Cdc42 Promotes G1 Progression through p70 S6 Kinase-mediated Induction of Cyclin E Expression

The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate by the cyclin E in the of or E was to but not of as the and constitutively active of were sufficient to activate the cyclin E the mechanism by which Cdc42 promotes The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate by the cyclin E in the of or E was to but not of as the and constitutively active of were sufficient to activate the cyclin E the mechanism by which Cdc42 promotes Rho family role in cellular proliferation and transformation. has that of family induce of the and anchorage-independent growth and in cell These effects the of the to such as cell cycle and Furthermore, for Rho in and in to such as and cell cycle progression of the Rho GTPase has the of in role for Cdc42 in cell cycle cell Cdc42 is sufficient to induce in the of it is not sufficient by it promotes proliferation and progression Cdc42 anchorage-independent growth is the mechanism by which Cdc42 for Cdc42 the family the family proteins and growth growth of Cdc42 that for to induce but has that that is the of the cell cycle that is by the as of Cdc42 role in the of the family which and expression of for such as cyclin of Cdc42 of in the of Cdc42 by is the of the that cyclin D-Cdk4 and cyclin E-Cdk2 that its to The that of Cdc42 induce in cyclin cyclin was not that Cdc42 expression of cyclin However, cyclin activation in it as the the cell cycle of Cdc42 the we the effects of Cdc42 the cell cycle and the to Using NIH3T3 cell that Cdc42 we show that Cdc42V12 is sufficient by itself to induce anchorage-independent but not mitogen-independent However, Cdc42V12 accelerates cyclin E-Cdk2 activation in response to growth the kinetics of cyclin D-Cdk4 activation was not by that Cdc42V12 by the cyclin E without affecting other of such as p27 or Cdc25A. of the cyclin E by Cdc42V12 was to but not to of as an the cyclin E was by of or constitutively active of which Cdc42 to and were the and in and The were in the which the and Cdc42V12 were by of cell were and in of was by the of and were of in the of were in or in the of as and The cyclin E by to to the in were in the was or of were for or were for in was to induce cell cycle and were cyclin E were in Cdk2 were E and for were in Cdk2 in in were in of and were by the of of Cdk2 of and of were for were by to cyclin were in were and for were in in in were in of and were by the of of and of by were for were by E or were the The the cyclin was were to in in the or of The were in in the or of and the were the to were in or to that of were in not of cyclin were and were and were as cyclin cyclin and p27 were E was was and were by the of the was for the was for of and were as and as and were to were the was and the was but the mechanism by which Cdc42 progression of the cell we NIH3T3 Cdc42V12 in an inducible of Cdc42V12 was by which expression of in the of in expression of Cdc42V12 was in the of that the was that of was sufficient to induce anchorage-independent as by the of Cdc42V12 to in in the of of in the of to to as in or in the of remained as in Cdc42V12 induce mitogen-independent was of cells. in was or of or not These were not Cdc42V12 is sufficient to induce of of the anchorage-independent but not mitogen-independent in NIH3T3 of but of Cdc42 to induce mitogen-independent growth is and Cdc42V12 However, other that Cdc42 that it induce that to of the cell cycle cyclin and cyclin or were in the or of of for for growth E or were by in or as of cyclin or cyclin was in the that Cdc42V12 is by itself to cell cycle in cyclin E-Cdk2 in was not However, of cyclin E-Cdk2 activation was in response to in cells. in in or in the of cyclin E to and the of in cyclin E-Cdk2 activation was and Cdc42V12 accelerates activation of cyclin cyclin E-Cdk2 activation but not cyclin D-Cdk4 or were in the or of as and for the E or were and to or as cell were to expression of were not that activation of cyclin cyclin we cyclin D-Cdk4 was in cells. the kinetics of cyclin activation were in and of the of These that by Cdc42 the cell cycle of cyclin activation and activation of cyclin E-Cdk2 the E in and activation of cyclin E-Cdk2 is that the of cyclin E and of Cdk2 as as of the p27 Cdk of is for Cdc42 in cyclin E-Cdk2 expression of cyclin E by and cyclin E were and of Cdc42V12 expression in the of was sufficient to cyclin E expression to by the cyclin E in and or were for in the or of were for to E or were by the cyclin E were or not for in the of as were and was as