(3) Lecture 20: Cell Cycle #2 (G1- Cdk, G1/S- Cdk, S-Cdk)

pre-replication complex

*G1* 1) ORC attaches to origin of replication 2) E2F synthesizes cdc6 3) cdc6 bind to ORC to recruit *MCM helicase*, which make the *pre-RC* (ready to fire) 4) Activation of *S-Cdk* marks start of S-phase 5) S-Cdk *phosphorylates* cdc6 and causes its destruction by *ubiquitination* 6) ORC is also phosphorylated to inhibit cdc6 from binding 7) *S phase* -- repliaction happens 8) Everything is reset after M-phase

CAK

- Cdk Activating Kinase -- phosphorylated CDK at it's active site

wee1

- Cdk- *inhibitory* kinase - places a phosphate on M-*CDK* to inhibit it -- is removed by cdc25

*unsure of this* If a chromosome is unattached to spindle, what is repressed

- activity of E3 ubiquitin ligase APC/ C

In all 3 major events that occur in the cell cycle how are the cyclin - Cdks used to activate multiple proteins needed for those stages

- as mentioned before, as the cell goes through the cell cycle, it is producing an abundance of enzymes (*S-Cdk, M- Cdk, APC/ C and others*) to be activated at their respective moment this way whenever the cell enters that phase the multiple cyclin - cdk complex will be ready to activate thousands of other proteins that are needed to ensure success of that phase

G1- Cdk: what is it? what does it phosphorylate? consequence of its activaiton ?

- produced in response to growht signals -- phorphorylates *Rb* -- *E2F* activation at low levels *&* triggers synthesis of G1/S- cyclin

cdc25

-- *phosphatase* -- removes inhibitory phosphate from M-cdk to *activate* it -- however cdc25 has to be *activated* by *M-cdk* -- how does this work ? next card

E2F proteins that constitute positive feedback are

-- E2F (activates more transcription factors for E2F) -- G1/S- Cdk -- S-Cdk * last two activates more transcription factors for E2F to trasncribe more cyclin

how is Rb inactivated to stop inhibiting E2F

-- G1 - Cdk phosphorylates it to make it *inactive*

Some of the important genes Myc transcribes are

-- G1- Cyclin (leads to activation of CdK) -- SCF gene (increases CKI degradation) -- E2F gene (increase in E2F)

CKI for S-Cdk is phosphorylated by..... to activate S-Cdk. What happens to CKI after it is completely phosphorylated?

-- G1/S- Cdk -- it becomes inactive and then it is *ubiquitylated* by *SCF* to be degraded through the ubiquitin-proteosome pathway

____ prevents entry into S phase, while ____pushes entry into S phase

-- Rb -- E2F

what ensures that wee1 does not rephosphorylate M-cdk after cdc25 (note cdc25 vs cdc6 are different)

-- active M-Cdk inhibits wee1 through *positive feedback*

SCF transcription by Myc leads to what

-- degradation of CKI by ubiquitylating it then sending to protesome -- leads to G1/S- Cdk activation (*start*)

Myc

-- important transcription factor -- *master* regulator of the cell cycle -- drives the transcription of multiple genes that push the cell into the *S phase*

M-Cdk: what is it? what does it phosphorylate? consequence of its activation ?

-- marks beginning of M-phase -- its *degradation* marks the end of *Metaphase* -- phosphorylates *ORC, Cdc6, Kamin, Condensin & more* -- Mitosis

what does S- Cdk do for S-phase ?

-- phosphorylates/activates DNA polymerase, Primase, etc. that's why you need a large stockpile of S-Cdk

Proto oncogene

-- protein that is inducing cell cycle --E2F

how is it that chromosomes are only replicated once and not twice?

-- there's a pre-replication complex at *each origin* that can only be used once

E2F is what? produces what? inhibited by what?

-- transcription factor -- produces *G1/S- cyclin* and *S-cyclin* which *activate* G1/S-Cdk and S- Cdk and cdc6 -- inhibited by *Rb*

how is it that cdc25 activates M-Cdk by dephosphorylating the phosphate placed by *wee1*, but cdc25 needs M-Cdk to activate it?

