Molecular antagonisms and cell cycle regulation

Document Sample
Molecular antagonisms and cell cycle regulation Powered By Docstoc
					Molecular antagonisms and cell
       cycle regulation

               Andrea Ciliberto
   Istituto Firc di Oncologia Molecolare
                    Milano
       The cell division cycle

                                    G1
                                      St
                             n          ar
                         isio             t
                 ll   div
               ce
                                                   S
                                                   (DNA Replication)
           h
      Finis



   M                                          G2
                             G2/M
(mitosis)
Major phases of Mitosis




         Kops and Cleveland, 2005
Kohn, Mol. Biol. Cell., 1999
The entry into mitosis
       The cell division cycle

                                    G1
                                      St
                             n          ar
                         isio             t
                 ll   div
               ce
                                                   S
                                                   (DNA Replication)
           h
      Finis



   M                                          G2
                             G2/M
(mitosis)
Murray and Kirschner, Science, 1989
Xenopus and the clock paradigm




Cell mass decreases during early divisions




                              Alberts et a., Molecular Biology of the Cell.2002
In Xenopus oscillations progress independently
    of DNA presence and cell cycle events
                Autonomous oscillations!




                                    Alberts et a., Molecular Biology of the Cell.2002
MPF, the mitosis promoting factor




                        Murray and Kirschner, Science, 1989
            MPF

            CDK          cyclin dependent kinase
            Cyc          cyclin (regulatory subunit)




- Cyclin dependent kinase without cyclin is inactive.

- Cyclin dependent kinase is present in excess.

- The binding of Cyclin and CDK is very spontaneous.

- Amount of cyclin approximately corresponds to MPF.
      CDK


            CDK
Cyc         Cyc



                  CDK
Cyclin is continuously synthesized and periodically degraded



           CDK


                   CDK                       cyclin
     Cyc           Cyc



                          CDK
                                      CDK
        How about MPF, i.e.                   ?
                                      Cyc



- Cyclin dependent kinase without cyclin is inactive.

- Cyclin dependent kinase is present in excess.

- The binding of Cyclin and CDK is very spontaneous.

- Amount of cyclin approximately corresponds to MPF.
Alberts et a., Molecular Biology of the Cell.2002
MPF activity (not relative concentration) increases abruptly




                                              Solomon et al, Cell, 1990



                          Anything strange?
Solomon used a non degradable cyclin...



                CDK


                        CDK
          Cyc           Cyc



                              CDK
There must be more than cyclin synthesis and degradation
Cyclin threshold for Cyclin activation




 CycThr




   MPF




                    CycThr               Cyclin
                MPF
         MPF


Cyclin




         time         CycThr
                                Cyclin

                MPF
         MPF


Cyclin




         time          CycThr
                                 Cyclin
                MPF
         MPF


Cyclin




         time         CycThr
                                Cyclin

                MPF
         MPF


Cyclin




         time          CycThr
                                 Cyclin
                MPF
         MPF


Cyclin




         time         CycThr
                                Cyclin

                MPF
         MPF


Cyclin




         time          CycThr
                                 Cyclin
Importance of irreversible transitions in the cell cycle:
                    re-replication



                                                G1
                                                  St
                                        on          ar
                                     isi              t
                             ll   div
                           ce
                                                               S
                                                               (DNA Replication)
                       h
                  Finis




                 M                                        G2
                                         G2/M
              (mitosis)
                MPF
         MPF


Cyclin




         time         CycThr
                               Cyclin
                MPF
         MPF


Cyclin




         time         CycThr
                               Cyclin
How to make an irreversible switch?
      CDK


            CDK
Cyc         Cyc



                  CDK
Yeast and the domino paradigm



                           G1



   Anaphase                          S




      Metaphase                 G2
Cell division cycle (cdc) mutants are temperature sensitive




                                                           Hartwell, Genetics, 1991




                                   Alberts et a., Molecular Biology of the Cell.2002
                             Balanced growth and division



                       Cell cycle                                      Cytoplasmic
                        engine                                           growth
                                        TC                TD
                                             Size control
                   4
Cytoplasmic mass




                   3
                                                          T C > TD


                   2
                                                                 T C = TD       exponential
                                                                                 balanced
                   1
                                                                                  growth

                                               T C < TD
                   0
                         0          1             2          3              4
Wee1 controls a rate limiting step in the cell cycle
        Cell division and cell growth are coupled


          wild type      wee1          cdc25




                                               Nurse, Noble lecture, 2000
Dominoes and clocks: Cdc28 is the
budding yeast homologous of MPF’s
         catalytic subunit




