Activation of Cyclin-dependent Kinase 2 by Full Length and Low Molecular Weight Forms of Cyclin E in Breast Cancer Cells

Abstract

Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G1 to S phase. Deregulation of the G1-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E·CDK2 complexes leading to G1/S deregulation and tumor progression. Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G1 to S phase. Deregulation of the G1-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E·CDK2 complexes leading to G1/S deregulation and tumor progression. Cyclin E has an important role in the regulation of the restriction point transition in the cell cycle. In normal cells, cyclin E expression occurs during a brief window of time from late G1 into early S phase, with a peak expression level near the restriction point (1Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (655) Google Scholar). Kinase activity of cyclin E·CDK2 1The abbreviations used are: CDK, cyclin-dependent kinase; CAK, cyclin-activating kinase; KAP, CDK-associated phosphatase; LMW, low molecular weight; ID, immunodepleted. 1The abbreviations used are: CDK, cyclin-dependent kinase; CAK, cyclin-activating kinase; KAP, CDK-associated phosphatase; LMW, low molecular weight; ID, immunodepleted. complexes are also at maximum levels near the restriction point (2Koff A. Giordano A. Desia D. Yamashita K. Harper J.W. Elledge S.J. Nishomoto T. Morgan D.O. Franza R. Roberts J.M. Science. 1992; 257: 1689-1694Crossref PubMed Scopus (908) Google Scholar, 3Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1841) Google Scholar). Cyclin E activation of CDK2 is an essential step in cell through the restriction are that the of cyclin E·CDK2 complexes in transition from G1 to S phase. For of cyclin E by of E into cells cell in G1 of the cell Roberts J.M. Cell. PubMed Scopus Google Scholar, K. Google Scholar). the overexpression of cyclin E in a in the of along with a cell K. Google Scholar, D. Reed S.I. Cell. PubMed Scopus Google Scholar). expression of a which is in ability to in or cells, G1 Science. PubMed Scopus Google Scholar). Cyclin E binding is not the only step for CDK2 The activity of CDK2 is also phosphorylation at the Morgan D.O. 1992; PubMed Scopus Google of the CDK2 kinase activity in as a of activating phosphorylation Morgan D.O. 1992; PubMed Scopus Google Scholar). of the or kinase activity, that are the of phosphorylation Morgan D.O. 1992; PubMed Scopus Google Scholar). at is by cyclin-activating kinase a complex of cyclin and Cell. PubMed Scopus Google Scholar). was as a present in cell extracts that activate in vitro D. Morgan D.O. Cell. 1992; PubMed Scopus Google Scholar, T. Cell. 1992; PubMed Scopus Google Scholar). of the complex to the of cyclin and Harper J.W. PubMed Scopus Google Scholar, Morgan D.O. Cell. Full Text PDF PubMed Scopus Google Scholar). is the of CAK, which Harper J.W. PubMed Scopus Google Scholar, Yamashita K. T. PubMed Scopus Google Scholar). not activity binding with cyclin to Roberts J.M. Cell. PubMed Scopus Google Scholar, Morgan D.O. Cell. Full Text PDF PubMed Scopus Google Scholar, Yamashita K. T. PubMed Scopus Google Scholar). In vitro cyclin complexes were shown to both CDK2 and Morgan D.O. Cell. Full Text PDF PubMed Scopus Google Scholar). regulation of phosphorylation at is by the CDK-associated T. Science. PubMed Scopus Google Scholar). has shown to of CDK2 in by with the T. Science. PubMed Scopus Google Scholar). The is by cyclin but is still able to with the complex as by T. Science. PubMed Scopus Google Scholar). phosphorylation of and of CDK2 is a of of the D.O. PubMed Scopus Google Scholar). cells also have a to which is a that the phosphorylation at and D.O. PubMed Scopus Google Scholar). the phosphorylation of CDK2 at the of activating and activating the cell levels of CDK2 activity and cell cyclin are the positive of the cell and of activation the Furthermore, have shown to have a in tumor E, and Science. PubMed Scopus Google Scholar, R. R. PubMed Scopus Google Scholar). The role of cyclin E as a CDK2 in of restriction point transition in the cell