A link between MAP kinase and p34cdc2/cyclin B during oocyte maturation: p90rsk phosphorylates and inactivates the p34cdc2 inhibitory kinase Myt1
1998; Springer Nature; Volume: 17; Issue: 17 Linguagem: Inglês
10.1093/emboj/17.17.5037
ISSN1460-2075
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoArticle1 September 1998free access A link between MAP kinase and p34cdc2/cyclin B during oocyte maturation: p90rsk phosphorylates and inactivates the p34cdc2 inhibitory kinase Myt1 Amparo Palmer Amparo Palmer European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Anne-Claude Gavin Anne-Claude Gavin European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Angel R. Nebreda Corresponding Author Angel R. Nebreda European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Amparo Palmer Amparo Palmer European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Anne-Claude Gavin Anne-Claude Gavin European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Angel R. Nebreda Corresponding Author Angel R. Nebreda European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Search for more papers by this author Author Information Amparo Palmer1, Anne-Claude Gavin1 and Angel R. Nebreda 1 1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5037-5047https://doi.org/10.1093/emboj/17.17.5037 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34cdc2. In G2-arrested Xenopus oocytes, there is a stock of p34cdc2/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34cdc2 phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34cdc2 inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2→M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90rsk, a protein kinase that can be phosphorylated and activated by MAPK. p90rsk in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34cdc2/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK–p90rsk cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90rsk, and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90rsk and that p90rsk in turn down-regulates Myt1, leading to the activation of p34cdc2/cyclin B. Introduction Xenopus laevis oocytes are naturally arrested in late G2 of the first meiotic division and are induced to enter into M-phase of meiosis upon exposure to progesterone. The events of meiotic maturation, including germinal vesicle breakdown (GVBD), chromosome condensation and formation of the metaphase spindles are associated with the activation of maturation-promoting factor (MPF), a regulatory factor involved in mitosis initiation in eukaryotic cells (Masui and Markert, 1971; Masui and Clarke, 1979). MPF is composed of a regulatory subunit, cyclin B, and a catalytic subunit, the protein kinase p34cdc2, whose phosphorylation state is very important for the activity of the p34cdc2/cyclin B complex. In particular, p34cdc2 phosphorylation on either Thr14 or Tyr15 mediated by Wee1 family kinases results in MPF inactivation (reviewed by Coleman and Dunphy, 1994; Morgan, 1995; Lew and Kornbluth, 1996). Translation of mRNA for the protein kinase Mos is required for the progesterone-induced maturation of Xenopus oocytes (Sagata et al., 1988, 1989; Kanki and Donoghue, 1991; Sheets et al., 1995). Mos is an efficient activator of MAPK kinase (Nebreda and Hunt, 1993; Nebreda et al., 1993b; Posada et al., 1993; Shibuya and Ruderman, 1993), and injection of neutralizing antibodies against MAPK kinase inhibits Mos-induced oocyte maturation (Kosako et al., 1994). This suggests that Mos activates p34cdc2/cyclin B through the MAPK cascade. Consistent with this hypothesis, injection of either constitutively active MAPK kinase or thiophosphorylated MAPK can trigger the maturation of Xenopus oocytes independently of any progesterone stimulation (Gotoh et al., 1995; Haccard et al., 1995; Huang et al., 1995). It should be noted, however, that although the Mos–MAPK pathway is likely to have an important role in oocyte maturation, there is also evidence that translation of mRNA(s) other than Mos is required to activate MPF during progesterone-induced maturation (Nebreda et al., 1995; Barkoff et al., 1998). Little