CELL CYCLE A D ITS REGULATIO Readings Karp chap

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					                                  CELL CYCLE A D ITS REGULATIO

Readings: Karp chap 14; esp. pp 570-579, 590-594 ( Exptl. History: 609-612)

I. Intro (dealing with euk cells only; will emphasize simple expts, "big picture", and "main themes". Thus,
        lecture will present a more simple version of a very complex and fast-moving field)
All cancer cells – regulation not working; continue to go through cell cycle; don’t respond to normal signals like
untransformed cells

A. Stages: defined by activity of nucleus (Fig 14-1)

                               Mitosis
                                                              G1 "sibling cell"

               G2                                             G1 <--------------------> "G0"

                               "S" ,DNA                                              non-dividing
Interphase                      synthesis                                            non-cycling

   dividing, cycling

Arbitrary how you define stages of cell = most common: what happens w/ regard to nucleus and DNA content
       - 50 yrs ago = advent of precursors
       - DNA incorporation didn’t occur in all cells; when analyzed time frame
               - S phase = DNA synthesis; doesn’t occur immediately after mitosis
               - G1 phase = gap 1
               - G2 phase = gap 2
               - G0 phase = cells withdrawn from cell cycle; highly differentiated; not permanent; cells can be
induced to reenter the cell cycle

B. How determined?
   1. mitosis: see in microscope
   2. S: =DNA synthesis, so incorporate DNA precursors          (e.g. 3H-thymidine; use autorad, or count)
   3. Amt of DNA: G1 = 2N , G2 = 4N , S = between 2N and 4N
                       (Assay single cells using fluorescent “cell sorter/analyzer” )
Mitosis can be seen in microscope
S = DNA synthesis, so look at incorporation of DNA precursors (3H-thymadine)
Look at amt of DNA in cell; amt of staining proportional to DNA (quantitate amt of DNA in cell)
In S phase, exp. measure amt of DNA in b/w 2N and 4N

How long cell cycle: briefly label population of cells and follow those as they progress thru cell cycle
Label cells w/ short pulse – only those that are in S and remove label precursor and allow cell to grow and go
thru cell cycle; assay cells that are in mitosis and how many are labeled
Start at 0 b/c all in S not M => more cells in mitosis that are labeled (50% = min time for G2); S is till 12 hrs
Entire cycle done w/ cell cycle at ~ 27

C. Properties:
   1. Timing: S,G2, and M usually fairly constant (e.g. S=6 hrs, human)
               G1 variable: very short/none to very long
                        G0 = no division
   2. Synthesis: all molecules, except DNA, synthesized in each stage except M. DNA only in S.
Stage very variable in length = G1 = decision to go thru cell cycle or not
Normal DNA replication: all stages except mitosis same
Some that depend on cell cycle = regulatory things

II. TRA SITIO S and their REGULATIO
What regulates and controls moving from one stage to next
Mitosis can start but not finish until all checkpoints completed

A. G1 (or G0) ---> S ; probably most impt "decision" pt for cells
              "start site", "restriction pt","to divide or not", etc

                Cancer cells = uncontrolled growth; do not stop here as do normal cells when "told to stop".
                      (also, some cancer due to cells not “dying” when supposed to; apoptosis)

Occurs in various stages of G1; cell deciding if going to stay in G1 or go thru S
If moving single nuclei, looking at what going on in DNA replication
If fusing cells together (mixing nuclei and cytoplasm) = have inhibitors; drive nuclear functions

G1 nucleus transplanted into S cytoplasm: capable of making DNA synthesis but something present/absent that
keeps it from doing it
S nucleus transplanted into G1 cytoplasm: DNA synthesize in S nucleus stops
Fuse G1 cell w/ S cell = both nuclei synthesizes DNA
S nucleus transplanted into G2 cytoplasm = DNA synthesis continues in S nucleus
Block protein synthesis in G1 cells = block transition of G1 to S

B. G2 ---> M Transition ("Why and when to begin mitosis?")

   1. Expt suggesting a "checkpoint" before M :
       a. Fuse G2 cell with S cell (e.g. tissue culture cells)
       b. results: S nuclei finishes DNA synthesis; but G2 nucleus does OT start DNA syn.

           also: this cell with both nuclei waits until S nucleus has finished its DNA syn, BEFORE starting M.

       c. Interp: There is a checkpoint before M can start, monitoring whether DNA syn is complete

Gives info to whether or not cell monitors everything before going on (checkpoint)
Fuse G2 cells w/ S cell nuclei = S nuclei makes DNA replication but G2 nucleus doesn’t start DNA replication;
cell doesn’t go to mitosis at normal time of G2 but cell waits until S phase finishes DNA replication
So, some type of checkpoint that tells it that it is done finishing all DNA replication