         Cdc28   =   Cdc2    =   CDK1
 MPF =
          Clb2       Cdc13       CycB
Phosphorylation also controls MPF activity




                Wee1           Wee1P
                                       G2
         P

        Cdc2            Cdc2
        Cyc             Cyc
                                       M


               Cdc25P          Cdc25
Phosphorylation as well as cyclin binding controls MPF activity




                          Wee1           Wee1P
                                                 G2
                   P

                  Cdc2            Cdc2
                  Cyc             Cyc
                                                 M


                         Cdc25P          Cdc25
Putting it all together




                 Wee1P           Wee1
           CDK
                                    P

                    CDK            CDK
    Cyc              Cyc           Cyc



                           CDK
            Wee1P   Wee1
      CDK
                       P

              CDK     CDK
Cyc           Cyc     Cyc
Wee1P   Wee1

           P

  CDK     CDK
  Cyc     Cyc
Wee1P   Wee1

           P

  CDK     CDK
  Cyc     Cyc




                Solomon et al, Cell, 1990
Step-by-step building of a molecular switch
                Law of Mass Action: forward reaction
                                         ka
                              pMPF             MPF



       dMPF                                   Steady State solution (MPFSS)
            = ka ! pMPF
        dt                                         dMPF
       pMPF = MPFtot - MPF                                =0
                                                    dt
       dMPF
            = ka ! (MPFtot - MPF)                  MPF SS = MPFtot
        dt


Notice: no dimer, only MPF. Cdk is supposed to be present in excess throughout
the cycle. Increasing MPF total mimics an increase in cyclin total.
                           dMPF
                                = ka ! (MPFtot -MPF)
                            dt

                                                                        MPFtot
  0.2                                                      1




                                                    MPF
                         dMPF
                              >0                          0.8

  0.1
                          dt
dMPF                                                      0.6

 dt                                                       0.4
   0                                      dMPF
                                               =0
                                           dt             0.2

  -0.1
                                                           0
         0   0.2   0.4    0.6   0.8   1
                                                                0   4    8     12   16   20
                   MPF                                                       time
Law of Mass Action: reversible reaction
                    ka
           pMPF           MPF
                     ki
     dMPF
          = ka ! pMPF - ki ! MPF
      dt


       Steady State solution
          dMPF
                 =0
           dt
                  ka ! MPFtot
          MPF =
              SS

                    ka + ki
                           ka
         pMPF                      MPF
                           ki
 dMPF
      = ka ! (MPFtot " MPF) - ki ! MPF
  dt
                  dephosphorylation            ph’n
                         +                      -

                                 dMPF
                                      =0
       0.25                       dt
dMPF
                          dMPF    dMPF
 dt    0.2                     >0      <0
                           dt      dt
       0.15


       0.1


       0.05


         0
              0     0.2    0.4     0.6   0.8   1
                                 MPF
       1
MPF
      0.8


      0.6


      0.4


      0.2


       0
            0   4   8      12   16   20

                    time




                                          t
                   Law of Mass Action:
               catalyzed reversible reaction
       ka
                              dMPF
pMPF        MPF                    = ka ! (MPFtot " MPF) - ki ! MPF ! Wee1
       ki                      dt
                                              dephosphorylation           ph’n
                                                     +                     -
        Wee1

                       rate
                 0.25                         4       3

                 0.2                5                       2
                 0.15


                 0.1                                        1

                 0.05


                   0
                        0     0.2       0.4   0.6     0.8       1   MPF
                                543 2             1
                                                    Nullclines

                      ka
     pMPF                                         dMPF
                                MPF                    = ka ! (MPFtot " MPF) - ki ! MPF ! Wee1
                  ki                               dt
                                                            dephosphorylation                  ph’n
                                                                   +                            -
                      Wee1

     rate
0.25                                3                       MPFSS
                            4                                              dMPF
                                                                  1
                                                                                <0
0.2               5                       2                                 dt
                                                                                              dMPF
0.15
                                                                                                   =0
                                                                                               dt
0.1                                       1                      0.5

                                                                           dMPF
0.05                                                                            >0
                                                                            dt
 0                                                MPF
       0    0.2       0.4   0.6     0.8       1                   0
                                                                       0            2.5               5
              543 2             1                                           1   2         3    4
                                                                                    Wee1
                       What happens if MPF total increases?

