Thr-161 Phosphorylation of Monomeric Cdc2

Abstract

Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B·Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B·Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. M-phase promoting factor cyclin-dependent kinase CDK-activating kinase S. cerevisiae CDK-activating kinase 1 germinal vesicle breakdown protein phosphatase 2C dithiothreitol endoplasmic reticulum glutathioneS-transferase reduced protein kinase phosphatase The grown Xenopus oocyte is arrested at the of contains a store of Cdc2 or The of the protein is present as a monomer in the of the whereas a minor as by is found to be associated with and The cyclin B·Cdc2 is by two on and of Cdc2 by the kinase S. S. phosphorylation of Cdc2, on the Thr-161 residue located in the of the is to be required for Cdc2 kinase activation The cyclin B·Cdc2 present in grown as contains a Cdc2 on and Thr-161 be the and the of Cdc2 phosphorylation on Thr-161 and the on cyclin with Cdc2 in the the is to the a of a CDK-activating kinase of cyclin and the factor a to be located the Xenopus oocyte S. a for for monomeric To activation of Cdc2, the to be by on and of Cdc2 by the kinase to as in the Cdc2 is a of the is a monomeric called or to the monomeric a activity in S. an is monomeric Cdc2 be phosphorylated on Thr-161 in the its with cyclin in the of the to its by the to the activation of into MPF an the protein phosphatase the on and of Cdc2 as protein phosphatase and Cdc2 to is the and Cdc2 is could be that an or a phosphatase, as is and as a threshold for the in is upon a or Cdc2 with free the by kinase and as a threshold to initiate MPF the presence of monomeric Cdc2 already phosphorylated on Thr-161 the of amount of phosphorylation of Cdc2 on Thr-161 a key regulation at two of the oocyte in prophase-arrested and at the time is is to the kinase and phosphatase, that a first to the Thr-161 phosphorylation of Cdc2 is we to a of cyclin Cdc2 and its Thr-161 phosphorylation The of that grown oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161, phosphorylation is negatively by a Mg2+-dependent phosphatase, and was from from grown Xenopus prophase as in of 1 or 1 with and at and the and or of by and as in a and at for and from in of or to and on a at of 1 and to and kinase and phosphatase on and to and cyclin and cyclin from with Xenopus and and The Cdc2 of and in a The and S. and and from and antibody and from The with and the from Activation and to and in kinase 1 and for at in kinase in the presence of and or kinase and 1 of for a or at The was by and by for was from and by with an The amount of in was by oocyte to and of in and Activation of Cdc2 present in or was the as for Cdc2, by or by binding for at on in or or in kinase the to or kinase of was by the protein for 1 at in the presence of in kinase and 1 of was on a to free or on for at was in the presence of of and in and at for and on The in was by and from the in a for was phosphorylated by of the of for at in the presence of and of was phosphorylated by for at in the presence of and of by addition of and at with an of by on or was for at in the presence of of Xenopus PP2C, or from the in the presence of and of and by addition of of for and the was activity was phosphorylated by and phosphorylated by as The a phosphatase activity that is on and to 1 The was at from in of the 1 1 The was at for and the was and at for to a and of was in the was in as by The was in 1 of 1 1 and with of in by in and 1 The fraction, in was on and on a in The was on and on a in with a from to 1 in activity was was to in the fraction, was on a in with a from to in and the phosphatase activity was at The was on with with and on a with with a from to in and the was at by against and the was at on the and of two of and to the protein phosphatase two to the of the protein and as and to the of the protein and as was with a of and and a of The was in the and the on with a and The at the the of and and as in a the of Xenopus Xenopus and Xenopus Thr-161 of cerevisiae protein and protein by and B. Xenopus is and a was by 1 of or Thr-161 in of prophase oocyte as for at was isolated was into the of was with The was in