is known of how MAPK triggers MPF activation and the maturation of oocytes. A well characterized substrate for MAPK is the 90 kDa ribosomal S6 kinase (p90rsk or S6KII), a serine/threonine protein kinase originally identified in Xenopus oocytes as a kinase that phosphorylates the S6 protein of the ribosomal 40S subunit in vitro (Erikson and Maller, 1986). Xenopus p90rsk is most similar to the mammalian Rsk1/Rsk2 protein kinases (also referred to as MAPKAP kinase-1a and -1b) (Alcorta et al., 1989), which can be phosphorylated and activated in vitro and in vivo by p42Erk2 and p44Erk1 MAPKs (Sturgill et al., 1988; Chung et al., 1991; Alessi et al., 1995; Zhao et al., 1996). In G2-arrested Xenopus oocytes, inactive p90rsk is associated with inactive MAPK in a heterodimer which dissociates upon phosphorylation and activation of the two kinases (Hsiao et al., 1994), suggesting that p90rsk has a role in oocyte maturation. In Xenopus oocytes, there is a pre-formed stock of inactive p34cdc2/cyclin B complexes (pre-MPF) in which p34cdc2 is phosphorylated on Thr14 and Tyr15 as well as Thr161 (Cyert and Kirschner, 1988; Gautier and Maller, 1991; Kobayashi et al., 1991). Thus, the activation of pre-MPF during oocyte maturation requires the dephosphorylation of p34cdc2 on Thr14 and Tyr15, probably catalysed by the dual-specificity phosphatase Cdc25C (Dunphy and Kumagai, 1991; Gautier et al., 1991; Kumagai and Dunphy, 1991; Strausfeld et al., 1991). In addition to an increased activity of Cdc25C, inhibition of the kinases that phosphorylate p34cdc2 on Thr14 and Tyr15 may also trigger the activation of p34cdc2/cyclin B complexes (Atherton-Fessler et al., 1994). The prototype of the p34cdc2 inhibitory kinases is Wee1, the negative regulator of mitosis in Schizosaccharomyces pombe. However, the first cloned human and Xenopus Wee1 homologues phosphorylate p34cdc2 only on Tyr15 but not Thr14, supporting the existence of a separate Thr14 kinase (Parker and Piwnica-Worms, 1992; McGowan and Russell, 1993; Mueller et al., 1995a). A kinase activity that can phosphorylate p34cdc2 on Thr14 was later detected in Xenopus extracts; this enzyme is associated with membranes and can be separated from a Tyr15-specific kinase activity in the soluble fraction (Atherton-Fessler et al., 1994; Kornbluth et al., 1994). A candidate cDNA for this activity, named Myt1, has been cloned from Xenopus (Mueller et al., 1995b) and humans (Booher et al., 1997; Liu et al., 1997). Myt1 encodes a membrane-associated Wee1 homologue that can phosphorylate p34cdc2 on both Thr14 and Tyr15. As expected, and in contrast to Cdc25C whose activity increases during mitosis, the activity of both Wee1 and Myt1 declines during mitosis, therefore contributing to the fall in the inhibitory phosphorylation of p34cdc2 at this stage of the cell cycle. Cdc25C, Wee1 and Myt1 all become heavily phosphorylated during mitosis (M phase) and these phosphorylations correlate with the increased activity of Cdc25C (Izumi et al., 1992; Kumagai and Dunphy, 1992, 1996; Izumi and Maller, 1993; Hoffmann et al., 1993) and the inhibition of Wee1 and Myt1 (Tang et al., 1993; McGowan and Russell, 1995; Mueller et al., 1995a,b; Booher et al., 1997). A possible connection between MAPK and the activation of pre-MPF in oocytes comes from the recent observation that MAPK down-regulates an activity which in turn inactivates p34cdc2/cyclin B in G2-arrested oocytes (Abrieu et al., 1997). This inhibitory activity targeted by MAPK could correspond to Thr14–Tyr15 p34cdc2 kinases such as Wee1 and Myt1. Since Wee1 is not present in G2-arrested oocytes and is synthesized only upon progesterone stimulation (Murakami and Vande Woude, 1998), it is not likely to keep pre-MPF in an inactive state. Thus, Myt1 may be the MPF inhibitory activity down-regulated by MAPK in Xenopus oocytes. Here we report that Myt1 is a target of MAPK-activated p90rsk. Thus, p90rsk phosphorylates the C-terminal regulatory domain of Myt1 and down-regulates the inhibitory activity of Myt1 on p34cdc2/cyclin B1 complexes in vitro. We also show that Myt1 becomes phosphorylated