                                                   ka
                                    pMPF                MPF
                                                  ki

                                                   Wee1

     rate
0.25                                3                     MPFSS
                            4                                           dMPF
                                                               1
                                                                             <0
0.2               5                       2                              dt
                                                                                           dMPF
0.15
                                                                                                =0
                                                                                            dt
0.1                                       1                   0.5

                                                                        dMPF
0.05                                                                         >0
                                                                         dt
 0                                                MPF
       0    0.2       0.4   0.6     0.8       1                0
                                                                    0            2.5            5
              543 2             1                                        1   2         3    4
                                                                                 Wee1
            Michaelis Menten kinetics

                 k1                            k2
     Wee1P                  PPase:Wee1P              Wee1
                      k1r
               Ppase                        PPase



PPase=PPase TOT -PPase:Wee1p
Wee1=Wee1 TOT -Wee1p-PPase:Wee1p


dPPase:Wee1p
               =k1 (PPase TOT -PPase:Wee1p)Wee1p-PPase:Wee1p(k1r +k 2 )
     dt
dWee1p
        =k1r PPase:Wee1p-k1 (PPase TOT -PPase:Wee1p)Wee1p
  dt


                   dWee1 k 2 PPase TOT Wee1p
                        =
                     dt    k1r +k 2
                                    +Wee1p
                              k1
             Michaelis-Menten: forward reaction
                               kwa
                       Wee1P         Wee1



  k wa =PPase TOTk 2


dWee1 kwa ! Wee1P                     Steady State solution
     =                                  dWee1
 dt    J + Wee1P                               =0
dWee1 kwa ! (Wee1tot - Wee1)             dt
     =                                  Wee1SS = Wee1tot
 dt    J + (Wee1tot - Wee1)
    0.2                                                  1
                                                                      Wee1tot
dWee1                                               b
 dt             dWee1                                   0.8

    0.1
                      >0
                 dt                                     0.6


                                                        0.4
        0
                                         dWee1
                                               =0       0.2
                                          dt
    -0.1                                                 0
            0   0.2   0.4    0.6   0.8   1                    0   4   8     12   16   20
                            Wee1                                          time
 Michaelis-Menten: reversible reaction

                        kwa
              Wee1P            Wee1
                         kwi

         k1                           k2
Wee1P               PPase:Wee1P             Wee1
              k1r
        PPase                      PPase



         k3                           k4
Wee1                 kinase:Wee1            Wee1P
              k3r
        kinase                     kinase
    if [PpaseTOT], [kinaseTOT] << [Wee1TOT]

           k wa =[PPase TOT ]k 2
           k wi =[kinase TOT ]k 4


dWee1 kwa ! (Wee1tot " Wee1) kwi ! Wee1
     =                      -
  dt    J + Wee1tot -Wee1     J + Wee1
         dephosphorylation           ph’n
                +                     -
              Michaelis-Menten: reversible reaction

        kwa                       dWee1 kwa ! (Wee1tot " Wee1) kwi ! Wee1
Wee1P          Wee1                    =                      -
        kwi                        dt     J + Wee1tot -Wee1     J + Wee1
                                                dephosphorylation   ph’n
                                                       +             -
                                      dWee1
                                            =0
                      rate             dt
                      0.5
                              dWee1        dWee1
                                    >0           <0
                      0.4      dt           dt

                      0.3


                      0.2


                      0.1


                      0.0
                            0.0     0.2   0.4     0.6   0.8   1.0


                                          Wee1*
                                    Nullclines
             kwa                 dWee1 kwa ! (Wee1tot " Wee1) kwi ! Wee1
Wee1P               Wee1              =                      -
             kwi                  dt     J + Wee1tot -Wee1     J + Wee1
                                              dephosphorylation                 ph’n
                                                     +                           -

                dWee1                           Wee1SS
                      =0
 rate            dt                                1
                                                                dWee1
 0.5
         dWee1       dWee1                                            <0
               >0          <0                                    dt
 0.4      dt          dt

 0.3                                              0.5

                                                                 dWee1
 0.2
                                                                       >0
                                                                  dt
 0.1


 0.0                                               0
                                                        0             2.5              5
       0.0    0.2   0.4    0.6    0.8   1.0
                                                            1     2         3   4
                    Wee1SS                                            MPF
Phase plane analysis




Wee1P         Wee1
        kwi
                 ki
         MPF          pMPF
                 ka
MPFSS                                    MPF
            dMPF
   1
                 <0




                                           5
             dt
                               dMPF
                                    =0               dWee1
                                                           >0
                                                                  dWee1
                                                                        <0
                                dt                    dt           dt
  0.5




                                           2.5
            dMPF
                 >0
             dt
   0




                                           0
        0            2.5            5
             1   2         3    4




                                                            0.5
                                                 0




                                                                      1
                     Wee1                                  Wee1SS
            dMPF
   1
                 <0




                                          5
MPFSS
             dt




                                        MPF
                        dMPF
                             =0                     dWee1
                                                          >0
                                                                    dWee1
                                                                          <0
                         dt                          dt              dt
  0.5




                                          2.5
            dMPF
                 >0
             dt
   0




                                          0
        0         2.5      1        5




                                                            0.5
                                                0




                                                                           1
                 Wee1                                             Wee1SS
                         MPF




                          0.5




                           0
                                0       2.5     Wee1    5
           How does MPF increases with Cyclin total?