-- when cdc25 is dephosphorlated, at *low* concentrations of M-Cdk, cdc25 *cannot* bind M-Cdk at all, -- however, at *high* concentrations of M-Cdk, cdc25 can bind M-Cdk sowly -- once M-Cdk is activated it can activate cdc25 which dephosphorlates M-Cdk at faster rates

Regulation of S-Cdk Activation (6) describe the process of stockpile of S-Cdk until their activation

1) G1/S-Cdk promotes synthesis of S-Cyclin 2) S-Cdk complex is formed but blocked by its own *CKI* 3) The cell builds a large *stockpile* of inactivated S-Cdk 4) *G1/S-Cdk phosphorylates CKI* (at multiple sites). This takes time that allows for the stockpile of S-Cdk to build up. 5) Once CKI has fully phosphorylated it releases S-Cdk (which is then free and active). 6) Phosphorylated CKI is ubiquitylated by SCF and destroyed in the proteasome 7) S phase begins . yay

3 major transitions states are:

1) Initiation of DNA replication 2) Entry into M- phase 3) Metaphase - Anaphase transition

whole process of *M-Cdk* activation is...

1) M-cyclin is produced during *G2* 2) M-cyclin binds to M-Cdk to form inactive M-Cdk 3) M-cyclin is phosphorylated by CAK(at an active site) and Wee1 (at an inactive site) 4) A stockpile of double phosphorylated M-Cdk builds up during *G2* phase 5) At the right moment M-Cdk gets activated by a *phosphatase* called cdc25 (removes wee1 phosphate)

Just remember *phosphatase* are always involved when a *kianse* is involved Also, the cell is constantly synthesizing proteins

:P

You study a cell where G1-cdk has been activated and see that Rb is phosphorylated. If you then inhibit the activity of G1-Cdk you observe that the cell contains unphosphorylated Rb protein. Why? A) The phosphorylation is degraded B) The phosphate is removed by a phosphatase C) Phosphorylated Rb is degraded D) Unphosphorylated Rb is synthesized E) G1/S-Cdk replaced the activity of G1-Cdk

B) D)

CKI is an inhibitor of

Cdk activation

What would be the effect of having a reduced amount of Rb protein in a cell? A) G1-Cdk would not be active B) S-Cdk would not be activated C) G1/S cyclin would not be synthesized D) The cell would enter S-phase prematurely E) The E2F factor would be inactivated

D) less Rb > less E2F needed to go into the cell cycle

What would happen if a cell did not have Wee1? A) M-Cdk would not be activated B) M-cyclin would not be synthesized C) M-cyclin would not be phosphorylated D) The activity of M-Cdk would rise proportionally to the synthesis of M-cyclin E) M-Cdk would be phosphorylated by CAK but it would not trigger entry into M-phase

D) remember Wee1 and cdc25 affect *Cdk* not cyclin!!

MCMs =

DNA helicase

What would be the effect of making a mutant of Cdc-6 protein that cannot be phosphorylated? A) Cdc-6 would not bind to ORC B) DNA replication could not initiate C) DNA replication would start multiple times from one origin D) The cell would enter S-phase prematurely E) Cdc-6 would not be degraded

E) can't be B or C becaues ORC is phosphorylated before replication, so causes cdc6 to dissociate

(T/F) Right after G1 phase, a lot of S- Cdk are produced and activated to begin "S" phase

FALSE; the whole time the cell is building up S-Cdk in the off state, and then once "S" phase begins, they are all turned on.

positive feedback

Feedback that tends to magnify a process or increase its output.

Growth hormones + favorable extracellular environment activate what

G1 - Cdk

If a favorable extracellular enviornment is present and growth hormones are present ...... production increase.

G1- Cdk

The *start* point (AKA the commitment point) of the cell cycle occurs in the production of ..... which makes .....

G1/S Cyclin G1/S Cdk * at this point the cell is committed to go forward through cell division!

M-cylcin is synthesized during what phase

G2

a stockpile of double-phosphorylated M-Cdk accumulates during what phase

G2

Ras activation leads to an activation of ...

MAP kinase

MAP Kinase leads to the production of what transcription factor

Myc

so is cdc6 an inhibitor of DNA replication ?