activation in the of were the and and in the of or were in the or of and for or without for cell were and to were as in was for were to that the effects we the of Cdc42V12 to activate by the cyclin E were in the or of for in was in the of in cells. The of Cdc42V12 the cyclin E was specific, as activation of the cyclin was not that Cdc42V12 is sufficient to induce cyclin E expression in the of Cdc42V12 anchorage-independent growth we to its to induce cyclin E expression in the of of the for in the of is to its to which the that Cdc42V12 was able to activate the cyclin E in in was Cdc42V12 activation of the cyclin E in an and mitogen-independent Cdc42V12 other cyclin E-Cdk2 that Rho family to the of the Cdk2 inhibitor p27 However, of Cdc42V12 expression the of p27 in expression of the that of Cdk2 expression is in response to in Cdc42V12 or expression of Cdc25A. that the effects of Cdc42V12 cyclin E-Cdk2 of cyclin E the E for Cdc42 and the and of Cdc42V12 of the cyclin E that the activation of were were the cyclin the which the activation of but not the activation of the cyclin E by Cdc42V12 which activation by its cyclin E activation of the was in the of the inhibitor as the of Cdc42V12 in cyclin E activation of the cyclin E is by were the cyclin E were or not and for in the of the inhibitor and were and was as activation in the of was of was and was were the cyclin E and the constitutively active the E is an inhibitor of is that and in to to that is the of activation of the cyclin E were for to activate the of the cyclin E an of that has regulatory in the cyclin E as of Cdc42 in activation of the cyclin E and Cdc42 to the cell cycle in and in the of that cyclin E activation is by Cdc42V12 and is to to the activation of that Cdc42V12 and promotes its and activation as of of the of the the or in the of the was the of The of to to an in the of the of the effects were to the of were activation of was by of in its Furthermore, of its Cdc42V12 in mitogen-independent of the cyclin E by Cdc42V12 its to activate in the of is in cells. or were for in the or of were or of and were for in the or of and for were or and to Cdc42V12 or is able to activate the cyclin E in the of as by its to of that was not by an for in the of were was of or was by that the of its in the in was in and cells. activation of and was in the activation of and cyclin E of by Cdc42V12 in the of were in and in the or of were to to or in were or as The of and mechanism by which Cdc42 to to progression has remained the we and cell cycle for Rho GTPase in NIH3T3 cells. that Cdc42V12 expression of cyclin E as Cdk2 and p27 the of growth Cdc42V12 by itself induce mitogen-independent activation of cyclin E-Cdk2 However, the of and cyclin E-Cdk2 activation is by to cells. These that of cyclin E is the in in NIH3T3 cells. the kinetics and of by Cdc42V12 is that by expression of cyclin E itself constitutively cyclin cyclin in activation of cyclin and that is for the is cell has to cyclin E-Cdk2 activation by in cell of cyclin E of and of that in NIH3T3 Cdc42V12 the for in by inducing cyclin E that Cdc42 induce cyclin and cyclin expression in cell cyclin in an in the of cyclin was not Cdc42 induce anchorage-independent growth by activation of cyclin and cyclin E The of Cdc42 to induce expression of the in cell we to activation of the cyclin E by Cdc42V12 in or cells. is in the role of Rho in progression we that Cdc42 cyclin E cyclin expression and Rho cyclin E-Cdk2 p27 and expression of Cdc42 in cell cycle and anchorage-independent growth and to to Cdc42 is to cyclin E expression as as cyclin and cyclin expression The of Cdc42 to induce in cell by the growth and for expression of Cdc42V12 the for inducing expression of E and of Cdk2 that sufficient to the active that the effects of the cell cycle in cell such as and in such as Using cell it has that of the cell cycle in and cell of cyclin However, the cyclin that of that or the of the show that of cyclin E expression in but the of the cyclin E-Cdk2 that of the Cdk2 inhibitor p27 that in response to growth of the cell cycle in cell has other that the of the it was to that activate the cyclin E show that is to induce the cyclin E the cyclin E as of the to cyclin in of cell cycle that by cyclin E expression of progression in of cyclin D-Cdk4 activation but expression of cyclin E was that the role in progression of cyclin E Rho family role in cellular proliferation and transformation. has that of family induce of the and anchorage-independent growth and in cell These effects the of the to such as cell cycle and