Phosphorylated Claspin Interacts with a Phosphate-binding Site in the Kinase Domain of Chk1 during ATR-mediated Activation

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated or UV-damaged DNA. The activated form of Claspin contains two repeated phosphopeptide motifs that mediate its binding to Chk1. We show that these phosphopeptide motifs bind to Chk1 by means of its N-terminal kinase domain. The binding site on Chk1 involves a positively charged cluster of amino acids that contains lysine 54, arginine 129, threonine 153, and arginine 162. Mutagenesis of these residues strongly compromises the ability of Chk1 to interact with Claspin. These amino acids lie within regions of Chk1 that are involved in various aspects of its The on Chk1 of the Claspin to the of in various that the of form a with Claspin in these that Claspin with a site in the of Chk1 activation by Claspin of Chk1 Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated or UV-damaged DNA. The activated form of Claspin contains two repeated phosphopeptide motifs that mediate its binding to Chk1. We show that these phosphopeptide motifs bind to Chk1 by means of its N-terminal kinase domain. The binding site on Chk1 involves a positively charged cluster of amino acids that contains lysine 54, arginine 129, threonine 153, and arginine 162. Mutagenesis of these residues strongly compromises the ability of Chk1 to interact with Claspin. These amino acids lie within regions of Chk1 that are involved in various aspects of its The on Chk1 of the Claspin to the of in various that the of form a with Claspin in these that Claspin with a site in the of Chk1 activation by Claspin of Chk1 to the of the of is the the kinase Chk1 to activation of the a of the Chk1 the and the kinase of on The activation of Chk1 in to in the is on a kinase in the that and Xenopus egg extracts to the of incompletely replicated the Xenopus Chk1 of of and activation in to the activation of The activation of on motifs in its by the Xenopus of and activation of a that to a of Claspin in and of the and Claspin with and the activation of and Claspin is in a of the that Claspin with in a The binding of Claspin that a that the of in the of a in the of Claspin that to interact with Claspin contains a the is and for The contains two repeated motifs that are amino acids in in the of and a of and is for binding of Claspin to and binding is essential for the activation of by the of these that a site that to with Claspin. the of that mediate its with of of with and the the various of that are in the of by with and the of by with the containing or These the The and of The and of by the and The and of with the with the in and of containing amino acids and the by the for the a of amino acids of contains the by and that with and in with and with containing for of Claspin to and of and various to to of Xenopus egg containing and of of and The extracts for the the by and with and and various in egg extracts the the the extracts with of and a The with of for The and with to Claspin and with the in for the of with a with and of with the N-terminal of that Claspin to a form of in Xenopus egg extracts containing of and and a that is to We to of is for binding to Claspin. various of and We that containing amino acids and of binding to Claspin in with containing amino acids to Claspin the and of the to the kinase in that the of to Claspin various a that a bind to Claspin these These that Claspin with the kinase of of or of the of the binding of to the in the kinase that with Claspin and a of and N-terminal of the kinase these to of the kinase that interact with Claspin and These that and kinase to with of Claspin with the of the the binding of Claspin to the kinase of involves the that for with We a containing the Claspin bind to the kinase of a that contains the and residues of Claspin. We the in egg extracts containing or We that to the with that with for binding to Claspin. a interact with in We the binding of Claspin to the kinase of on and of Claspin. of Claspin or the contains and in egg extracts with or in the of in and the to Claspin to the binding of the of to form of Claspin. these that the kinase of to the of Claspin in a that on and of Claspin to the kinase of involves the and of Claspin on and and in egg extracts containing and the of in the of and The and the for with Claspin and and the of and egg of Claspin and in egg extracts containing and and and and in the of and The and binding of the Claspin and with a and the kinase of a with Claspin with the kinase to positively charged residues lysine and on the of that form a site for the motifs and The of residues Chk1 to of is that contains a the of Chk1. are in and with in the of the in a positively charged cluster of amino acids that contains and of in the kinase of on binding to of the kinase and and of are with and various of with the in its kinase in egg extracts in the and or and of The the for with Claspin and of the the various for kinase a The to and with a of the site in of residues Chk1. The for and are in are The of the a of Claspin is to that of the in the of the of and and with a are in these residues interact with a these amino acids in regions of Chk1 that are for various aspects of in a of is a in the of The amino in the kinase is the that a the of the kinase and lie in a of Chk1 that to the activation in various the of and for a that to the in the in the of Chk1. The in the of is a with and to and the and of the of the in the of Chk1 to the of the in and Chk1 to of on its and to in the We that of the or with Claspin the of binding is a positively charged that to the We that the of to Claspin. We the Claspin binding of a of in to in the of that the with these of with in the site and activation the of on in the and of is a site on for