                                            ka ! MPFtot
                              MPF   SS
                                         =
                                           ki ! Wee1+ka


                                          MPF
MPF




 0.5




  0
       0         2.5   Wee1    5                    0

                                                          MPFtot
Not quite the same!




                      Solomon et al, Cell, 1990
Wee1P         Wee1
        kwi
                 ki
         MPF          pMPF
                 ka
              Michaelis-Menten:
         catalyzed reversible reaction
                    kwa
            Wee1P          Wee1
                     kwi


                      MPF


dWee1 kwa ! (Wee1tot " Wee1) kwi ! Wee1! MPF
     =                      -
  dt    J + Wee1tot -Wee1         J + Wee1
          dephosphorylation         ph’n
                 +                   -
                                             Nullclines


 Wee1P                        Wee1     dWee1 kwa ! (Wee1tot " Wee1) kwi ! Wee1! MPF
                  kwi                       =                      -
                                         dt    J + Wee1tot -Wee1         J + Wee1

                   MPF                                 dephosphorylation              ph’n
                                                              +                        -

                                                            Wee1SS           dWee1
rate                                                         1
                                                                                   <0
0.5                                                                           dt
                                             5
                                                            0.8
0.4
                                             4                                   dWee1
                                                                                       =0
0.3                                          3              0.6                   dt
0.2                                          2              0.4       dWee1
                                                                            >0
0.1
                                             1                         dt
                                                            0.2
                                        .1
0.0
      0.0   0.2         0.4      0.6   0.8       1.0         0
                                                                  0      1   2    3    4     5
                        Wee1SS                                               MPF
              Phase plane analysis




                                           dWee1
                                                 =0
MPFSS                         Wee1SS        dt
1                             1

          dMPF       dMPF    0.8
               <0         =0                  dWee1
           dt         dt                            <0
                             0.6
0.5                                            dt
                              0.4 dWee1
          dMPF                          >0
               >0             0.2
                                   dt
           dt
0                              0
      0      2.5      5            0   1     2    3    4   5
              Wee1                               MPF
                Phase plane analysis



                                             dWee1
                                                   =0
Wee1                           Wee1SS         dt
5             dMPF
                   <0           1
               dt
                               0.8
                    dMPF                        dWee1
                         =0    0.6                    <0
2.5                  dt                          dt
                               0.4 dWee1
                                         >0
          dMPF                      dt
               >0              0.2
           dt
0                               0
      0       0.5         1          0   1     2    3    4   5
               MPFSS                               MPF
First solution, MPF wins, Wee1 loses


         1
                               MPFtot=1.5

        0.8
 Wee1
        0.6



        0.4



        0.2



         0
              0   0.4   0.8     1.2    1.6


                              MPF
Second solution, Wee1 wins, MPF loses

           1

                                  MPFtot=0.5
          0.8



   Wee1   0.6



          0.4



          0.2



           0
                0   0.4   0.8     1.2    1.6

                                MPF
Third solution, both can win: hysteresis


        1
                              MPFtot=1

       0.8



       0.6
Wee1
       0.4



       0.2



        0
             0   0.4   0.8   1.2   1.6


                       MPF
How does MPF increases with Cyclin total?
Wee1                Wee1               Wee1




       MPFtot=0.5
                            MPFtot=1          MPFtot=1.5




       MPF                 MPF                 MPF
This network is compatible with the threshold of cyclin for
                    MPF activation


              Wee1P         Wee1
                      kwi
                               ki
                       MPF          pMPF
                               ka
  This network is also consistent with the irreversible
                   activation of MPF

MPF                                   MPF


      Threshold down




             MPFtot                         MPFtot   Threshold
                       Threshold up
But is it true?
Hysteresis in the Xenopus early cycles: simulation
             of an experimental result




              From Sha et al, PNAS, 2003
The threshold for going ‘down’ is different from ‘going up’
MPF

      Threshold down




             MPFtot
                       Threshold up
               According to modelers:

- Mitotic transitions should better be irreversible.
- Experimentally verified for the entry into mitosis.
- Irreversibility is given by antagonism and positive feedbacks.
                 According to modelers:

  - Mitotic transitions should better be irreversible.
  - Experimentally verified for the entry into mitosis.
  - Irreversibility is given by antagonism and positive feedbacks.