A.B. Google cyclin E an as a in tumor Cyclin E are in tumor cell as as tumor from breast cancer R. R. PubMed Scopus Google Scholar, K. T. in Scopus Google Scholar). regulation of cyclin E expression is in tumor cells, leading to constitutive cyclin E expression the cell K. D. Google Scholar, A. Google Scholar, A. R. A. Google Scholar). cells cyclin E K. Google which is accompanied by the appearance of low molecular weight which are not present in normal cells K. Google Scholar, K. A.B. Google Scholar, PubMed Scopus Google Scholar). Our suggest that forms are in breast tumor cells by of the full-length cyclin E by the of K. Google Scholar, D. R. K. Cell. PubMed Scopus Google Scholar). In addition to the full-length at we have forms that are by at in cyclin E, which in of and These in complex with are as by ability to and D. R. K. Cell. PubMed Scopus Google with full-length cyclin E·CDK2 into normal cells, the forms have cells to the cell K. Google Scholar, D. R. K. Cell. PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar). we have also shown that the of the forms in breast cancer is with and K. A.B. Google as as a K. PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar). The we in this is overexpression of cyclin E in tumor cells has the ability to directly activate CDK2. The we is to in vitro translated cyclin E or the with tumor cell extracts and the role of the cyclin E in activating endogenous CDK2. we that the addition of cyclin E full-length and in the activation of CDK2 in both normal or tumor cell extracts. and or containing cyclin E, or was used to cyclin E in The of was previously K. Google Scholar). of were by in in to were by by at The was and were the and of the cyclin E was examined a in vitro containing the cyclin E cyclin E, or or was to the at with for at were used in For of in vitro cyclin cyclin E, or or with was to tumor or normal cell extracts. or of cell extract was used as were at The amount of by the was as for a with of with a activity of was to the of of was also to a a of of of into of of is an of the amount of by the of the of and Kinase and kinase or of cell extract was used with to or CDK2 D. R. K. Cell. PubMed Scopus Google Scholar). of CDK2 was to or as for For the of was to for The binding was in The was with for and with and with kinase and were by or kinase For from were in and were a as previously K. Google Scholar). was with E or For the kinase were with kinase of of in a of at for kinase were a The was and to For the were and was by a the were to used for E of cyclin of or were with for to the to the were with and to of cell or were with cell extracts for at for binding to and with for a of of were with and with kinase to of cell extracts were the cell extracts were used in with cyclin and of in vitro cyclin and was mixed with of cell with and to with E, A, or kinase of and CDK2 in CDK2 was the as previously D. R. K. Cell. PubMed Scopus Google Scholar). CDK2 was as a from the of Harper of cells were with at a of of and at extracts were to and kinase as The CDK2 was from the ability of the forms of cyclin E cyclin to activate a series of or N-terminal cyclin E that to forms were These previously K. Google cyclin cyclin E, and Cyclin and to the forms of cyclin E in tumor cells, which we to as and D. R. K. Cell. PubMed Scopus Google Scholar). Cyclin E to the (1Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (655) Google Scholar, A. A. K. Roberts J.M. Cell. 1991; 66: Full Text PDF PubMed Scopus Google Scholar). We that this an of cyclin not to in cell D. R. K. Cell. PubMed Scopus Google Scholar). In vitro expression of the cyclin and the of forms in tumor cells from to as we have K. Google Scholar). N-terminal deleted cyclin E forms are able to to and activate CDK2 in the in vitro translated of the cyclin E forms were mixed with tumor cell The cell extract was with and to both CDK2 and kinase The that cyclin E forms were able to with was able to to CDK2 only E, and were able to activate as shown by ability to binding with was to activation can only to the of CDK2 The of the of CDK2 has previously Morgan D.O. 