during oocyte maturation and that p90rsk and Myt1 are associated in mature oocytes. Our results indicate that p90rsk–Myt1 provides a link between the activation of MAPK and MPF during Xenopus oocyte maturation. Results Identification of protein kinases in Xenopus cell-free extracts that phosphorylate Myt1 in vitro In addition to the catalytic domain, Myt1 contains a region which includes many potential phosphorylation sites and is therefore likely to have a regulatory role. To identify protein kinases that can phosphorylate and potentially regulate Xenopus Myt1, we fused the non-catalytic regions of Myt1 (C-terminal half) to glutathione S-transferase (GST) and used the bacterially produced fusion protein as a substrate in kinase assays with Xenopus cell-free extracts. As shown in Figure 1A, GST–Myt1 can be phosphorylated efficiently in vitro by cell-free extracts prepared from either eggs (lane 3) or progesterone-matured oocytes (lane 2), but not from control, G2-arrested oocytes (lane 1). We then used GST–Myt1 to screen Xenopus egg extracts fractionated by Mono Q chromatography and we detected two peaks of kinase activity that phosphorylate GST–Myt1 in vitro (Figure 1B, GST–Myt1). As a control we also fused GST to the non-catalytic region of Wee1 (N-terminal half) and found that GST–Wee1 can also be phosphorylated in vitro by cell-free extracts prepared from unfertilized eggs (Figure 1A, lane 5) but not from control, G2-arrested oocytes (Figure 1A, lane 4). When GST–Wee1 was used as a substrate to analyse the Mono Q-fractionated egg extracts, we found that most of the activity phosphorylating GST–Wee1 eluted in different fractions from those containing the major peak of activity that phosphorylates GST–Myt1 (Figure 1B). The same Mono Q fractions were also analysed by immunoblot with anti-p34cdc2 and anti-MAPK antibodies to identify the positions where these two protein kinases elute. We found that the major peak of kinase activity phosphorylating GST–Myt1 overlaps with the fractions where the p42Mpk1 MAPK is detected (Figure 1B, anti-MAPK), whereas the major kinase activity phosphorylating GST–Wee1 overlaps with some of the fractions where p34cdc2 eluted (Figure 1B, anti-p34cdc2). Figure 1.Identification of protein kinase activities in Xenopus cell-free extracts that phosphorylate GST–Myt1 and GST–Wee1 in vitro. (A) Cell-free extracts prepared from either eggs (in vivo mature oocytes, lanes 3 and 5), oocytes matured in vitro by progesterone (lane 2) or control, G2-arrested oocytes (lanes 1 and 4) were used to phosphorylate GST–Myt1 (lanes 1–3) or GST–Wee1 (lanes 4 and 5). (B) Egg extracts were ultracentrifuged and then fractionated by Mono Q chromatography. The fractions were either used in kinase assays with GST–Myt1 or GST–Wee1 as the substrate, or analysed by immunoblotting using anti-p34cdc2 or anti-Mpk1 MAPK antibodies, as indicated. Download figure Download PowerPoint To gain further information on the protein kinase(s) that phosphorylates GST–Myt1, we used the fusion protein for pull-down experiments followed by a kinase assay. For these experiments, GST–Myt1 was first coupled to glutathione–Sepharose beads and then incubated with Xenopus egg extracts. The beads were recovered from the extracts, washed and then incubated in a kinase reaction with [γ-32P]ATP and Mg2+. The phosphorylated proteins were finally analysed by polyacrylamide gel electrophoresis and autoradiography. We found that protein kinases from the extracts were able to bind to and efficiently phosphorylate the GST–Myt1 fusion protein (Figure 2, lane 5 in upper panel). Moreover, the electrophoretic mobility of the GST–Myt1 recombinant protein shifted upwards upon phosphorylation in the pull-downs (Figure 2, lanes 4 and 5 in lower panel). We also detected in the GST–Myt1 pull-downs additional phosphorylated bands of ∼34 and 90 kDa (Figure 2, arrowheads in upper panel). In the same experiments, GST–Wee1 was also phosphorylated but more weakly than GST–Myt1 (Figure 2, lane 3 in upper panel), although some variability was observed depending on the extracts. Moreover, we