EXPERIMENTAL
CELL
RESEARCH
202, 549-551 (19%)
SHORT NO osphorylation of Xenopus Elongation tein Kinase: Identification of the Ph OD~LEM~LNER-LORILLON,*PATRICKCORMIER,*JEAN-CLA~DECAVA JULIAMORALES," ROBERT~OULHE,*AND ROBERT BELL~%**~ *Laboratoire de Physiologie de la Reproduction, UPMC, TCentre de Recherche de Biochimie Macromol~culaire,
CNRS URA 1449, INRA, UPR CNRS 41, INSERM
The cdc2 protein kinase phosphorylates elongation factor-l? (EF-I-/) during meiotic maturation of Xenopus ooeytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EFPy was au in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-ly. Tryptie hydrolysis of EF-17 and the P2 peptide, both phosphorylated by ede2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-ly corn&rated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-17 therefore contains one unique phosphoacceptor for cd& protein kinase, identified as threonine230. o 1982 Academic Press, hc.
INTRODUCTION The cd& protein kinase is universally implicated in the regulation of cell division [I]. Many substrates of the kinase have now been characterized [2]. One of the first, characterized in viva, was a protein ~47, stoichiometrically phosphorylated during meiotic cell division of Xenopus socytes [3-63. The p47 protein is in a dephosphorylated form in prophase oocytes and fully phosphorylated in metaphase oocytes [3]. It is present in a macromolecular complex, identified as components of elongation factor-l [4-6] 0 Elongation factors of protein biosynthesis are implicated in the elongation of polypeptide chains [7]. Elongation factor-la (EF-la) is responsible for the binding of aminoacyl-tRNA to the ribosome with concomitant hydrolysis of GTP. EF-Q3Sy complex catalyzes the IGTP exchange on EF-la!. Both fi and 6 proteins
1 To whom reprint
requests should be addressed.
4 place Jussieu> 75256 Paris Cedex 05, France; and 249, BP 5051, 34033 Montpellier Cede& France
possess the guanine nueleotide activity, at least in Artemia [8]. In Xenopus oocytes, the p47 complex, purified as a cdc2 protein kinase substrate, was identified as EFl@y, the p47 protein corresponding to EF-17 14-61. have cloned the three Xenopus oteins and have recently sequenced EF-1y cDNA ] We identify in this report the site of in EF-17 by cdc2 protein kinase at the level of threonine-230, and analyze the co~se~~e~ce ofthe phos~borylation on the GDPGTP exchange reaction performed with purified Xenopus EF-ICY.
ATERIA$S AND Protein purification. El?-1/3Sy complex was purified from Xenopus oocytes as described [3]. EF-la was also purified from Xenopus oocytes [IQ]. The cdc2 protein kinase (Pli ~bos~bo~al~u~ase fraction, standard activity 300 pmol/mn/& [il.]) was a gift from Dr. Labh6, Montpellier. Peptides synthesis and purification. Solid-phase peptide synthesis was performed (Milligen 9050 synthesizer) with the use of O-Auorenylmethyloxycarbonyl as temporary a-amino group protection. The peptide was cleaved from the resin (Pep Syn KG; &ii&pore) with trifluoroacetic acid/water (95:5) change chromatography on a Mono nn (Pharmacia). Phosphorylation of EF-I and p 1 EF-WY (5 ed was phosphorylated in. vitro by 0.05 ~1 cdc‘2 p .inase in the presence of 50 p&f [Y-~‘P]ATP [2]. EF-1~ was isolated by SDS-PAGE and eluted from the gel [12]. Peptides (2 mII& were phosphorylated under the same conditions. Reaction was stopped by acetic acid and phosphopeptides were isolated by thin-layer eleetsophoresis at pll[ 3.5 on cellulose-coated plates from Kodak. Autoradiographies were on XOMAT-AR films (Kodak). Results were q;lantified by Cerenkov counting of gel slices or of scraped cellulose. Tryptic phosphopeptide mapping. Lyopbihzed protein and peptides were treated twice with TPCK-trypsin (Sigma), 30 pg for 18 h and then 20 pg for 4 h in LO mM NH,HCO, (500 ALP final volume), The resulting phosphopeptides were analyzed by eleetrophoresis at pH 3.5 for the first dimension and by chromatography in isobutyric acid buffer for the second dimension [12]. Guanine nucleotide exchange activity. change on RF-la was performed f13] usi phosphate (11.6 Wmmol; Amersham).