and The was on a and the was on a was with from to 1 of and in and at or Cdc2 protein kinase Xenopus oocyte is by the of cyclin and The activating phosphorylation on Thr-161 the is required for kinase activity and be at of the of oocyte at the into I MPF is first and the cyclin the addition of a to Thr-161 of Cdc2 or cyclin binding at of show that a significant of monomeric Cdc2 in a from prophase is in by cyclin that free Cdc2 is present in the the presence of phosphorylated of Cdc2 by an antibody specifically directed against Cdc2. antibody is to monomeric Cdc2, in the of To the of the or Cdc2 phosphorylated in on or Thr-161, by in and the detection of the phosphorylated activating of the of and Cdc2 by two is to that monomeric Cdc2 is present in the oocyte a of Cdc2 by cyclin that Xenopus oocyte contains the that the addition and of on residue Thr-161 of monomeric Cdc2. in is required for the of phosphorylated monomeric Cdc2, to be by cyclin to for the presence of a Mg2+-dependent protein phosphatase of Cdc2 in the Xenopus to the of a Mg2+-dependent phosphatase, is by an antibody against and the of was to as as Xenopus Cdc2. the Xenopus a Mg2+-dependent phosphatase activity to the Thr-161 residue of monomeric Cdc2. to Cdc2, is to Thr-161 that is a a and that an from Xenopus contains a activity that monomeric phosphatase activity present in phosphatase could be found in from prophase in the presence of to be could be that a phosphatase or in prophase is or of a phosphatase with monomeric Cdc2 Cdc2 in Xenopus oocytes. the to or MPF contains a activity of Mg2+-dependent and to of and and and in S. cerevisiae as the two 2C that monomeric the of monomeric in two and that monomeric in new by the that phosphorylated monomeric Cdc2 isolated oocyte could be by a monomeric and that a phosphatase 2C the of on of is at present the Xenopus oocyte contains an that the phosphorylation of Thr-161 in monomeric Cdc2. of monomeric Cdc2 phosphorylated on Thr-161 with the of for monomeric the that an be present and in the for the of kinase is to or to the Mg2+-dependent phosphatase activity that to kinase show for the first time that monomeric Cdc2 be isolated from and that is a of an the Thr-161 kinase PP2C, the presence of two monomeric Cdc2 in the phosphorylated on Thr-161 and by cyclin binding the is is the of phosphorylated monomeric Cdc2 in the of the MPF in cyclin be to with and to Cdc2 already phosphorylated on Thr-161 and to a threshold Cdc2 kinase activity to MPF an that the of and is required in for the of MPF oocyte be that cyclin or an of monomeric Cdc2 could be in the on of its kinase be to the of monomeric Cdc2 and to the phosphatase 2C and its kinase to regulation the The grown Xenopus oocyte is arrested at the of contains a store of Cdc2 or The of the protein is present as a monomer in the of the whereas a minor as by is found to be associated with and The cyclin B·Cdc2 is by two on and of Cdc2 by the kinase S. S. phosphorylation of Cdc2, on the Thr-161 residue located in the of the is to be required for Cdc2 kinase activation The cyclin B·Cdc2 present in grown as contains a Cdc2 on and Thr-161 be the and the of Cdc2 phosphorylation on Thr-161 and the on cyclin with Cdc2 in the the is to the a of a CDK-activating kinase of cyclin and the factor a to be located the Xenopus oocyte S. a for for monomeric To activation of Cdc2, the to be by on and of Cdc2 by the kinase to as in the Cdc2 is a of the is a monomeric called or to the monomeric a activity in S. an is monomeric Cdc2 be phosphorylated on Thr-161 in the its with cyclin in the of the to its by the to the activation of into MPF an the protein phosphatase the on and of Cdc2 as protein phosphatase and Cdc2 to is the and Cdc2 is could be that an or a phosphatase, as is and as a threshold for the in is upon a or Cdc2 with free the by kinase and as a threshold to initiate MPF the presence of monomeric Cdc2 already phosphorylated on Thr-161 the of amount of The phosphorylation of Cdc2 on Thr-161 a key regulation at two of the oocyte in prophase-arrested and at the time is is to the kinase and phosphatase, that a first to the Thr-161 phosphorylation of Cdc2 is we to a of cyclin Cdc2 and