by We the kinase of a the of the and that the of on The kinase in the and of The a kinase to that of the in to in in to the of the on activation of the in the of the on in in the of for the to on and kinase binding of the and to these of with Claspin. binding to of by these that residues in the site of and are for the activation of of Claspin for Claspin to the N-terminal kinase of the of to in the with Claspin. of or of the binding to Claspin the that the of a binding site for Claspin or to the site in the N-terminal kinase domain. to binding of Claspin to the of the of the of in the and of Claspin with that a of containing its acids in the of and in extracts Claspin by the of the by the of to the The of the in extracts the the of the by with these that Claspin with the of of Claspin for its of Claspin. extracts with or and to the a of with the extracts in in the or of The extracts of the of a the of by and the of the Claspin and in the binding of Claspin to the form of of the of binding Claspin and in egg extracts containing and in the binding of Claspin to these the form of that the form of the activated form of of Claspin and in in egg extracts in the or of the the and binding of Claspin by with with Claspin strongly with the form of egg extracts with or and for of a containing and in to the to its in the or of the and to its a with The to and by with and egg the of bind to Claspin. and various of and in egg extracts containing and The and binding of Claspin by with with that of and the for means to egg extracts with and to the of Claspin and and to these extracts in the of the form of in extracts to the Claspin that to the of in the of binding of to Claspin. These are with the that to Claspin and a of and that in the in the of in a in kinase to in egg extracts that a the that of with a the ability of the of the of a for and the The the We that binding of the to Claspin in with the of to the with Claspin. a in the of that its kinase in the of a binding to Claspin. of binding of its motifs by of the that Claspin a for activated for the of that to interact with Claspin a that the N-terminal kinase of interact with Claspin in various of residues in the kinase and that are involved in the with Claspin. these residues a site in the of Chk1 the that these amino acids involved in a in the a Claspin on two and that in the of two repeated in the of Claspin. of and of the is for binding of Claspin to the kinase of These that the phosphopeptide motifs in the of Claspin interact with the in that the of a in by the activated form of Claspin that on and with by means of the site in its kinase of The of two motifs in Claspin two of to with of Claspin. the of the kinase Chk1 that the the motifs of Claspin the binding of two to a Claspin that two of bind to Claspin are that a binding site for the motifs or that two motifs Claspin to interact with a binding site in the for the of with Claspin. on its two or of of on its motifs by and to the activation of The form of Claspin to and is for the activation of a is that Claspin to The of the motifs of Claspin the kinase of and the of by two bind to of these of that Claspin. in the form of to Claspin the activated of of activated Claspin to for of in that the form with the of Claspin and with the a site in its kinase to with that a for the on a with by means of and in the kinase domain. The of these residues to of and in and a in kinase to a the two is that Claspin is and a of to in of Claspin by and is Claspin with its is that the kinase of Chk1 with a of residues that are for The of the Chk1 with and a that a the of the kinase domain. The for the is in Claspin is that residues in to the in interact with of in the of the is that the of that its Claspin and that Claspin with by means of a site in its kinase domain. is for the activation of a to the of the of is the the kinase Chk1 to activation of the a of the Chk1 the and the kinase of on The activation of Chk1 in to in the is on a kinase in the that and We Xenopus egg extracts to the of incompletely replicated the Xenopus Chk1 of of and activation in to the activation of The activation of on motifs in its by the Xenopus of Xenopus Chk1 Xenopus of kinase Xenopus Chk1 Xenopus of kinase The and activation of a that to a of Claspin in and of the and Claspin with and the activation of and Claspin is in a of the that Claspin with in a The binding of Claspin that a that the of in the of a in the of Claspin that to interact with Claspin contains a the is and for The contains two repeated motifs that are amino acids in in the of and a of and is for binding of Claspin to and binding is essential for the activation of by the of these that a site that to with Claspin. the of that mediate its with Claspin. of of with and the the various of that are in the of by with and the of by with the containing or These the The and of The and of by the and The and of with the with the in and of containing amino acids and the by the for the a of amino acids of contains the by and that with and in with and with containing for of Claspin to and of and various to to of Xenopus egg containing and of of and The extracts for the the by and with and and various in egg extracts the the the extracts with of and a The with of for The and with to Claspin and with the in for the of with a with and of of of with and the the various of that are in the of by with and the of by with the containing or These the The and of The and of by the and The and of with the with the in and of containing amino acids and the by the for the a of amino acids of contains the by and that with and in with and with containing for of Claspin to and of and various to to of Xenopus egg containing and of of and The extracts for the the by and with and and various in egg extracts the the the extracts with of