could not detect any significant size shift of the GST–Wee1 recombinant protein upon phosphorylation in pull-downs (Figure 2, lanes 2 and 3 in lower panel). Figure 2.Identification of protein kinase activities in egg extracts that bind to and phosphorylate GST–Myt1 and GST–Wee1 in pull-downs. Bacterially produced GST (lane 1), GST–Wee1 (lane 3) and GST–Myt1 (lane 5) proteins were incubated with egg extracts, recovered by centrifugation, incubated with [γ-32P]ATP and analysed by polyacrylamide gel electrophoresis and autoradiography. Purified GST–Wee1 (lane 2) and GST–Myt1 (lane 4) were also incubated with [γ-32P]ATP and analysed by polyacrylamide gel electrophoresis in parallel. The positions where the two purified recombinant proteins run are indicated. Arrowheads indicate two additional bands of ∼34 and 90 kDa which are phosphorylated in GST–Myt1 pull-downs (lane 5). The upper panel shows an autoradiograph and the lower panel the same gel stained with Coomassie Blue. Download figure Download PowerPoint p34cdc2-dependent and independent kinase activities can phosphorylate Myt1 Many regulatory and structural proteins become phosphorylated or hyperphosphorylated during M-phase, and p34cdc2/cyclin complexes are known to play an important role in these phosphorylations. To investigate whether p34cdc2 could be detected in GST–Myt1 pull-downs, we analysed, by immunoblot with anti-p34cdc2 antibodies, GST and GST–Myt1 pull-downs prepared from Xenopus egg extracts. We found that in GST–Myt1 pull-downs there was significantly more p34cdc2 than in those with GST alone (Figure 3A, compare lanes 2 and 4 with lanes 3 and 6, respectively), indicating that p34cdc2 can associate specifically either directly or indirectly with the C-terminal regulatory region of Myt1. We also found p34cdc2 associated with GST–Wee1 pull-downs (Figure 3A, lane 5). Figure 3.p34cdc2 binds to Myt1. (A) Egg extracts (lane 1) and either GST (lanes 2 and 4), GST–Myt1 (lanes 3 and 6) or GST–Wee1 (lane 5) pull-downs of the extracts were analysed by immunoblotting with anti-p34cdc2 antibodies. (B) CSF-arrested egg extracts were incubated in the presence (lanes 2, 4 and 6) or absence (lanes 1, 3 and 5) of Ca2+ for 30 min and then used for in vitro kinase assays using as substrates either histone H1 + MBP (lanes 1 and 2) or GST–Myt1 (lanes 3 and 4). Aliquots of the extracts were also used for GST–Myt1 pull-downs (lanes 5 and 6) and then incubated with [γ-32P]ATP and analysed by polyacrylamide gel electrophoresis and autoradiography. Download figure Download PowerPoint To investigate the possible connection between p34cdc2/cyclin B and the kinase activity responsible for GST–Myt1 phosphorylation in egg extracts, we used Ca2+-treated egg extracts. Treatment of cytostatic factor (CSF)-arrested egg extracts with Ca2+ triggers cyclin B degradation, leading to MPF inactivation after 20–30 min as indicated by the absence of H1 kinase activity in the extracts (Figure 3B, lane 2). These H1 kinase-negative egg extracts can still phosphorylate GST–Myt1, albeit less efficiently than the untreated CSF-arrested egg extracts (Figure 3B, lanes 3 and 4). Moreover, in the Ca2+-treated extracts, there is still a kinase activity that can bind to and phosphorylate GST–Myt1 in pull-down experiments, but interestingly in this case the electrophoretic mobility of the GST–Myt1 protein does not shift as it does in the pull-downs from CSF-arrested egg extracts (Figure 3B, lanes 5 and 6). We conclude from this experiment that there are at least two protein kinase activities that can bind to and phosphorylate the C-terminal regulatory region of Myt1 in vitro. One of the kinase activities is dependent on MPF and may be either p34cdc2/cyclin B or another p34cdc2/cyclin complex. The other protein kinase activity can bind to and phosphorylate Myt1 independently of MPF activity. To gain more information on this MPF-independent kinase activity that phosphorylates Myt1, we carried out in-gel kinase assays using GST–Myt1 as a substrate. As shown in Figure 4A, both in egg extracts (lane 3) and in lysates prepared from