549
0014-4827/92
Copyright 0 1932 hy Academic All
rights
of reproduction
in any form
$5.00
Press, Inc. reserved.
550
SHORT
IB
EF-1~
1 Coomassie blue 47kDa
Autoradiography
36kDa 3OkDa
No
Pl
P2
FIG. 1. Effects of peptides Pl and P2 on the phosphorylation of EF-1-r by cdc2 protein kinase. (A) Coomassie blue-stained SDSPAGE (10% acrylamide) of EF-1fiBy complex purified from Xenopus oocytes. (B) SDS-PAGE at the level of EF-17 after phosphorylation by cdc2 protein kinase in the presence of peptides Pl or P2 (2 mM). Top, Coomassie blue staining of the gel; bottom, corresponding autoradiography,
RESULTS
AND
Identification of cdc2 Protein Site in EF-1~
DISCUSSION
Kinase Phosphorylation
The cDNA-deduced protein sequence of EF-1~ [9] contains two putative phosphoacceptors at positions 46 and 230, both containing threonine amino acid, the target for cdc2 protein kinase [3]. Two peptides were synthesized, Pl and P2, EFQFGVTNK46TPEFLKK and PKKEz3’TPKKEKPA, respectively, matching the cDNA-deduced protein sequence. We first analyzed peptide Pl and P2 phosphorylation by cdc2 protein kinase. Peptide P2 and not peptide Pl was phosphorylated by the kinase, suggesting that it could contain the target threonine. Phosphorylation of EF-17 was analyzed in the presence of peptides Pl and P2 (2 m&f). Purified EF-1/3&y was incubated in the pres-
EF-1 y
NOTE
ence of cdc2 protein kinase and separated into & 6, and y subunits by SDS-PAGE (Fig. 1A). Peptide Pl was totally ineffective on the phosphorylation of EF-17, whereas peptide P2 was found to be inhibitory (Fig. lB, bottom). Inhibition by peptide P2 was 88%. Therefore, peptide P2 was likely to contain a phosphorylation site of EF-1~. We then performed proteolytic digestion of EF-ly and peptide P2, both phosphorylated by cdc2 protein kinase. Two-dimensional analysis of phosphopeptides derived from EF-1~ trypsic hydrolysis repeatedly showed the presence of two phosphopeptides as illustrated on Fig. 2A. This result indicated the presence of either two phosphorylation sites in the protein or a bipeptide generation due to the presence of hardly hydrolyzable lysines, as has been reported for sequences containing lysine-lysine, lysine-glutamate, or lysine-proline [12]. When phosphopeptide P2 was analyzed, several hydrolysis products were obtained (see Fig. 2B), indicating incomplete hydrolysis of the phosphopeptide by trypsin, in spite of the drastic experimental conditions (see Materials and Methods). This result was consistent with the presence in the peptide sequence of barely hydrolyzable lysines [ 121. When two-dimensional separation of EF-1~ and peptide P2 hydrolysates were compared (see Figs. 2A and 2B), the major phosphorylated product of each hydrolysate migrated to identical positions and the second peptide from EF-17 hydrolysis was found to have a peptide P2 counterpart. To ascertain the coincidence between the two EF-1-y products and peptide P2 products, they were mixed and analyzed in two-dimensional separation. The two EFly phosphopeptides comigrated exactly with two of the peptide P2 products (Fig. 2C), therefore demonstrating that they derive from a unique cdc2 protein kinase phosphoacceptor in EF-1~ sequence which corresponds to threonine-230.