1992; PubMed Scopus Google Scholar). the that the activating phosphorylation of CDK2 in the appearance of the CDK2 also that the phosphorylated in CDK2 can be the through of cells with of CDK2 were able to the as the of CDK2. The with are with cyclin binding to CDK2 is an from and phosphorylation D.J. PubMed Scopus Google Scholar). The in the to the in vitro of expression of both full-length cyclin E which can activate and an N-terminal deleted cyclin E which activate normal and tumor cell extracts. The kinase for was with to the kinase activity of the in vitro translated cyclin E from the endogenous E and A, which also complex with CDK2. the of cyclin and the kinase activity in normal and tumor cells, an to CDK2 was to the complexes For of in vitro translated cyclin and were mixed with of tumor and normal cell with and to and kinase The level of phosphorylated CDK2 is by the addition of cyclin Cyclin Cyclin and to a by the addition of in both normal and tumor extracts. cells have a level of endogenous phosphorylated CDK2 normal cells addition of cyclin E Addition of cyclin an increase in CDK2 activity from to in tumor extracts Cyclin and from to in normal cell extracts Cyclin Addition of the in of an increase in CDK2 activity, to in tumor cell extracts and to in normal extracts only a of CDK2 activity binding to CDK2 also an increase in CDK2 activity, at a which may the low levels of CDK2 kinase activity with the that not complex to phosphorylated CDK2. These suggest that cyclin E protein, in the of in vitro cyclin was to the cell the amount of phosphorylated CDK2 was in normal and tumor cells cyclin E, and the CDK2 activity levels in normal and tumor cells. In both tumor cells have a level of CDK2 levels and activity normal cells. These as to is the increase in phosphorylated CDK2 by and is the increase in CDK2 activity. are for the increase in CDK2 activity addition of cyclin E into cell extracts. These are an activity in the binding of cyclin E to binding of cyclin E to CDK2 endogenous cyclin or cyclin E of CDK2 because of cyclin The and of kinase activity examined was a activity the which was used in The used to cyclin E in vitro a cell extract cell that to this or mixed with cyclin or was with and to kinase of tumor cell extract the of or mixed with cyclin or was also with CDK2 and to kinase in the to by of to CDK2 activity from cell extracts or mixed with only activity at mixed with cyclin a increase an increase in CDK2 activity from extracts to from addition of cyclin to tumor extracts. Addition of to extracts an increase from to The in activity by addition of cyclin to cell extracts is the activity in mixed with cyclin Cyclin that the activity in the for the increase in CDK2 activity. the of for the increase in CDK2 activity, we examined the of endogenous cyclin the increase in CDK2 activity. CDK2 can in at complex to cyclin A, to cyclin E, or cyclin E to cell extracts to CDK2 from of the complex such as binding to CDK2 that has from cyclin A. The used to the of cyclin the increase in activity was to cyclin from extracts by and CDK2 activity with cyclin Cyclin was by of cyclin from cell and the extracts were mixed with full-length cyclin E Cyclin were with to with and kinase that of the cyclin was ID, the cyclin cell along with the extracts with were examined by with amount of cyclin was during the with which was because of cyclin was detected in the cell extracts Cyclin The cyclin that was from the cell extracts can be detected from the cyclin used in the These that the cyclin cyclin from the cell extracts and that the only a amount of cyclin in the extracts. Cyclin was to the extracts and with cyclin to CDK2 levels and CDK2 kinase levels with and the of cyclin CDK2 that of cyclin A, and kinase activity, can be in and CDK2 that cyclin is in complex with endogenous cyclin E or in CDK2 kinase activity from to the with ID, which is a of the CDK2 in the ID, of cyclin a in kinase activity to in the cell extracts addition of cyclin E cyclin was to extracts of cyclin A, the CDK2 activity to and Cyclin Addition of cyclin to extracts to of cyclin in a increase