   2. Expt suggesting a "mitosis promoting factor" ( MPF ) which is dominant and positive
       a. fuse M cells with cells in other stages (G1,S,G2)
       b. result: cells driven into mitosis; "premature chromosome condensation", nucl envel breakdown, etc.
               EVEN THO DNA replication had not begun (G1),or not complete (S).
                                        Figure 14-3

Expt confirmed that there is a + super strong factor that promotes cell to go thru mitosis
Took cells in mitosis and fused w/ cells in other stages; (expt figure out which cells in what stage)
Results = G1 + mitosis cell = see metaphase human CH but also cell premature CH condensation
M + S phase = some replicated and duplicated and some not and when hit w/ condensation signal, in a mess

+ promoting factor that drives cells into mitosis

III. Properties of Factors regulating transition: MPF, as example

A. Factors have been cloned, purified;    are active when injected into cells -- e.g. inject MPF, mitosis induced

   1. MPF composed of 2 subunits:
       a. protein kinase (= "Cdk", cyclin-dependent kinase)
                               (= "cdc2 protein", and other names)
               its concentration does not change during cell cycle, BUT

               its activity is influenced by phosphorylation,       A D             the presence of:

    b. "cyclin" protein
          its conc varies during cell cycle; mitosis cyclin is quickly destroyed in mitosis (metaphase); and
                resynthesized during next interphase
                                       (Fig 14-4,5)
         destruction via ubiquination/ proteasome pathway and specific SCF, APC complexes

Activity capable of driving cells into mitosis (premature condensenation) made up of two diff. subunits: protein
kinase and cyclin protein
       -       CDK; CDC2 protein = cyclin dependent kinase
               o       When analyzed concentration of this protein, it was constant; didn’t vary in concentration
                   thru cell cycle (contrast to the other component of complex = cyclin)
       -       Cyclin = concentration does change thru cell cycle
               o       Concentration gradually increases
       -       Activity of MPF = 0 at G1, S and starts increasing at G2 = what expect to drive cell to mitosis

   2. Situation even more complex: each transition can have its own "promoting factor" composed up of the
        same or diff Cdk and a specific "cyclin-type" subunit. For example: (Fig 14-8)
                      mammals:
                       a. "Start point"       has: D-cyclin + Cdk4,6
                       b. G1--> S                    E- cyclin   Cdk 2
                       c. S phase                    A- cyclin    Cdk 2
                       d. G2-->M (MPF)               B-cyclin + Cdk1 (=cdc2 of yeast)

   so diff Cdk's (1 in yeast, >3 in mammals)
      diff cyclins (1 in yeast, >8 in mammals)
      diff kinase (promoting factor) complexes

               (Some partial redundancy possible for Cdk’s and cyclins)
               (e.g., too much D-cyclin reported to act as an oncogene, "car gas petal depressed")
In humans, many CDK and cyclin
Variations = subtle
Too much of D cyclin = drive cells thru start cycle; doesn’t stop (act as oncogene = tumor more active)
B. Activity of these cell cycle kinases (factors) regulated by other proteins:

       1. "CDI" -- cyclin-dependent kinase inhibitor:                   (Fig 14-10)
             --binds to complex, blocking its kinase activity
             --so can act as a tumor suppressor (p21,p27) = "car brake"

Protein binds to CDK-cyclin complex and prevents it from acting
Not get fully active MPF if this is bound
Acts as tumor suppressor (prevents CDK from active = prevents cells from going thru cycle) = eq. to break on
car

       2. protein kinases that phosphorylate specific amino acids in CDK:
                                    (Fig 14-6)
              yeast gene "Mo15" phosphorylates threonine#161 which activates, if no tyr-14,15-P
                      (called “CAK” for Cdk-activating kinase)

               But:

               yeast gene "Wee1" phosphorylates tyrosines #14,15 (close to ATP binding site),
                       so causes dominant inhibition of MPF activity

                so if this Wee1 kinase is mutant/defective (but CAK is OK), CDK more active or active earlier,
                        so cells divide earlier and so are smaller when divide.
Activity also influenced by state of phosphorylation: 2 sites on CDK that can get phosphorylated
CDK + cyclin still not active w/ 2 sites: thr and tyr
161 Tyr phosphprylated = active
Phosphorylation at 14, 15 tyr messes up activity of Wee1

       3. phosphatases -- remove specific PO4 groups
              ( e.g. yeast gene “cdc25", removes P from tyr-14,15-P, so activates MPF if threo-161P present).
              So if this defective/mutant, MPF never activated, and cell never enters mitosis.