its Thr-161 phosphorylation The of that grown oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161, phosphorylation is negatively by a Mg2+-dependent phosphatase, and was from from grown Xenopus prophase as in of 1 or 1 with and at and the and or of by and as in a and at for and from in of or to and on a at of 1 and to and kinase and phosphatase on and to and cyclin and cyclin from with Xenopus and and The Cdc2 of and in a The and S. and and from and antibody and from The with and the from Activation and to and in kinase 1 and for at in kinase in the presence of and or kinase and 1 of for a or at The was by and by for was from and by with an The amount of in was by oocyte to and of in and Activation of Cdc2 present in or was the as for Cdc2, by or by binding for at on in or or in kinase the to or kinase of was by the protein for 1 at in the presence of in kinase and 1 of was on a to free or on for at was in the presence of of and in and at for and on The in was by and from the in a for was phosphorylated by of the of for at in the presence of and of was phosphorylated by for at in the presence of and of by addition of and at with an of by on or was for at in the presence of of Xenopus PP2C, or from the in the presence of and of and by addition of of for and the was activity was phosphorylated by and phosphorylated by as The a phosphatase activity that is on and to 1 The was at from in of the 1 1 The was at for and the was and at for to a and of was in the was in as by The was in 1 of 1 1 and with of in by in and 1 The fraction, in was on and on a in The was on and on a in with a from to 1 in activity was was to in the fraction, was on a in with a from to in and the phosphatase activity was at The was on with with and on a with with a from to in and the was at by against and the was at on the and of two of and to the protein phosphatase two to the of the protein and as and to the of the protein and as was with a of and and a of The was in the and the on with a and The at the the of and and as in a the of Xenopus Xenopus and Xenopus Thr-161 of cerevisiae protein and protein by and B. 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by and by for was from and by with an The amount of in was by oocyte to and of in and Activation of Cdc2 present in or was the as for Cdc2, by or by binding for at on in or or in kinase the to or kinase of was by the protein for 1 at in the presence of in kinase and 1 of was on a to free or on for at was in the presence of of and in and at for and on The in was by and from the in a to and in kinase 1 and for at in kinase in the presence of and or kinase and 1 of for a or at The was by and by for was from and by with an The amount of in was by oocyte to and of in and Activation of Cdc2 present in or was the as for Cdc2, by or by binding for at on in or or in kinase the to or kinase of was by the protein for 1 at in the presence of in kinase and 1 of was on a to free or on for at was in the presence of of and in and at for and on The in was by and from the in a to and in kinase 1 and for at in kinase in the presence of and or kinase and 1 of for a or at The was by and by for to and in kinase 1 and for at in kinase in the presence of and or kinase and 1 of for a or at The was by and by for was from and by with an The amount of in was by oocyte to and of in and Activation of Cdc2 present in or was the as for Cdc2, by or by binding for at on in or or in kinase the to or kinase was from and by with an The amount of in was by oocyte to and of in and Activation of Cdc2 present in or was the as for Cdc2, by or by binding for at on in or or in kinase the to or kinase of was by the protein for 1 at in the presence of in kinase and 1 of was on a to free or on for at was in the presence of of and in and at for and on The in was by and from the in a of was by the protein for 1 at in the presence of in kinase and 1 of was on a to free or on for at was in the presence of of and in and at for and on The in was by and from the in a for was phosphorylated by of the of for at in the presence of and of was phosphorylated by for at in the presence of and of by addition of and at with an of by on or was for at in the presence