of CDK2 activity to the CDK2 kinase levels of cell extracts mixed with cyclin and cyclin that the kinase activity that is because of cyclin cyclin for CDK2 binding is The of CDK2 activity because of complexes only a of CDK2 to cyclin addition of cyclin to extracts of cyclin can still an increase in CDK2 activity with Cyclin that the increase CDK2 activity is from as of the increase in CDK2 activation is because of the with endogenous cyclin E to binding to cyclin E was from cell and the extracts were mixed with in vitro translated cyclin were with to with and kinase in the cyclin cyclin was to extracts and endogenous cyclin E was as a to CDK2 levels and kinase activity with and the of cyclin E·CDK2 a of cyclin E was during the cyclin E with of the endogenous cyclin E was and cyclin the cyclin cyclin E cyclin was a amount of cyclin E the ID, because the high levels of cyclin E the ability of the to cyclin The cyclin E that was from the cell extracts can be from the cyclin E used in the The amount of cyclin E from the cell extracts by the with can be in These that the cyclin E endogenous cyclin E from the cell extracts and that the only a amount of cyclin E in the extracts. CDK2 that of cyclin E and kinase activity can be in was a in CDK2 levels in the cyclin extracts which was because of the of cyclin E·CDK2 complexes from the extracts. of CDK2 kinase activity from to the with which is a of the amount of CDK2 in the ID, of cyclin E CDK2 activity with addition of cyclin to cyclin E CDK2 activity and Cyclin is with the cyclin as as kinase from 2. addition of cyclin cyclin E in a increase in kinase activity to which was addition of cyclin The addition of cyclin to cell extracts cyclin E still an increase in CDK2 activity, that cyclin binding to CDK2 from endogenous cyclin E is for a of CDK2 activity. The addition of cyclin to cell extracts of endogenous cyclin E an increase in kinase activity, that the CDK2 activity is from as from cyclin may to the CDK2 cyclin E can this activity. Our suggest that the increase in CDK2 activity addition of cyclin to cell extracts is in because of binding of CDK2 from endogenous cyclin and cyclin E and CDK2. Furthermore, addition of cyclin to cell extracts has also shown to the increase in phosphorylated that phosphorylated CDK2 may be to the increase in CDK2 activity. The that CDK2 at a CDK2 a is Cell. PubMed Scopus Google Scholar, Morgan D.O. Cell. Full Text PDF PubMed Scopus Google Scholar). is that is the for the increase in CDK2 phosphorylation cyclin E is to extracts. this we the CDK2 phosphorylation were present the cyclin E·CDK2 was by of cyclin and with tumor cell extracts and of tumor cell extract with has shown to to complexes not not with CDK2. were to CDK2 and kinase and Addition of cyclin or to cell extracts the increase in phosphorylated CDK2 or as with extracts which was previously in cyclin or was CDK2 the increase in phosphorylated CDK2 was not detected cyclin CDK2 phosphorylation occurs only in the cell and not in the that the the phosphorylation increase of CDK2 is not of the cyclin has to in cyclin which the of CDK2 phosphorylation was addition of cyclin or not an increase in CDK2 the of the increase in CDK2 activity still Cyclin and were mixed with tumor cell extracts and CDK2 and to kinase cyclin and were mixed with extracts in an increase in activity to for cyclin and an increase in activity to for cyclin or from an activity of is cyclin and were mixed with extracts still in an activity increase in activity from to for cyclin and to for cyclin or cyclin and were able to the increase in CDK2 activity an increase in CDK2 that cyclin E can still increase the kinase activity to unphosphorylated CDK2. The levels of CDK2 activity were in the that were the was the of activating or with may in kinase activity because the is from the the cell These suggest that cyclin and are able to the increase in CDK2 activity by binding to of the increase in phosphorylated CDK2. we that was the increase in phosphorylated CDK2 cyclin E to cell extracts. this was tumor extracts of of from cell extracts CDK2 phosphorylation from