progesterone-matured oocytes (lane 2), we detected a protein kinase activity of ∼90 kDa which can phosphorylate GST–Myt1 upon renaturation in the gel. This activity, however, was not detected in lysates prepared from unstimulated oocytes arrested in G2 (lane 1). A protein kinase activity of 90 kDa was also strongly enriched in GST–Myt1 pull-downs prepared from egg extracts when compared with the same pull-downs using GST alone (∼12-fold increase by PhosphorImager quantification; Figure 4A, compare lanes 5 and 6). In addition, we detected in GST–Myt1 pull-downs another kinase activity of 50–60 kDa (Figure 4A, dot), but we do not know at present whether this activity is related to the 90 kDa activity or corresponds to a totally different protein kinase. When a duplicate of these samples was analysed in gels cast with GST alone, we could detect a kinase of ∼90 kDa which was able to renature, and presumably autophosphorylate, only in the GST–Myt1 pull-downs, but not in any of the other samples (Figure 4A, arrowhead in lower panel). This observation is consistent with the idea that the 90 kDa kinase that phosphorylates Myt1 is enriched in GST–Myt1 pull-downs, and confirms the previous detection of a phosphorylated band of 90 kDa in kinase assays using GST–Myt1 pull-downs (Figure 2, lane 5 in upper panel). Figure 4.A 90 kDa protein kinase binds to and phosphorylates Myt1. In-gel kinase assays using as substrates GST–Myt1 (A, upper), GST–Wee1 (B, upper) or GST alone (A and B, lower). (A) Control, G2-arrested oocyte lysates (lane1), progesterone-matured oocyte lysates (lane 2), egg extracts (lane 3), purified GST–Myt1 (lane 4), GST (lane 5) and GST–Myt1 (lane 6) pull-downs from egg extracts. (B) GST (lane 1), GST–Wee1 (lane 2) and GST–Myt1 (lane 3) pull-downs from egg extracts. The arrowheads and the dot indicate kinase activities of 90 and 50–60 kDa, respectively. Download figure Download PowerPoint As a control, we performed in-gel kinase assays using GST–Wee1 polymerized in the gel. We observed that the 90 kDa protein kinase activity that binds to GST–Myt1 pull-downs did not phosphorylate GST–Wee1 (Figure 4B, lane 3 in upper panel). Moreover, using a gel with GST alone, we confirmed that the GST–Myt1-associated 90 kDa protein kinase is able to renature and autophosphorylate in the gel (Figure 4B, arrowhead in lower panel) but does not bind to GST–Wee1 (Figure 4B, lane 2), nor did we detect any protein kinase activity that is able to renature and phosphorylate GST–Wee1 (Figure 4B, lane 2 in upper panel). p90rsk strongly associates with Myt1 The protein kinase p90rsk is activated during Xenopus oocyte maturation at the same time as MAPK (Nebreda et al., 1993a; Hsiao et al., 1994). Thus, we decided to investigate the connection between p90rsk and the 90 kDa protein kinase that associates with and phosphorylates the C-terminus of Myt1. By immunoblotting using anti-p90rsk-specific antibodies, we found that p90rsk can be detected specifically in GST–Myt1 pull-downs prepared from Xenopus egg extracts but not in the pull-downs prepared with GST alone (Figure 5A, p90rsk). In contrast, the p42Mpk1 MAPK is not present at detectable levels in the same GST–Myt1 pull-downs (Figure 5A, MAPK). Moreover, the C-terminus of Myt1 associates more strongly with the active and hyperphosphorylated p90rsk present either in egg extracts (Figure 5B, lane 9) or in lysates prepared from progesterone-matured oocytes (Figure 5B, lane 8) than with the inactive and hypophosphorylated p90rsk present in lysates from G2-arrested oocytes (Figure 5B, lane 7). Figure 5.p90rsk binds to Myt1. (A) Egg extracts (lane 1) and either GST (lane 2) or GST–Myt1 (lane 3) pull-downs of the extracts were analysed by immunoblotting with both anti-p90rsk and anti-Mpk1 MAPK antibodies. (B) GST–Myt1 pull-downs prepared from G2-arrested oocyte lysates (lane 7), progesterone-matured oocyte lysates (lane 8) or egg extracts (lane 9) were analysed by immunoblotting with anti-p90rsk antibodies. The extracts before (lanes 1–3) and after the GST pull-downs (lanes 4–6) were also analysed in the same