Furthermore, for Rho in and in to such as and cell cycle progression of the Rho GTPase has the of in role for Cdc42 in cell cycle cell Cdc42 is sufficient to induce in the of it is not sufficient by it promotes proliferation and progression Cdc42 anchorage-independent growth is the mechanism by which Cdc42 for Cdc42 the family the family proteins and growth growth of Cdc42 that for to induce but has that that is the of the cell cycle that is by the as of Cdc42 role in the of the family which and expression of for such as cyclin of Cdc42 of in the of Cdc42 by is the of the that cyclin D-Cdk4 and cyclin E-Cdk2 that its to The that of Cdc42 induce in cyclin cyclin was not that Cdc42 expression of cyclin However, cyclin activation in it as the the cell cycle of Cdc42 the we the effects of Cdc42 the cell cycle and the to Using NIH3T3 cell that Cdc42 we show that Cdc42V12 is sufficient by itself to induce anchorage-independent but not mitogen-independent However, Cdc42V12 accelerates cyclin E-Cdk2 activation in response to growth the kinetics of cyclin D-Cdk4 activation was not by that Cdc42V12 by the cyclin E without affecting other of such as p27 or Cdc25A. of the cyclin E by Cdc42V12 was to but not to of as an the cyclin E was by of or constitutively active of which Cdc42 to and were the and in and The were in the which the and Cdc42V12 were by of cell were and in of was by the of and were of in the of were in or in the of as and The cyclin E by to to the in were in the was or of were for or were for in was to induce cell cycle and were cyclin E were in Cdk2 were E and for were in Cdk2 in in were in of and were by the of of Cdk2 of and of were for were by to cyclin were in were and for were in in in were in of and were by the of of and of by were for were by E or were the The the cyclin was were to in in the or of The were in in the or of and the were the to were in or to that of were in not of cyclin were and were and were as cyclin cyclin and p27 were E was was and were by the of the was for the was for of and were as and as and were to were the was and the was and were the and in and The were in the which the and Cdc42V12 were by of cell were and in of was by the of and were of in the of were in or in the of as and The cyclin E by to to the in were in the was or of were for or were for in was to induce cell cycle and were cyclin E were in Cdk2 were E and for were in Cdk2 in in were in of and were by the of of Cdk2 of and of were for were by to cyclin were in were and for were in in in were in of and were by the of of and of by were for were by E or were the The the cyclin was were to in in the or of The were in in the or of and the were the to were in or to that of were in not of cyclin were and were and were as cyclin cyclin and p27 were E was was and were by the of the was for the was for of and were as and as and were to were the was and the was but the mechanism by which Cdc42 progression of the cell we NIH3T3 Cdc42V12 in an inducible of Cdc42V12 was by which expression of in the of in expression of Cdc42V12 was in the of that the was that of was sufficient to induce anchorage-independent as by the of Cdc42V12 to in in the of of in the of to to as in or in the of remained as in Cdc42V12 induce mitogen-independent was of cells. in was or of or not These were not Cdc42V12 is sufficient to induce of of the anchorage-independent but not mitogen-independent in NIH3T3 of but of Cdc42 to induce mitogen-independent growth is and Cdc42V12 However, other that Cdc42 that it induce that to of the cell cycle cyclin and cyclin or were in the or of of for for growth E or were by in or as of cyclin or cyclin was in the that Cdc42V12 is by itself to cell cycle in cyclin E-Cdk2 in was not However, of cyclin E-Cdk2 activation was in response to in cells. in in or in the of cyclin E to and the of in cyclin E-Cdk2 activation was and Cdc42V12 accelerates activation of cyclin that activation of cyclin cyclin we cyclin D-Cdk4 was in cells. the kinetics of cyclin activation were in and of the of These that by Cdc42 the cell cycle of cyclin activation and activation of cyclin E-Cdk2 the E in and activation of cyclin E-Cdk2 is that the of cyclin E and of Cdk2 as as of the p27 Cdk of is for Cdc42 in cyclin E-Cdk2 expression of cyclin E by and cyclin E were and of Cdc42V12 expression in the of was sufficient to cyclin E expression to by the cyclin E in and or were for in the or of were for to E or were by the cyclin E were or not for in the of as were and was as activation in the of were the and and in the of or were in the or of and for or