Peptide P2
EF-1 y +peptide P2
Chromatography FIG. 2. Analysis of the phosphorylation of EF-17 and peptide P2 by cdc2 protein kinase. Autoradiography of tryptic hydrolysates after two-dimensional separation. (A) Digestion products of EF-ly. (B) Digestion products of peptide P2. (C) Mix of EF-1~ and peptide P2 hydrolysates. The arrows indicate the position of the two common subproducts.
SHORT
551
NOTE
dicating that phosphory~atio~ of tbre~~~~e-2~~ ly did not affect tbe activity of fi or 6
100
in
We are very grateful to E. Ricquier for expert technical assistance. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, and Ministere de la Recberche et de I’Enseignement Superieur.
30 0
0.1
0.2
0.3
0.4
0.5
EF-1 pyS complex (pg) FIG. 3. Effect of phosphorylation of EF-r on the guanine nucleotide exchange activity of EF-1flS-y complex. Complex containing dephospho-EF-ly (open squares) was purified from prophase Xenopals oocytes. Complex containing cdc2-phospho-EF-1-y (black squares) was purified from metaphase Xenopus oocytes. Both complexes were purified in parallel. Guanine nucleotide exchange activity of EF-l@y complex was measured using Xenopus EF-la bound to [3H]GDP as a substrate. Results are expressed as the percentage of nucleotide remaining on EF-lol after a 3-min incubation in the presence of the indicated amount of EF-l@y.
Consequence of Threonine-230
Phosphorylation
EF-l&%y complexes were isolated from prophase and metaphase oocytes. The metaphase complex contains EF-17, in a cd& protein kinase-phosphorylated form, whereas the prophase complex does not [3]. Both were assayed on the GDPIGTP exchange reaction using EFla isolated from Xenopus oocytes as a substrate. Both complexes were found to possess GDP/GTP exchange activity (Fig. 3). Activity was 8 pmol of exchange/3 mini pmol protein, and showed no significant difference, inReceived April
21, 1992
1. 2. 3. 4. 5. 6.
Nurse, P. (1990) Nature 344, 503-508. Nigg, E. A. (1991) Sem. Mulner-Lorillon, O., P , P., Labbe, J.-C., Doree, M., and Belle, R. Belle, R., Derancourt, J., Podhe, FL, Capony, J. P., &on, R., and Mulner-Lorillon, 0. (1989) FEBS Lett. 255, 101-104. Belle, R., Cormier, P., Poulhe, R., Morales, J., Huchon, D., and Mulner-Lorillon, 0. (1999) Int. J. Deu. Biol. 34, 111-115. Janssen, G. M. C., Morales, J., Schipper, A., M.ui orillon, Q., Belle, R., and Moller, W. (1991) J. Biol. @hem. 1488514888.
7.
Riis, B., Rattan, S. I. S., Clark, B. F. C., and Merrick, W. C. (1990) Trends Biochem. Sci. 16, 420-424. 8. Van Damme, W. T. F., Amons, R., arssies, R., ‘Timmers, C. J., Janssen, 6. M. C., and Moller, W. (199G) Biochim. Biophys. Acta 1050, 241-247. 9. Cormier, P., Osborne, H. B., Morales, J., Bassez, T., Pouhle, R., Mazabraud, A., Mulner-Lorillon, O., and Belle, R. (1991) Mucleic Acids Res. 19, 6644. 10.
Morales, J., Mulner-Lorillon, Biochimie X3, 1249-1253.
11.
Labbe, J. C., Picard, A., Karsenti, Biol. 127, 157-159.
12.
Boyle, W. J., Van Der Geer, P., and Hunter, T. (1991) in Metbods in Enzymology (Hunter, T., and Sefton, B. M., Eds.), Vol. 201, pp. 110-149, Academic Press, New York.
13.
Janssen, 6. M. C., and Moller, 1773-1778.
Q., Denis, H., and Belle, R. (1991) E., and Doree, M. (1988) 13ev.