immunoblot. Download figure Download PowerPoint p90rsk is an unusual protein kinase in that it contains two catalytic (kinase) domains: the N-terminal D1 domain is related to protein kinase A (PKA) and p70 S6K, while the C-terminal D2 domain is related to the γ-subunit of phosphorylase kinase (Jones et al., 1988). To characterize further the association between p90rsk and the C-terminus of Myt1, we expressed both proteins in a yeast two-hybrid system (Figure 6A). We found that Myt1 and p90rsk can also associate in this assay. Moreover, the association between p90rsk and Myt1 was only detected with the full-length p90rsk; neither the D1 nor the D2 kinase domain alone were able to bind to the C-terminus of Myt1. MAPKAP K-2, a kinase that also can be phosphorylated and activated by MAPK in vitro (Stokoe et al., 1992), did not associate with Myt1. These results strongly suggest a direct interaction between p90rsk and the C-terminus of Myt1 in yeast. Interestingly, other p90rsk-interacting proteins such as the p42Mpk1 MAPK can interact in yeast both with the full-length p90rsk and with the D2 kinase domain alone (A.-C.Gavin and A.R.Nebreda, unpublished results). Association between the C-terminus of Myt1 and full-length p90rsk, but not the D1 or the D2 kinase domain, was also detected by expressing these proteins in Xenopus oocytes. In vitro transcribed mRNAs encoding either full-length p90rsk or the D1 and D2 kinase domains were microinjected into oocytes and, after overnight incubation to allow for expression of the proteins, the corresponding oocyte lysates were used for GST–Myt1 pull-downs. The results of this experiment shown in Figure 6B confirm that Myt1 associates more strongly with the active, hyperphosphorylated p90rsk than with the hypophosphorylated form. Figure 6.Myt1 interacts in yeast and in Xenopus oocytes with full-length p90rsk but not the D1 or D2 kinase domains independently. (A) Saccharomyces cerevisiae α cells expressing the Gal4 DNA-binding domain fused in-frame to the C-terminal half of Myt1 were mated with S.cerevisiae a cells expressing the Gal4 activation domain either alone (vector) or fused in-frame to MAPKAP K-2, full-length p90rsk, D1 kinase domain alone or D2 kinase domain alone, as indicated. Positive interactions were selected by plating the cells on medium lacking histidine. The five mating cultures grew to the same extent on medium supplemented with histidine. (B) In vitro transcribed mRNAs encoding myc-tagged full-length p90rsk or either myc-tagged D1 or D2 kinase domains alone were injected into Xenopus oocytes which, after overnight incubation, were either left untreated (lanes 1, 3, 5, 7, 9, 11, 13, 15 and 17) or matured by progesterone stimulation (lanes 2, 4, 6, 8, 10, 12, 14, 16 and 18). Total lysates prepared from these oocytes (lanes 13–18) together with GST pull-downs (lanes 1–6) and GST–Myt1 pull-downs (lanes 7–12) obtained from the same oocyte lysates were analysed by immunoblot with anti-myc antibodies. Download figure Download PowerPoint p90rsk phosphorylates Myt1 We next investigated whether p90rsk can phosphorylate the C-terminus of Myt1. Immunoprecipitates prepared from egg extracts with anti-p90rsk-specific antibodies can phosphorylate exogenously added GST–Myt1 efficiently in vitro (Figure 7A, lane 8). Moreover, the Myt1-phosphorylating kinase activity in the egg extracts can be immunodepleted using p90rsk antibodies (Figure 7A, compare lane 6 with lanes 1 and 2). As expected, GST–Myt1 pull-downs can also phosphorylate exogenously added GST–Myt1 efficiently (Figure 7A, lane 5). In addition, a significant reduction in Myt1-phosphorylating activity in the extract was observed following GST–Myt1 pull-downs (Figure 7A, lane 3). When the same set of samples was analysed by immunoblot with anti-p90rsk antibodies (Figure 7A, lower panel), we found that following GST–Myt1 pull-downs the total amount of p90rsk in the extract was strongly reduced (Figure 7A, compare lanes 2 and 3 in the lower panel). This suggests a strong interaction between the C-terminus of Myt1 and p90rsk, which would allow for the almost