If both sites phosphorylated, still inactive
Phosphatase activity = only Thr161 phosphorylated = active if no inhibitory activity
inhibits kinase wee1 but stimulates cdc25

       4. MPF acts autocatalytically to stimulate its own activation

               analogy: slow burning fuse -------> explosion
                      as [cyclin] increase       when cyclin > threshold

                result: strong, definite effect at precise time, "switch turned on"
Net result = activity of MPF turned on in sharp way
More available to stimulate = more active
So, have strong definite switch = integrating activity of kinase, phosphatase

       5. Destruction of cyclin crucial as well, for cells to move thru each stage:
              a. e.g., if MPF cyclin is not degraded, cell never leaves mitosis (stuck in metaphase)
                       e.g., by APC complex (Anaphase promoting complex; ubiquitination and proteasomes).
              b. MPF stimulates the destruction of its own cyclin, as well
Equally important = how to turn it off (in G1 and G2 don’t want MPF activity)
If MPF cycle not degraded, cell stuck in mitosis
       Make mutations of MPF cycle where ubiquitin activity = stuck in metaphase
       APC complex

C. Lots of inhibitory feedback controls to ensure that each stage is completed before starting the next stage.
        = “Checkpoints”              ( "washing machine control" analogy)

Enter M: G2 checkpoint = is cell big enough, is environment favorable, is all DNA replicated?
Exit M = Metaphase = are all CH aligned at metaphase

       1.e.g. --DNA replic completed before G2-->M
              --no damaged DNA for G2-->M
              --cells big enough, and enough nutrients for G1-->S1, G2-->M
              --mitotic spindle "OK", chromosomes at metaphase plate for cells to finish and exit M

       2. Nature of "signalling", "checkpoint monitoring" still unclear; probably complicated, redundant.
                       e.g. steps in checkpoints induced in response to ionizing radiation involve many
                       proteins, kinases(phosphorylation), inhibitions, stimulations, degradation, etc. at
                       several transitions and cell-cycle stages. (G1->S, thru S, G2->M)
                               (e.g. Fig 14-9 for 2 DNA damage checkpoints)

D. Substrates of the various cyclin-CDK complexes not fully known:

       For MPF, thought to include:
            nuclear lamins (for dissociation necessary for nucl.envel.breakdown)
            histone H1, etc (for chromo condensation ? )
            tubulin ( for mitotic spindle assembly)

MPF = kinase which phosphorylate: nuclear lamina breaks down (so, nuclear lamins); histone H1, etc (for CH
condensation); changes in tubulin population (mitotic spindle)

IV. Other Cell Cycle Topics/Questions:

A. More information about how “protein destruction” is an important part of the mechanism of cell cycle
      control:

APC (Anaphase promoting complex): is a multimeric protein complex with at least 2 different “adapter”
proteins that determine what it does.
What tells it to start separating CH at anaphase:

    This complex required for cells to begin Anaphase and finish Mitosis.
  HOW ? A S: it acts as an ubiquitin ligase, and so tags specific proteins for destruction by proteasomes.

Two known systems:

1. When the adapter protein is Cdc20, it allows the “sister” chromatid to separate at the beginning of Anaphase.
HOW?       (Fig 14.26)
      Cdc20 made late in cell cycle, bind to APC, and causes it to tag “securin” , a protein that inhibits the
       activity of a protease (“separase”) which is specific for the cleavage of the protein (“cohesin”) that
       holds the sister chromatid together. So when securin is destroyed, separase can work cutting cohesin and
       thus allowing the sister chromatids to “separate” / move apart.

   This form of APC is also involved in the spindle attachment checkpoint:
               Unattached chromosomes have a Mad2 protein present that inhibits APC-Cdc20 from working
               (allowing Anaphase to begin), so until all chromosomes are attached and all Mad2 is gone, APC-
               Cdc20 cannot work.
APC complex still links ubiquitin to tag for destruction
Which substrate it tags determined by adaptor protein (need Cdh1)
Cdc20 tags securin for destruction; securin = protease that destroys protein that links sister proteins together
(cohesion)

Monitoring check point if spindle fibers attached
Mad2 inhibits APC from active, when Mad2 all destroyed = go through whole

2. When adapter protein is Cdh1 (after Cdc20 gets destroyed), the APC-Cdh1 tags for destruction the mitotic
cyclins, and so the activity of the mitotic Cdk’s is gone ---- which is necessary for the cells to finish Mitosis,
and to “reset” themselves for G1, etc. Ultimately, Cdh1 is itself destroyed, eliminating the activity of APC until
the next Mitosis and new Cdc20 and Cdh1.