NP_001886), chicken (P21868), Xenopus (P28020), zebrafish (CAA68229) and Drosophila (P08181), which were obtained from PIR and GenBank databases (the numbers in parentheses indicated the accession numbers) using a software (GENETYX). Dots indicate identical amino acid. The nucleotide sequence data of cDNA encoded p36 will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession number AB024599.
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Fig. 5. Phosphorylation of the isolated MRLC and MRLC of intact myosin II by the purified myosin II kinase and the recombinant p36. The MLCs (0.06 mg/ml; lanes 1, 2, 5 and 6) or intact myosin II (0.32 mg/ml; lanes 3, 4, 7 and 8) were incubated with (lanes 2, 4, 6 and 8) or without (lanes 1, 3, 5 and 7) purified myosin II kinase (lanes 1–4) or the recombinant p36 (lanes 5–8) for 30 min under the conditions described in Section 1. Each sample was analyzed by SDS-PAGE followed by autoradiography. HC indicates myosin II heavy chains.
1999). Especially, as it was demonstrated that specific prevention of hatching is not sufficient to affect development at the level of the blastula to gastrula transition, the targets of CK2 are not restricted to the production and/or activation of the hatching enzyme. In the stage of blastula/ gastrula transition, cells dynamically change their shape and relocate in embryos, suggesting occurrence of dynamic reassembly of cytoskeletal structures inside those cells. As increasing numbers of cytoskeletal proteins, including spectrin, troponin T and smooth muscle and non-muscle myosins, have been reported to be phosphorylated by CK2 (Tuazon and Traugh, 1991), phosphorylation of these proteins by CK2 are supposed to be involved in regulating blastula/gastrula transition. Several reports suggest that CK2 is required for progression through the cell division cycle. Marshak and Russo (1994) reported that, in HeLa cells, high CK2 activity is necessary for a normal G1 phase but that progression through the S phase requires inhibition of CK2. A likely target of CK2 during G1 is p34 cdc2, which has been shown to be a substrate for CK2 at Ser 39 in HeLa cells (Litchfield et al., 1991; Russo et al., 1992). CK2 phosphorylation at Ser 39 inhibits kinase activities of p34 cdc2 through prevention of cyclin binding to p34 cdc2 during G1 phase and may contribute to maintaining the cell in G1. p34 cdc2 has also been known to phosphorylate MRLC at the sites phorphorylatable by PKC and to inhibit actin-activated ATPase activities of myosin II (Satterwhite et al., 1992). Thus, CK2 may play important roles in the maintenance of myosin ATPase activities in G1.
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Fig. 6. Identification of the phosphorylation site of MRLC by the purified myosin II kinase or the recombinant p36. One-dimensional tryptic peptide mapping of MRLC phosphorylated by MLCK (lane 1), PKC (lane 2), the purified myosin II kinase (lane 3), the recombinant p36 (lane 4), and human glioblastoma CK2 (lane 5), respectively. MRLC phosphorylated by each kinase was digested with trypsin and then processed on a cellulose plate for electrophoresis. ‘ori’ indicates the origin.
5. Conclusion 1. We purified a Ca 21/CaM-independent myosin II kinase from sea urchin egg extract. 2. The purified myosin II kinase was identified as protein kinase CK2 from sea urchin eggs. 3. Expressed protein (P36) of sea urchin CK2 in E. coli phosphorylated the same site of MRLC as did the purified myosin II kinase.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H.H. and M.M.H., and the Hayashi Memorial Foundation for Female Natural Scientists to M.M.H. and the Sasakawa Scientific Research Grant from The Japan Science Society to S.K. References Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., Kaibuchi, K., 1996. Phosphorylation and activation of
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