W. (1988) J. BLol. &err,.
Z(63,
CELL
RESEARCH
202, 549-551 (19%)
SHORT NO osphorylation of Xenopus Elongation tein Kinase: Identification of the Ph OD~LEM~LNER-LORILLON,*PATRICKCORMIER,*JEAN-CLA~DECAVA JULIAMORALES," ROBERT~OULHE,*AND ROBERT BELL~%**~ *Laboratoire de Physiologie de la Reproduction, UPMC, TCentre de Recherche de Biochimie Macromol~culaire,
CNRS URA 1449, INRA, UPR CNRS 41, INSERM
The cdc2 protein kinase phosphorylates elongation factor-l? (EF-I-/) during meiotic maturation of Xenopus ooeytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EFPy was au in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-ly. Tryptie hydrolysis of EF-17 and the P2 peptide, both phosphorylated by ede2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-ly corn&rated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-17 therefore contains one unique phosphoacceptor for cd& protein kinase, identified as threonine230. o 1982 Academic Press, hc.
INTRODUCTION The cd& protein kinase is universally implicated in the regulation of cell division [I]. Many substrates of the kinase have now been characterized [2]. One of the first, characterized in viva, was a protein ~47, stoichiometrically phosphorylated during meiotic cell division of Xenopus socytes [3-63. The p47 protein is in a dephosphorylated form in prophase oocytes and fully phosphorylated in metaphase oocytes [3]. It is present in a macromolecular complex, identified as components of elongation factor-l [4-6] 0 Elongation factors of protein biosynthesis are implicated in the elongation of polypeptide chains [7]. Elongation factor-la (EF-la) is responsible for the binding of aminoacyl-tRNA to the ribosome with concomitant hydrolysis of GTP. EF-Q3Sy complex catalyzes the IGTP exchange on EF-la!. Both fi and 6 proteins
1 To whom reprint
requests should be addressed.
4 place Jussieu> 75256 Paris Cedex 05, France; and 249, BP 5051, 34033 Montpellier Cede& France
possess the guanine nueleotide activity, at least in Artemia [8]. In Xenopus oocytes, the p47 complex, purified as a cdc2 protein kinase substrate, was identified as EFl@y, the p47 protein corresponding to EF-17 14-61. have cloned the three Xenopus oteins and have recently sequenced EF-1y cDNA ] We identify in this report the site of in EF-17 by cdc2 protein kinase at the level of threonine-230, and analyze the co~se~~e~ce ofthe phos~borylation on the GDPGTP exchange reaction performed with purified Xenopus EF-ICY.
ATERIA$S AND Protein purification. El?-1/3Sy complex was purified from Xenopus oocytes as described [3]. EF-la was also purified from Xenopus oocytes [IQ]. The cdc2 protein kinase (Pli ~bos~bo~al~u~ase fraction, standard activity 300 pmol/mn/& [il.]) was a gift from Dr. Labh6, Montpellier. Peptides synthesis and purification. Solid-phase peptide synthesis was performed (Milligen 9050 synthesizer) with the use of O-Auorenylmethyloxycarbonyl as temporary a-amino group protection. The peptide was cleaved from the resin (Pep Syn KG; &ii&pore) with trifluoroacetic acid/water (95:5) change chromatography on a Mono nn (Pharmacia). Phosphorylation of EF-I and p 1 EF-WY (5 ed was phosphorylated in. vitro by 0.05 ~1 cdc‘2 p .inase in the presence of 50 p&f [Y-~‘P]ATP [2]. EF-1~ was isolated by SDS-PAGE and eluted from the gel [12]. Peptides (2 mII& were phosphorylated under the same conditions. Reaction was stopped by acetic acid and phosphopeptides were isolated by thin-layer eleetsophoresis at pll[ 3.5 on cellulose-coated plates from Kodak. Autoradiographies were on XOMAT-AR films (Kodak). Results were q;lantified by Cerenkov counting of gel slices or of scraped cellulose. Tryptic phosphopeptide mapping. Lyopbihzed protein and peptides were treated twice with TPCK-trypsin (Sigma), 30 pg for 18 h and then 20 pg for 4 h in LO mM NH,HCO, (500 ALP final volume), The resulting phosphopeptides were analyzed by eleetrophoresis at pH 3.5 for the first dimension and by chromatography in isobutyric acid buffer for the second dimension [12]. Guanine nucleotide exchange activity. change on RF-la was performed f13] usi phosphate (11.6 Wmmol; Amersham).