of of Xenopus PP2C, or from the in the presence of and of and by addition of of for and the was for was phosphorylated by of the of for at in the presence of and of was phosphorylated by for at in the presence of and of by addition of and at with an of by on or was for at in the presence of of Xenopus PP2C, or from the in the presence of and of and by addition of of for and the was for was phosphorylated by of the of for at in the presence of and of was phosphorylated by for at in the presence of and of by addition of and at with an of by on was phosphorylated by of the of for at in the presence of and of was phosphorylated by for at in the presence of and of by addition of and at with an of by on or was for at in the presence of of Xenopus PP2C, or from the in the presence of and of and by addition of of for and the was or was for at in the presence of of Xenopus PP2C, or from the in the presence of and of and by addition of of for and the was Xenopus activity was phosphorylated by and phosphorylated by as The a phosphatase activity that is on and to 1 The was at from in of the 1 1 The was at for and the was and at for to a and of was in the was in as by The was in 1 of 1 1 and with of in by in and 1 The fraction, in was on and on a in The was on and on a in with a from to 1 in activity was was to in the fraction, was on a in with a from to in and the phosphatase activity was at The was on with with and on a with with a from to in and the was at by against and the was at activity was phosphorylated by and phosphorylated by as The a phosphatase activity that is on and to 1 The was at from in of the 1 1 The was at for and the was and at for to a and of was in the was in as by The was in 1 of 1 1 and with of in by in and 1 The fraction, in was on and on a in The was on and on a in with a from to 1 in activity was was to in the fraction, was on a in with a from to in and the phosphatase activity was at The was on with with and on a with with a from to in and the was at by against and the was at Xenopus on the and of two of and to the protein phosphatase two to the of the protein and as and to the of the protein and as was with a of and and a of The was in the and the on with a and The at the the Based on the and of two of and to the protein phosphatase two to the of the protein and as and to the of the protein and as was with a of and and a of The was in the and the on with a and The at the the of and and as in a the of Xenopus Xenopus and Xenopus Thr-161 of cerevisiae protein and protein by and B. Xenopus is and a was by 1 of or Thr-161 in of prophase oocyte as for at was isolated was into the of was with The was in and The was on a and the was on a was with from to 1 of and in and at and and as in a the of Xenopus Xenopus and Xenopus Thr-161 of cerevisiae protein and protein by and B. Xenopus is and a was by 1 of or Thr-161 in of prophase oocyte as for at was isolated Xenopus was into the of was with The was in and The was on a and the was on a was with from to 1 of and in and at or Cdc2 protein kinase Xenopus oocyte is by the of cyclin and The activating phosphorylation on Thr-161 the is required for kinase activity and be at of the of oocyte at the into I MPF is first and the cyclin the addition of a to Thr-161 of Cdc2 or cyclin binding at of show that a significant of monomeric Cdc2 in a from prophase is in by cyclin that free Cdc2 is present in the the presence of phosphorylated of Cdc2 by an antibody specifically directed against Cdc2. antibody is to monomeric Cdc2, in the of To the of the or Cdc2 phosphorylated in on or Thr-161, by in and the detection of the phosphorylated activating of the of and Cdc2 by two is to that monomeric Cdc2 is present in the oocyte a of Cdc2 by cyclin that Xenopus oocyte contains the that the addition and of on residue Thr-161 of monomeric Cdc2. in is required for the of phosphorylated monomeric Cdc2, to be by cyclin to for the presence of a Mg2+-dependent protein phosphatase of Cdc2 in the Xenopus to the of a Mg2+-dependent phosphatase, is by an antibody against and the of was to as as Xenopus Cdc2. the Xenopus a Mg2+-dependent phosphatase activity to the Thr-161 residue of monomeric Cdc2. to