without for cell were and to were as in was for were to that the effects we the of Cdc42V12 to activate by the cyclin E were in the or of for in was in the of in cells. The of Cdc42V12 the cyclin E was specific, as activation of the cyclin was not that Cdc42V12 is sufficient to induce cyclin E expression in the of Cdc42V12 anchorage-independent growth we to its to induce cyclin E expression in the of of the for in the of is to its to which the that Cdc42V12 was able to activate the cyclin E in in was Cdc42V12 activation of the cyclin E in an and mitogen-independent Cdc42V12 other cyclin E-Cdk2 that Rho family to the of the Cdk2 inhibitor p27 However, of Cdc42V12 expression the of p27 in expression of the that of Cdk2 expression is in response to in Cdc42V12 or expression of Cdc25A. that the effects of Cdc42V12 cyclin E-Cdk2 of cyclin E the E for Cdc42 and the and of Cdc42V12 of the cyclin E that the activation of were were the cyclin the which the activation of but not the activation of the cyclin E by Cdc42V12 which activation by its cyclin E activation of the was in the of the inhibitor as the of Cdc42V12 in cyclin E activation of the cyclin E is by were the cyclin E were or not and for in the of the inhibitor and were and was as activation in the of was of was and was were the cyclin E and the constitutively active the E is an inhibitor of is that and in to to that is the of activation of the cyclin E were for to activate the of the cyclin E an of that has regulatory in the cyclin E as of Cdc42 in activation of the cyclin E and Cdc42 to the cell cycle in and in the of that cyclin E activation is by Cdc42V12 and is to to the activation of that Cdc42V12 and promotes its and activation as of of the of the the or in the of the was the of The of to to an in the of the of the effects were to the of were activation of was by of in its Furthermore, of its Cdc42V12 in mitogen-independent of the cyclin E by Cdc42V12 its to activate in the of is in cells. or were for in the or of were or of and were for in the or of and for were or and to Cdc42V12 or is able to activate the cyclin E in the of as by its to of that was not by an for in the of were was of or was by that the of its in the in was in and cells. activation of and was in the activation of and cyclin E of by Cdc42V12 in the of were in and in the or of were to to or in were or as The of and Cdc42V12 but the mechanism by which Cdc42 progression of the cell we NIH3T3 Cdc42V12 in an inducible of Cdc42V12 was by which expression of in the of in expression of Cdc42V12 was in the of that the was that of was sufficient to induce anchorage-independent as by the of Cdc42V12 to in in the of of in the of to to as in or in the of remained as in Cdc42V12 induce mitogen-independent was of cells. in was or of or not These were not Cdc42V12 is sufficient to induce of of the anchorage-independent but not mitogen-independent in NIH3T3 cells. Cdc42V12 of but of Cdc42 to induce mitogen-independent growth is and Cdc42V12 However, other that Cdc42 that it induce that to of the cell cycle cyclin and cyclin or were in the or of of for for growth E or were by in or as of cyclin or cyclin was in the that Cdc42V12 is by itself to cell cycle in cyclin E-Cdk2 in was not However, of cyclin E-Cdk2 activation was in response to in cells. in in or in the of cyclin E to and the of in cyclin E-Cdk2 activation was and Cdc42V12 accelerates activation of cyclin that activation of cyclin cyclin we cyclin D-Cdk4 was in cells. the kinetics of cyclin activation were in and of the of These that by Cdc42 the cell cycle of cyclin activation and activation of cyclin E-Cdk2 Cdc42V12 the E in and activation of cyclin E-Cdk2 is that the of cyclin E and of Cdk2 as as of the p27 Cdk of is for Cdc42 in cyclin E-Cdk2 expression of cyclin E by and cyclin E were and of Cdc42V12 expression in the of was sufficient to cyclin E expression to by that the effects we the of Cdc42V12 to activate by the cyclin E were in the or of for in was in the of in cells. The of Cdc42V12 the cyclin E was specific, as activation of the cyclin was not that Cdc42V12 is sufficient to induce cyclin E expression in the of mitogen. Cdc42V12 anchorage-independent growth we to its to induce cyclin E expression in the of of the for in the of is to its to which the that Cdc42V12 was able to activate the cyclin E in in was Cdc42V12 activation of the cyclin E in an and mitogen-independent Cdc42V12 other cyclin E-Cdk2 that Rho family to the of the Cdk2 inhibitor p27 However, of Cdc42V12 expression the of p27 in expression of the that of Cdk2 expression