549
0014-4827/92
Copyright 0 1932 hy Academic All
rights
of reproduction
in any form
$5.00
Press, Inc. reserved.
550
SHORT
IB
EF-1~
1 Coomassie blue 47kDa
Autoradiography
36kDa 3OkDa
No
Pl
P2
FIG. 1. Effects of peptides Pl and P2 on the phosphorylation of EF-1-r by cdc2 protein kinase. (A) Coomassie blue-stained SDSPAGE (10% acrylamide) of EF-1fiBy complex purified from Xenopus oocytes. (B) SDS-PAGE at the level of EF-17 after phosphorylation by cdc2 protein kinase in the presence of peptides Pl or P2 (2 mM). Top, Coomassie blue staining of the gel; bottom, corresponding autoradiography,
RESULTS
AND
Identification of cdc2 Protein Site in EF-1~
DISCUSSION
Kinase Phosphorylation
The cDNA-deduced protein sequence of EF-1~ [9] contains two putative phosphoacceptors at positions 46 and 230, both containing threonine amino acid, the target for cdc2 protein kinase [3]. Two peptides were synthesized, Pl and P2, EFQFGVTNK46TPEFLKK and PKKEz3’TPKKEKPA, respectively, matching the cDNA-deduced protein sequence. We first analyzed peptide Pl and P2 phosphorylation by cdc2 protein kinase. Peptide P2 and not peptide Pl was phosphorylated by the kinase, suggesting that it could contain the target threonine. Phosphorylation of EF-17 was analyzed in the presence of peptides Pl and P2 (2 m&f). Purified EF-1/3&y was incubated in the pres-
EF-1 y
NOTE
ence of cdc2 protein kinase and separated into & 6, and y subunits by SDS-PAGE (Fig. 1A). Peptide Pl was totally ineffective on the phosphorylation of EF-17, whereas peptide P2 was found to be inhibitory (Fig. lB, bottom). Inhibition by peptide P2 was 88%. Therefore, peptide P2 was likely to contain a phosphorylation site of EF-1~. We then performed proteolytic digestion of EF-ly and peptide P2, both phosphorylated by cdc2 protein kinase. Two-dimensional analysis of phosphopeptides derived from EF-1~ trypsic hydrolysis repeatedly showed the presence of two phosphopeptides as illustrated on Fig. 2A. This result indicated the presence of either two phosphorylation sites in the protein or a bipeptide generation due to the presence of hardly hydrolyzable lysines, as has been reported for sequences containing lysine-lysine, lysine-glutamate, or lysine-proline [12]. When phosphopeptide P2 was analyzed, several hydrolysis products were obtained (see Fig. 2B), indicating incomplete hydrolysis of the phosphopeptide by trypsin, in spite of the drastic experimental conditions (see Materials and Methods). This result was consistent with the presence in the peptide sequence of barely hydrolyzable lysines [ 121. When two-dimensional separation of EF-1~ and peptide P2 hydrolysates were compared (see Figs. 2A and 2B), the major phosphorylated product of each hydrolysate migrated to identical positions and the second peptide from EF-17 hydrolysis was found to have a peptide P2 counterpart. To ascertain the coincidence between the two EF-1-y products and peptide P2 products, they were mixed and analyzed in two-dimensional separation. The two EFly phosphopeptides comigrated exactly with two of the peptide P2 products (Fig. 2C), therefore demonstrating that they derive from a unique cdc2 protein kinase phosphoacceptor in EF-1~ sequence which corresponds to threonine-230.