was or Cyclin and were to tumor cell extracts that were of of and with were to and CDK2 kinase that was from the was extracts and ID, the of in cell extracts and that was not present in extracts was detected in the CDK2 that not to complexes that was from cell extracts by cyclin or was mixed with extracts of increase in the levels of phosphorylated CDK2 that was for the CDK2 phosphorylation addition of cyclin or the of from the cell addition of cyclin or still an increase in CDK2 activity or cyclin or was the increase was from to for cyclin and to for was the increase was from to for cyclin and to for The CDK2 activity levels are extracts and because the CDK2 activity. The of this that is for an increase in CDK2 phosphorylation that occurs cyclin E is to extracts. The increase in CDK2 phosphorylation by CAK, contributes to only a of the increase in CDK2 activity cyclin E is to extracts. this a CDK2 was into the and kinase CDK2 this activating of The was the cell For this and cell were For in vitro translated cyclin was or and kinase activity was is in CDK2 is present in both because of a kinase activity is in both the and CDK2 and to be activity was in cyclin was used to that the CDK2 phosphorylation at the this the CDK2 was able to the addition of cyclin that cyclin is able to activate of the phosphorylation suggest that the increase in CDK2 activity from addition of cyclin E to cell extracts is a of cyclin E binding to endogenous CDK2 from cyclin A, cyclin E, and unbound which is phosphorylated and of the mechanism is shown in Furthermore, we have in vitro that cyclin E is able to kinase activity to CDK2 activating the in tumor cells that to CDK2 activity is the amount of cyclin cells cyclin E, which in the constitutive expression and kinase activity of cyclin E the tumor cell K. D. Google Scholar). The tumor-specific overexpression of cyclin E the as to the mechanism of increase kinase activity. Addition of in vitro translated cyclin the full-length cyclin E in tumor and normal cells, to tumor cell extracts the increase of CDK2 activity along with an increase in phosphorylated CDK2. The we examined in this study was that the increase in CDK2 activity was because of the increase in phosphorylated through a pathway involving that the increase in CDK2 activity from addition of cyclin E to cell extracts is through binding to CDK2 from endogenous cyclin CDK2 from endogenous cyclin E as as unbound CDK2. The increase in phosphorylated CDK2 was a of phosphorylation of CDK2. The increase in CDK2 activity the addition of cyclin or to cell extracts was to be of the of CAK, The forms of CDK2 in be to the activity of as only the shown by kinase to in a The were for the of in vitro translated cyclin E can only be because of the cyclin E that cyclin E has to CDK2 from by T. Science. PubMed Scopus Google is that this may be in the CDK2 is phosphorylation in the or the is to and the CDK2. The in and to as and both forms in but a of the only in the is by the kinase activity the cyclin E, and were shown to to and activate CDK2. was only shown to be in complex with unphosphorylated CDK2 and was not in vitro a was used in the CDK2 was used to kinase activity was the activity with extracts These that by complexes are not can increase the levels of CDK2 phosphorylation and activity. The mechanism by which can increase CDK2 activity was not in this that we was that CDK2. and CDK2 from an for to only CDK2 activity. is a normal tumor in CDK2 binding or CDK2 activity, cyclin or were mixed in with normal or tumor extracts CDK2 activity levels were in tumor cells normal cells, and activity with cyclin or were in tumor cells normal cells. cyclin E is to both normal and tumor in an increase in CDK2 activity the levels increase in activity was normal and tumor that both normal and tumor extracts have the to the CDK2 activation ability of an level of cyclin only was an increase in activity, but addition of cyclin E also an increase in CDK2 in both normal and tumor cells a normal cell were to cyclin E, suggest that increase level of CDK2 activity and which may in a We have this we