and a The with of for The and with to Claspin and with the in for the of with a with and of with the N-terminal of that Claspin to a form of in Xenopus egg extracts containing of and and a that is to We to of is for binding to Claspin. various of and We that containing amino acids and of binding to Claspin in with containing amino acids to Claspin the and of the to the kinase in that the of to Claspin various a that a bind to Claspin these These that Claspin with the kinase of of or of the of the binding of to the in the kinase that with Claspin and a of and N-terminal of the kinase these to of the kinase that interact with Claspin and These that and kinase to with of Claspin with the of the the binding of Claspin to the kinase of involves the that for with We a containing the Claspin bind to the kinase of a that contains the and residues of Claspin. We the in egg extracts containing or We that to the with that with for binding to Claspin. a interact with in We the binding of Claspin to the kinase of on and of Claspin. of Claspin or the contains and in egg extracts with or in the of in and the to Claspin to the binding of the of to form of Claspin. these that the kinase of to the of Claspin in a that on and of a with Claspin with the kinase to positively charged residues lysine and on the of that form a site for the motifs and The of residues Chk1 to of is that contains a the of Chk1. are in and with in the of the in a positively charged cluster of amino acids that contains and of in the kinase of on binding to of the kinase and and of are with and various of with the in its kinase in egg extracts in the and or and of The the for with Claspin and of the the various for kinase a The to and with a of the site in of residues Chk1. The for and are in are The of the a of Claspin is to that of the in the of the of and and with a are in these residues interact with a these amino acids in regions of Chk1 that are for various aspects of in a of is a in the of The amino in the kinase is the that a the of the kinase and lie in a of Chk1 that to the activation in various the of and for a that to the in the in the of Chk1. The in the of is a with and to and the and of the of the in the of Chk1 to the of the in and Chk1 to of on its and to in the We that of the or with Claspin the of binding is a positively charged that to the We that the of to Claspin. We the Claspin binding of a of in to in the of that the with these of with in the site and activation the of on in the and of is a site on for by We the kinase of a the of the and that the of on The kinase in the and of The a kinase to that of the in to in in to the of the on activation of the in the of the on in in the of for the to on and kinase binding of the and to these of with Claspin. binding to of by these that residues in the site of and are for the activation of of Claspin for Claspin to the N-terminal kinase of the of to in the with Claspin. of or of the binding to Claspin the that the of a binding site for Claspin or to the site in the N-terminal kinase domain. to binding of Claspin to the of the of the of in the and of Claspin with that a of containing its acids in the of and in extracts Claspin by the of the by the of to the The of the in extracts the the of the by with these that Claspin with the of of Claspin for its of Claspin. extracts with or and to the a of with the extracts in in the or of The extracts of the of a the of by and the of the Claspin and in the binding of Claspin to the form of of the of binding Claspin and in egg extracts containing and in the binding of Claspin to these the form of that the form of the activated form of of Claspin and in in egg extracts in the or of the the and binding of Claspin by with with Claspin strongly with the form of egg extracts with or and for of a containing and in to the to its in the or of the and to its a with The to and by with and egg the of bind to Claspin. and various of and in egg extracts containing and The and binding of Claspin by with with that of and the for means to egg extracts with and to the of Claspin and and to these extracts in the of the form of in extracts to the Claspin that to the of in the of binding of to Claspin. These are with the that to Claspin and a of and that in the in the of in a in kinase to in egg extracts that a the that of with a the ability of the of the of a for and the The the We that binding of the to Claspin in with the of to the with Claspin. a in the of that its kinase in the of a binding to Claspin. of binding of its motifs by of the that Claspin a for activated for Claspin with the N-terminal of that Claspin to a form of in Xenopus egg extracts containing of and and a that is to We to of is for binding to Claspin. various of and We that containing amino acids and of binding to Claspin in with containing amino acids to Claspin the and of the to the kinase in that the of to Claspin various a that a bind to Claspin these These that Claspin with the kinase of of or of the of the binding of Claspin. to the in the kinase that with Claspin and a of and N-terminal of the kinase these to of the kinase that interact with Claspin and These that and kinase to with Claspin. of Claspin with the of the the binding of Claspin to the kinase of involves the that for with We a containing the Claspin bind to the kinase of a that contains the and residues of Claspin. We the in egg extracts containing or We that to the with that with for binding to Claspin. a interact with in We the binding of Claspin to the kinase of on and of Claspin. of Claspin or the contains and in egg extracts with or in the of in and the to Claspin to the binding of the of to form of Claspin. these that the kinase of to the of Claspin in a that on and The of a with Claspin with the kinase to positively charged residues lysine and