Peptide P2
EF-1 y +peptide P2
Chromatography FIG. 2. Analysis of the phosphorylation of EF-17 and peptide P2 by cdc2 protein kinase. Autoradiography of tryptic hydrolysates after two-dimensional separation. (A) Digestion products of EF-ly. (B) Digestion products of peptide P2. (C) Mix of EF-1~ and peptide P2 hydrolysates. The arrows indicate the position of the two common subproducts.
SHORT
551
NOTE
dicating that phosphory~atio~ of tbre~~~~e-2~~ ly did not affect tbe activity of fi or 6
100
in
We are very grateful to E. Ricquier for expert technical assistance. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, and Ministere de la Recberche et de I’Enseignement Superieur.
30 0
0.1
0.2
0.3
0.4
0.5
EF-1 pyS complex (pg) FIG. 3. Effect of phosphorylation of EF-r on the guanine nucleotide exchange activity of EF-1flS-y complex. Complex containing dephospho-EF-ly (open squares) was purified from prophase Xenopals oocytes. Complex containing cdc2-phospho-EF-1-y (black squares) was purified from metaphase Xenopus oocytes. Both complexes were purified in parallel. Guanine nucleotide exchange activity of EF-l@y complex was measured using Xenopus EF-la bound to [3H]GDP as a substrate. Results are expressed as the percentage of nucleotide remaining on EF-lol after a 3-min incubation in the presence of the indicated amount of EF-l@y.
Consequence of Threonine-230
Phosphorylation
EF-l&%y complexes were isolated from prophase and metaphase oocytes. The metaphase complex contains EF-17, in a cd& protein kinase-phosphorylated form, whereas the prophase complex does not [3]. Both were assayed on the GDPIGTP exchange reaction using EFla isolated from Xenopus oocytes as a substrate. Both complexes were found to possess GDP/GTP exchange activity (Fig. 3). Activity was 8 pmol of exchange/3 mini pmol protein, and showed no significant difference, inReceived April
21, 1992
1. 2. 3. 4. 5. 6.
Nurse, P. (1990) Nature 344, 503-508. Nigg, E. A. (1991) Sem. Mulner-Lorillon, O., P , P., Labbe, J.-C., Doree, M., and Belle, R. Belle, R., Derancourt, J., Podhe, FL, Capony, J. P., &on, R., and Mulner-Lorillon, 0. (1989) FEBS Lett. 255, 101-104. Belle, R., Cormier, P., Poulhe, R., Morales, J., Huchon, D., and Mulner-Lorillon, 0. (1999) Int. J. Deu. Biol. 34, 111-115. Janssen, G. M. C., Morales, J., Schipper, A., M.ui orillon, Q., Belle, R., and Moller, W. (1991) J. Biol. @hem. 1488514888.
7.
Riis, B., Rattan, S. I. S., Clark, B. F. C., and Merrick, W. C. (1990) Trends Biochem. Sci. 16, 420-424. 8. Van Damme, W. T. F., Amons, R., arssies, R., ‘Timmers, C. J., Janssen, 6. M. C., and Moller, W. (199G) Biochim. Biophys. Acta 1050, 241-247. 9. Cormier, P., Osborne, H. B., Morales, J., Bassez, T., Pouhle, R., Mazabraud, A., Mulner-Lorillon, O., and Belle, R. (1991) Mucleic Acids Res. 19, 6644. 10.
Morales, J., Mulner-Lorillon, Biochimie X3, 1249-1253.
11.
Labbe, J. C., Picard, A., Karsenti, Biol. 127, 157-159.
12.
Boyle, W. J., Van Der Geer, P., and Hunter, T. (1991) in Metbods in Enzymology (Hunter, T., and Sefton, B. M., Eds.), Vol. 201, pp. 110-149, Academic Press, New York.
13.
Janssen, 6. M. C., and Moller, 1773-1778.
Q., Denis, H., and Belle, R. (1991) E., and Doree, M. (1988) 13ev.
W. (1988) J. BLol. &err,.
Z(63,