normal cells with the forms of cyclin E D. R. K. Cell. PubMed Scopus Google Scholar). cells a increase in CDK2 activity addition of cyclin E into extracts. The increase in CDK2 activity in cyclin E overexpression to at the and cells to leading to tumor was that cyclin E to normal or tumor cell extracts a increase in CDK2 activity The increase in CDK2 activity may from of the an activity to the an increase in phosphorylated binding to CDK2 from cyclin A, binding to CDK2 from endogenous cyclin E, or binding to CDK2. we examined the for activity or with the of cyclin E was a increase in kinase activity cyclin was present in the not to for increase cyclin is to cell extracts. of CDK2 activation addition of cyclin are the binding of cyclin to endogenous which can in complex with endogenous or in a We examined cell extracts of endogenous cyclin or cyclin E the increase in CDK2 activity and the increase in CDK2 activity is a of cyclin binding to CDK2 from cyclin or E, endogenous cyclin or E a increase in CDK2 activity cyclin was Our this and cyclin E or was from the level of activation was cyclin was These that a of the increase in CDK2 activity was a of cyclin binding to CDK2 from endogenous cyclin and In tumor cells that have a high level of cell cyclin E, the binding of CDK2 from cyclin may cell at late and cyclin E is not to be with CDK2 from endogenous cyclin and E, we an of unbound CDK2 by the levels of cyclin E to CDK2 during the with endogenous levels of cyclin E and A. Addition of cyclin or to cell extracts in an increase in the levels of phosphorylated along with an increase in the levels of CDK2 activity The by this are the increase in CDK2 and is the increase in phosphorylated CDK2 to the increase in CDK2 We was for the phosphorylation of because is to CDK2 at which CDK2 to a Morgan D.O. A. 1992; PubMed Scopus Google Scholar). The in was to was for the increase in phosphorylated CDK2 and increase in CDK2 activity. Addition of cyclin to of complexes not increase the levels of phosphorylated that the CDK2 is not of the complex cyclin was mixed with cell extracts that have of the increase of CDK2 phosphorylation not that is the activity for CDK2 cyclin or were mixed with of CDK2 in the increase in CDK2 activity the of phosphorylation of CDK2 Furthermore, cyclin and were mixed with tumor cell extracts of the increase in CDK2 activity was not with is These suggest that is for the increase in CDK2 phosphorylation addition of cyclin E to cell the increase in CDK2 activity is not the increase in phosphorylated CDK2 or the of is by the CDK2 The CDK2 was able to addition of cyclin the activating the N-terminal deleted forms of cyclin E were shown to to and activate that endogenous N-terminal deleted forms of cyclin E are also of binding and activating CDK2 in tumor cells. forms of cyclin E have shown to have prognostic for the and of breast cancer K. PubMed Scopus Google Scholar). The prognostic data with data from this study suggest that the forms of cyclin E are able to activate CDK2 and that may be in tumor levels of cyclin E in tumor extracts in the increase in phosphorylated CDK2 and CDK2 activity. which by not to is able to increase CDK2 activity through a mechanism by which unphosphorylated CDK2. an addition of cyclin E to cell extracts an increase in the levels of CDK2 phosphorylation by CAK, the that cyclin E and may in a Cyclin may increase the activity of by cyclin E levels in cells increase activity, which CDK2 and activity by In the is shown that in vitro CDK2 is kinase of phosphorylation has shown that the of CDK2 to cyclin E in cells is the cyclin E was also in complex with unphosphorylated and phosphorylated CDK2 PubMed Scopus Google Scholar). In the for of is to the cyclin may is be the or and activity. In an cell the CDK2 be as cyclin E the In we that addition of in vitro cyclin E to cell extracts a of leading to an increase in CDK2 activity. In cells are with cyclin E, the overexpression leads to a kinase activity with suggest that overexpression of cyclin E a in the activation of cell cycle, which lead to uncontrolled cell We Harper for the CDK2