No, cdc6 is the *one way* ticket he's talking about. That's why replication only occurs once. cdc6 recruits MCMs (DNA helicases). Then ORC and cdc6 get phosphorylated so no more cdc6 will bind and recruit DNA helicases. *BOOM*

every origin of replication is labeled by a large protein complex (kind of like a histone complex) called

ORC *O*rigin *R*ecognition *C*omplex

Growth hormones lead to the production of *Myc* through what pathway ?

Ras/ MAP Kinase Signaling Pathway

A tumor supressor protein is....

Rb -- becaue it prevents cell cycle from continuing

G1- Cdk phosphorylates to inactivate _____ which activates _______ to create ______ which ultimately activated what Cdk?

Rb; E2f; G1/S-cyclin; G1/S Cdk

E2F marks entry into what phase

S phase

after G1/S- Cdk has passed, what is activated next?

S- Cdk

DNA re-replication is inhibited by what

S- Cdk M- Cdk

what is the job of S- Cdk?

S- Cdk phosphorylates (activates) proteins that are needed in DNA replication i.e. DNA polymerase Ligase

Active S-Cdk promotes entry into

S-phase

Ras

a GTPase -- activated when bound to GTP -- inactive when bound to GDP

cdc6

binds to origin or replication

pre-ORC can *ONLY* be formed when

cdc6 binds *AND* then recruits MCM helicase

(T/F) Once production of G1-Cdk has begun, the growth hormone can go away and the production of G1- Cdk will still continue

false; needs to be a stable signal until the production of G1- Cdk induces *G1/S cyclin* production which will eventually create *G1/S- Cdk*

(T/F) *ONLY* G1- Cdk can phosphorylate Rb

false; once enough G1/S- Cdk is produced, it can take over and phosphorylate Rb too

(T/F) Whether inactive or active, M-Cdk inhibits wee1

false; only *active* M-Cdk inhibits wee1 activity

in G2 we observe a (low/high) concentration of wee1 and a (low/high) concentration of cdc25

high; low

in M-phase we observe a (low/high) concentration of cdc25 and a (low/high) concentration of wee1

high; low

when is the pre-replication complex formed?

in G1!!!!!!!!

DNA damage does what

inactivates or represses G/ S - Cdk S- Cdk M- Cdk

unphosphorylated cdc25 has (low/high) affinity for M-Cdk

low

DNA replication starts at replication ------ and proceeds (uni/bi) directionally

origins, bidirectionally

What happens when G1 - Cdk is activated?

synthesis of: G1 / S cyclin & S cyclin

(T/F) Bacteria chromosomes have only *ONE* origin of replication while humans have *MORE* than one because they have larger chromosomes

true

(T/F) Both E2F and G1/S-Cdk boost the expression and activation of more E2F (and inhibition of Rb), leading to transcription of *S-cyclin* & *cdc6*

true

(T/F) In order for a cell to undergo cell division it has to be in the *right place* (favorable extracellular environment) or receive a signal from *growth hormoneS*

true

(T/F) Rb defects makes it more susceptible to getting cancer

true

(T/F) There's enough S-cdk to start Sphase and to prevent a second round of replication

true

(T/F) Unreplicated DNA does not go through Mitosis because M- CDK prevents it

true

(T/F) only a little bit of E2F is needed to transcribe *G1/S- Cyclin* but *a lot* more E2F is needed to transcribe *S-cyclin* & *cdc6*

true

(T/F) ubiquitination only occurs on *phosphorylated* molecules

true

(T/F) when cdc25 is phosphorylated (active), it can dephosphorylate M-Cdk at high *or* low concentrations of M-Cdk

true

(T/F) Yeast have only one Cdk protein while animals have many

true; as mentioned before cdk's on yeast can be used all throughout the cycle while only the cyclin changes but animals have different Cdks

(T/F) ORC exists all the way through the cell cycle

true; but after S phase it remains *phosphorylated* the whole way through so . *M-Cdk & S-Cdk* makes sure it is phosphorylated

Rb phosphorylation by G1- Cdk has to occur until what point

until G1/S- Cdk takes over and phosphorylates Rb too! * because remember positive feedback