is in response to in Cdc42V12 or expression of Cdc25A. that the effects of Cdc42V12 cyclin E-Cdk2 of cyclin E Cdc42V12 the E for Cdc42 and the and of Cdc42V12 of the cyclin E that the activation of were were the cyclin the which the activation of but not the activation of the cyclin E by Cdc42V12 which activation by its cyclin E activation of the was in the of the inhibitor as the of Cdc42V12 in cyclin E the E is an inhibitor of is that and in to to that is the of activation of the cyclin E were for to activate the of the cyclin E an of that has regulatory in the cyclin E as of Cdc42 in activation of the cyclin E and Cdc42 to the cell cycle in and in the of that cyclin E activation is by Cdc42V12 and is to to the activation of that Cdc42V12 and promotes its and activation as of of the of the the or in the of the was the of The of to to an in the of the of the effects were to the of were activation of was by of in its Furthermore, of its Cdc42V12 in mitogen-independent of the cyclin E by Cdc42V12 its to activate in the of mitogen. Cdc42V12 is able to activate the cyclin E in the of as by its to of that was not by an for in the of were was of or was by that the of its in the in was in and cells. activation of and was in the activation of and cyclin E mechanism by which Cdc42 to to progression has remained the we and cell cycle for Rho GTPase in NIH3T3 cells. that Cdc42V12 expression of cyclin E as Cdk2 and p27 the of growth Cdc42V12 by itself induce mitogen-independent activation of cyclin E-Cdk2 However, the of and cyclin E-Cdk2 activation is by to cells. These that of cyclin E is the in in NIH3T3 cells. the kinetics and of by Cdc42V12 is that by expression of cyclin E itself constitutively cyclin cyclin in activation of cyclin and that is for the is cell has to cyclin E-Cdk2 activation by in cell of cyclin E of and of that in NIH3T3 Cdc42V12 the for in by inducing cyclin E that Cdc42 induce cyclin and cyclin expression in cell cyclin in an in the of cyclin was not Cdc42 induce anchorage-independent growth by activation of cyclin and cyclin E The of Cdc42 to induce expression of the in cell we to activation of the cyclin E by Cdc42V12 in or cells. is in the role of Rho in progression we that Cdc42 cyclin E cyclin expression and Rho cyclin E-Cdk2 p27 and expression the effects of the cell cycle in cell such as and in such as Using cell it has that of the cell cycle in and cell of cyclin However, the cyclin that of that or the of the show that of cyclin E expression in but the of the cyclin E-Cdk2 that of the Cdk2 inhibitor p27 that in response to growth of the cell cycle in cell has other that the of the it was to that activate the cyclin E show that is to induce the cyclin E the cyclin E as of the to cyclin in of cell cycle that by cyclin E expression of progression in of cyclin D-Cdk4 activation but expression of cyclin E was that the role in progression of cyclin E The mechanism by which Cdc42 to to progression has remained the we and cell cycle for Rho GTPase in NIH3T3 cells. that Cdc42V12 expression of cyclin E as Cdk2 and p27 the of growth Cdc42V12 by itself induce mitogen-independent activation of cyclin E-Cdk2 However, the of and cyclin E-Cdk2 activation is by to cells. These that of cyclin E is the in in NIH3T3 cells. the kinetics and of by Cdc42V12 is that by expression of cyclin E itself constitutively cyclin cyclin in cells. The activation of cyclin and that is for the is cell has to cyclin E-Cdk2 activation by in cell of cyclin E of and of that in NIH3T3 Cdc42V12 the for in by inducing cyclin E that Cdc42 induce cyclin and cyclin expression in cell cyclin in an in the of cyclin was not Cdc42 induce anchorage-independent growth by activation of cyclin and cyclin E The of Cdc42 to induce expression of the in cell we to activation of the cyclin E by Cdc42V12 in or cells. is in the role of Rho in progression we that Cdc42 cyclin E cyclin expression and Rho cyclin E-Cdk2 p27 and expression the effects of the cell cycle in cell such as and in such as Using cell it has that of the cell cycle in and cell of cyclin However, the cyclin that of that or the of the show that of cyclin E expression in but the of the cyclin E-Cdk2 that of the Cdk2 inhibitor p27 that in response to growth of the cell cycle in cell has other that the of the it was to that activate the cyclin E show that is to induce the cyclin E the cyclin E as of the to cyclin in of cell cycle that by cyclin E expression of progression in of cyclin D-Cdk4 activation but expression of cyclin E was that the role in progression of cyclin E for of the