on the of that form a site for the motifs and The of residues Chk1 to of is that contains a the of Chk1. are in and with in the of the in a positively charged cluster of amino acids that contains and these residues interact with a these amino acids in regions of Chk1 that are for various aspects of in a of is a in the of The amino in the kinase is the that a the of the kinase and lie in a of Chk1 that to the activation in various the of and for a that to the in the in the of Chk1. The in the of is a with and to and the and of the of the in the of Chk1 to the of the in and Chk1 to of on its We and to in the We that of the or with Claspin the of binding is a positively charged that to the We that the of to Claspin. We the Claspin binding of a of in to in the of that the with Claspin. these of with in the site and activation the of on in the and of is a site on for by We the kinase of a the of the and that the of on The kinase in the and of The a kinase to that of the in to in in to the of the on activation of the in the of the on in in the of for the to on and kinase binding of the and to these of with Claspin. binding to of by these that residues in the site of and are for the activation of The of Claspin for Claspin to the N-terminal kinase of the of to in the with Claspin. of or of the binding to Claspin the that the of a binding site for Claspin or to the site in the N-terminal kinase domain. to binding of Claspin to the of the of the of in the and of Claspin with that a of containing its acids in the of and in extracts Claspin by the of the by the of to the The of the in extracts the the of the by with these that Claspin with the of Claspin the of the Claspin and in the binding of Claspin to the form of of the of binding Claspin and in egg extracts containing and in the binding of Claspin to these the form of that the form of Claspin. means to egg extracts with and to the of Claspin and and to these extracts in the of the form of in extracts to the Claspin that to the of in the of binding of to Claspin. These are with the that to Claspin and a of and that in the in the of in a in kinase to in egg extracts that a the that of with a the ability of the of the of a for and the The the We that binding of the to Claspin in with the of to the with Claspin. a in the of that its kinase in the of a binding to Claspin. of binding of its motifs by of the that Claspin a for activated for the of that to interact with Claspin a that the N-terminal kinase of interact with Claspin in various of residues in the kinase and that are involved in the with Claspin. these residues a site in the of Chk1 the that these amino acids involved in a in the a Claspin on two and that in the of two repeated in the of Claspin. of and of the is for binding of Claspin to the kinase of These that the phosphopeptide motifs in the of Claspin interact with the in that the of a in by the activated form of Claspin that on and with by means of the site in its kinase of The of two motifs in Claspin two of to with of Claspin. the of the kinase Chk1 that the the motifs of Claspin the binding of two to a Claspin that two of bind to Claspin are that a binding site for the motifs or that two motifs Claspin to interact with a binding site in the Claspin to and is for the activation of a is that Claspin to The of the motifs of Claspin the kinase of and the of by two bind to of these of that Claspin. in the form of to Claspin the activated of of activated Claspin to for of in that the form with the of Claspin and with the a site in its kinase to with that a for the on a with by means of and in the kinase domain. The of these residues to of and in and a in kinase to a the two is that Claspin is and a of to in of Claspin by and is Claspin with its is that the kinase of Chk1 with a of residues that are for The of the Chk1 with and a that a the of the kinase domain. The for the is in Claspin is that residues in to the in interact with of in the of the is that the of that its Claspin and that Claspin with by means of a site in its kinase domain. is for the activation of a the of that to interact with Claspin a that the N-terminal kinase of interact with Claspin in various of residues in the kinase and that are involved in the with Claspin. these residues a site in the of Chk1 the that these amino acids involved in a in the a Claspin on two and that in the of two repeated in the of Claspin. of and of the is for binding of Claspin to the kinase of These that the phosphopeptide motifs in the of Claspin interact with the in that the of a in by Claspin. the activated form of Claspin that on and with by means of the site in its kinase of The of two motifs in Claspin two of to with of Claspin. the of the kinase Chk1 that the the motifs of Claspin the binding of two to a Claspin that two of bind to Claspin are that a binding site for the motifs or that two motifs Claspin to interact with a binding site in the Claspin to and is for the activation of a is that Claspin to The of the motifs of Claspin the kinase of and the of by two bind to of these of that Claspin. in the form of to Claspin the activated of of activated Claspin to for of in that the form with the of The Claspin and with the a site in its kinase to with that a for the on a with by means of and in the kinase domain. The of these residues to of and in and a in kinase to a the two is that Claspin is and a of to in of Claspin by and is Claspin with its is that the kinase of Chk1 with a of residues that are for The of the Chk1 with and a that a the of the kinase domain. The for the is in Claspin is that residues in to the in interact with of in the of the is that the of that its Claspin and that Claspin with by means of a site in its kinase domain. is for the activation of a We are to for with the of Chk1 in We the of for on the