quantitative removal of p90rsk from the egg extract. We then analysed Xenopus egg extracts, fractionated using Mono Q chromatography, by immunoblot with anti-p90rsk antibodies, and found that the fractions where p90rsk was detected overlapped with the major peak of kinase activity phosphorylating GST–Myt1 in vitro (data not shown). Figure 7.p90rsk phosphorylates Myt1 on serine residues. (A) Upper panel: in vitro kinase assay using GST–Myt1 as a substrate (except for lane 7 where no substrate was added). Lane 1, egg extracts; lane 2, egg extracts after GST pull-down; lane 3, egg extracts after GST–Myt1 pull-down; lane 4, GST pull-down from egg extracts; lane 5, GST–Myt1 pull-down from egg extracts; lane 6, egg extracts after p90rsk immunoprecipitation; lanes 7 and 8, p90rsk immunoprecipitates from egg extracts incubated alone or in the presence of GST–Myt1, respectively. Lower panel: immunoblot with anti-p90rsk antibodies of the same samples. (B) In vitro kinase assay using p90rsk immunoprecipitates from egg extracts and as a substrate either GST–Cdc25 Nt (lane 1), GST–Wee1 Nt (lane 2), malE–Cdc25 Nt (lane 3), malE–Wee1 Nt (lane 4), GST–Myt1 Ct (lane 5) or GST–Myt1 Nt (lane 6), as indicated. (C) GST–Myt1 was in vitro phosphorylated by p90rsk immunoprecipitates in the presence of [γ-32P]ATP and then subjected to phosphoamino acid analysis. Download figure Download PowerPoint To assess the specificity of p90rsk for the C-terminus of Myt1, we investigated the ability of p90rsk immunoprecipitates to phosphorylate GST fused to the N-terminus of Myt1 (Myt Nt). GST–Myt1 Nt was not phosphorylated by p90rsk in vitro (Figure 7B, lane 6). We also used recombinant proteins containing the N-terminal regulatory regions of either Wee1 or Cdc25C, but neither was phosphorylated significantly by p90rsk in vitro (Figure 7B, lanes 1–4). Phosphoamino acid analysis of in vitro phosphorylated GST–Myt1 showed that phosphorylation by p90rsk only involves serine residues (Figure 7C), consistent with the known substrate specificity of p90rsk (Leighton et al., 1995). To investigate which of the two p90rsk kinase domains is responsible for Myt1 phosphorylation, mRNAs encoding myc-tagged p90rsk proteins mutated in either one or both domains (Figure 8A) were microinjected into Xenopus oocytes. The expressed p90rsk mutant proteins were then recovered by immunoprecipitation (Figure 8B, lower panel) and tested for their ability to phosphorylate GST–Myt1 in vitro (Figure 8B, upper panel). As for the endogenous p90rsk present in oocytes, we found that activation of the myc-tagged p90rsk required that the oocytes are first stimulated by progesterone treatment (Figure 8B, lanes 3 and 4). Moreover, mutation of both p90rsk kinase domains (D1 and D2) totally abolished the activity of p90rsk in progesterone-treated oocytes (Figure 8B, lane 10). However, mutation of the D2 kinase domain alone only slightly reduced the activity of p90rsk on GST–Myt1 (Figure 8B, lane 8), whereas mutation of only D1 totally impaired the ability of p90rsk to phosphorylate GST–Myt1 in vitro (Figure 8B, lanes 6). This is consistent with results published for chicken Rsk2 and rat p90rsk1 which showed that the D1 kinase domain is required for the phosphorylation of exogenous substrates (Leighton et al., 1995; Fisher and Blenis, 1996). These results together with the in-gel kinase assay strongly suggest that the activity phosphorylating Myt1 is p90rsk itself rather than a p90rsk-associated protein kinase. Figure 8.The D1 but not the D2 kinase domain of p90rsk is required for phosphorylation of Myt1. (A) Schematic representation of the lysine to arginine mutations introduced in p90rsk. (B) In vitro transcribed mRNAs encoding the myc-tagged p90rsk mutants were injected into Xenopus oocytes which then were either left untreated (lanes 1, 3, 5, 7 and 9) or matured by progesterone stimulation (lanes 2, 4, 6, 8 and 10). The p90rsk proteins expressed in the oocytes were recovered by immunoprecipitation with anti-myc antibodies and the immunoprecipitates were th
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