Cdc2, is to Thr-161 that is a a and that an from Xenopus contains a activity that monomeric phosphatase activity present in phosphatase could be found in from prophase in the presence of to be could be that a phosphatase or in prophase is or of a phosphatase with monomeric Cdc2 Cdc2 in Xenopus oocytes. the to or MPF contains a activity of Mg2+-dependent and to of and and and in S. cerevisiae as the two 2C that monomeric the of monomeric in two and that monomeric in new by the that phosphorylated monomeric Cdc2 isolated oocyte could be by a monomeric and that a phosphatase 2C the of on of is at present the Xenopus oocyte contains an that the phosphorylation of Thr-161 in monomeric Cdc2. of monomeric Cdc2 phosphorylated on Thr-161 with the of for monomeric the that an be present and in the for the of kinase is to or to the Mg2+-dependent phosphatase activity that to kinase show for the first time that monomeric Cdc2 be isolated from and that is a of an the Thr-161 kinase PP2C, the presence of two monomeric Cdc2 in the phosphorylated on Thr-161 and by cyclin binding the is is the of phosphorylated monomeric Cdc2 in the of the MPF in cyclin be to with and to Cdc2 already phosphorylated on Thr-161 and to a threshold Cdc2 kinase activity to MPF an that the of and is required in for the of MPF oocyte be that cyclin or an of monomeric Cdc2 could be in the on of its kinase be to the of monomeric Cdc2 and to the phosphatase 2C and its kinase to regulation the MPF or Cdc2 protein kinase Xenopus oocyte is by the of cyclin and The activating phosphorylation on Thr-161 the is required for kinase activity and be at of the of oocyte at the into I MPF is first and the cyclin the addition of a to Thr-161 of Cdc2 or cyclin binding at of show that a significant of monomeric Cdc2 in a from prophase is in by cyclin that free Cdc2 is present in the the presence of phosphorylated of Cdc2 by an antibody specifically directed against Cdc2. antibody is to monomeric Cdc2, in the of To the of the or Cdc2 phosphorylated in on or Thr-161, by in and the detection of the phosphorylated activating of the of and Cdc2 by two is to that monomeric Cdc2 is present in the oocyte a of Cdc2 by cyclin that Xenopus oocyte contains the that the addition and of on residue Thr-161 of monomeric Cdc2. in is required for the of phosphorylated monomeric Cdc2, to be by cyclin to for the presence of a Mg2+-dependent protein phosphatase of Cdc2 in the Xenopus to the of a Mg2+-dependent phosphatase, is by an antibody against and the of was to as as Xenopus Cdc2. the Xenopus a Mg2+-dependent phosphatase activity to the Thr-161 residue of monomeric Cdc2. to Cdc2, is to Thr-161 that is a a and that an from Xenopus contains a activity that monomeric phosphatase activity present in phosphatase could be found in from prophase in the presence of to be could be that a phosphatase or in prophase is or The of a phosphatase with monomeric Cdc2 Cdc2 in Xenopus oocytes. the to or MPF contains a activity of Mg2+-dependent and to of Cdc2. and and and in S. cerevisiae as the two 2C that monomeric the of monomeric in two and that monomeric in new by the that phosphorylated monomeric Cdc2 isolated oocyte could be by a monomeric and that a phosphatase 2C the of on of is at present the Xenopus oocyte contains an that the phosphorylation of Thr-161 in monomeric Cdc2. of monomeric Cdc2 phosphorylated on Thr-161 with the of for monomeric the that an be present and in the for the of kinase is to or to the Mg2+-dependent phosphatase activity that to kinase show for the first time that monomeric Cdc2 be isolated from and that is a of an the Thr-161 kinase PP2C, the presence of two monomeric Cdc2 in the phosphorylated on Thr-161 and by cyclin binding the is is the of phosphorylated monomeric Cdc2 in the of the MPF in cyclin be to with and to Cdc2 already phosphorylated on Thr-161 and to a threshold Cdc2 kinase activity to MPF an that the of and is required in for the of MPF oocyte be that cyclin or an of monomeric Cdc2 could be in the on of its kinase be to the of monomeric Cdc2 and to the phosphatase 2C and its kinase to regulation the the of the for and to for and Cdc2 and to for cyclin to for to B. for and to for