Experimentalists have a different view though...
                                  G1
                                    St
                           n          ar
                       isio             t
               ll   div
             ce
                                                 S
                                                 (DNA Replication)
         h
    Finis



   M                                        G2
                           G2/M
(mitosis)
                                  G1
                                  St
                           n        ar
                       isio           t
               ll   div
             ce
                                               S
                                               (DNA Replication)
         h
    Finis



   M                                      G2
                           G2/M
(mitosis)
  MPF ‘going up’ and ‘coming down’ are governed by
  two different processes.




Entry into mitosis occurs when cyclin crosses a critical threshold

Mitotic exit is allowed only when mitosis is completed and most of cyclin is
degraded.
            Wee1P         Wee1
      CDK
                             P

              CDK           CDK
Cyc           Cyc           Cyc



                    CDK
                                      cdc20
                     cyc


             Cdk

                                      cdc20
                     Cdk                      Cdk
                     cyc
sic1




                                       cdh1
       cyc

                                  P
              sic1




                           sic1

                                       cdh1
        Cdk1

cdc20
                cdh1




        Cdk1

        cyc




         sic1
        Cdk1

cdc20
                cdh1




        Cdk1

        cyc




         sic1
        Cdk1

cdc20
                cdh1




        Cdk1

        cyc
         sic1




                       time (min)
        Cdk1

cdc20
                cdh1



        Cdk1

        cyc


         sic1
        Cdk1

cdc20
                cdh1



        Cdk1

        cyc


         sic1
        Cdk1

cdc20
                cdh1



        Cdk1

        cyc


         sic1
Any experimental validation here?
        An inhibitor of Cdk/Cyc accelerates the exit from mitosis


            Cdk1

cdc20
                      cdh1




            Cdk1

            cyc
             sic1




                    Flavopiridol introduced at time zero
        An inhibitor of Cdk/Cyc accelerates an irreversible exit
                             from mitosis

            Cdk1

cdc20
                       cdh1




            Cdk1

             cyc
              sic1




                     Flavopiridol introduced at time zero
                          and removed at time 25
        An inhibitor of Cdk/Cyc induces the exit from mitosis in
                     absence of cyclin degradation


          Cdk1

cdc20
                    cdh1




          Cdk1

          cyc
           sic1




                  Flavopiridol introduced at time zero
        An inhibitor of Cdk/Cyc induces a reversible exit from mitosis
                        in absence of cyclin degradation


             Cdk1

cdc20
                        cdh1




             Cdk1

              cyc
               sic1




                      Flavopiridol introduced at time zero
                           and removed at time 25
Is cyclin degradation required for irreversibility?
     = Cdk inhibitor

                                                                 Ub
                                                             Ub       Cdk1
                                           APC
                                                            Ub

                                    CycB                 CycB
AA                    CycB                                               CycB

                             Cdk1
                                    Cdk1
                                                                      X
                                                         Cdk1 proteasome




                                             inhibitor       CycB
      Cdk1 activity




                                                             Cdk1
                                             inhibitor




                                                            CycB
                                                            Cdk1
        Cdk1

cdc20
                cdh1



        Cdk1

        cyc




         sic1
                                         Cdh1                h1
                                                           Cd
                                     inactive




                                                                                                                                                     Cdk inhibitor
                                                                                                                                                     washed away
                                                                                                                                     Cdk inhibitor
                                                +      +
                                                                                             Ub




                                                                                                                   microtubule
                                                                                         Ub       Cdk1    Ub
                                                                                                              Ub

                                                                                        Ub
                                                                                                         Ub

                                                CycB                                 CycB




                                                                                                                   poison
                                                       X
AA                                CycB                                                               CycB
                                                Cdk1                                 Cdk1 proteasome
                                                                     0
                                                                c2
                                         Cdk1              Cd

                                                       SAC                                                                       X
     CycB level & Cdk1 activity




                                                                         inhibitor




                                                                     Cdh1




                                    OFF                                  ON
                                                Cdh1
                            Conclusions

- Several different positive feedback loops are at place during the

eukaryotic cell cycle.

- They are required to guarantee the irreversibility during key cell cycle

transitions (G1->S, G2->M, exit from M).

- Irreversibility is due to the generation of bistable systems (?).

- Bistability has been experimentally demonstrated in the G1->S and in

the G2->M. No clear data available, yet, on the exit from mitosis.
  Bela Novak
Oxford University
Major phases of Mitosis




                          from www.sparknotes.com