APC = anaphase-promoting complex

Not to be confused with:APC (adenomatous polyposis coli)

APC (antigen-presenting cell)

The different cyclins are degraded by two different E3 ligases

e.g. cyclin B, the G2/M cyclin is degraded by

How does APC function?

APC

The cyclin must be degraded in order for anaphase to take place

M

M

ProteinLevel

Time

cyclin A cyclin B

M M M

Remember?cyclins must be removed for mitosis to

be completed

Negative feedback generates a repeating oscillator

CDKs are positively regulated by cyclins

A Cyclin promotes synthesis of the next cyclin that in turn, promotes destruction of the previous one

These regulatory activities are indirect

Mechanisms of CDKs regulation

1. Abundance of cyclins

2. CDK phosphorylation

3. Binding to CKIs (inhibitory proteins)

CDK

Cyclin

active

p21+

inactiveCDK

Cyclinp21

Activating phosphorylation is catalyzed by Cdk-Activating Kinases (CAKs). However, they are

abundant and not regulated

Cyclin

Cdk

Phosporylation of Thr by CAK

Substrate binding to the kinase

1

2 4

3

Inhibitory phosphorylation is also involved in CDKs regulation

- The Wee1/Cdc25 switch event is regulated by substrates and extrinsic signals

- e.g. Phosphorylation by Wee1 Tyr kinase blocks the CDK’s active site

- e.g. Cdc25 is a phosphatase that removes this inhibitory block

M-CDK

Mechanisms of CDKs regulation

1. Abundance of cyclins

2. CDK phosphorylation

3. Binding to CKIs (inhibitory proteins)

CDK

Cyclin

active

p21+

inactiveCDK

Cyclinp21

Cdk inhibitor proteins (CKIs)

- Discovered by asking : “what binds to CDKs”?

- The INK4 family proteins (e.g. p16) bind to CDK4/6, blocking cyclin D binding

- The Cip/Kip family proteins (e.g. p21, p27) bind to blocking active site of multiple CDKs

- CKIs normally regulate entry into S phase

CKIs Regulate the G1-S Transition

(p16)

(p21, p27)

p16 is Frequently Mutated in Human Tumors

Tumor t ype L ines (n) Del etion s (n) D eleti ons (%) Astrocytoma 17 14 82 Bladder 15 5 33 Breast 10 6 60 Colon 20 0 0 G lioma 35 25 71 L eukemia 4 1 25 L ung 59 15 25 Melan oma 99 57 58 N euro blast oma 10 0 0 O steosarcoma 5 3 60 O vary 7 2 29 Renal 9 5 56 Total 29 0 13 3 46

Table 1. D eleti ons i n tumor cells and p rimary tumors.

See Kamb et al. (1994) Science 264: 436; Nobori et al. (1994) Nature 368:753 for detail

9p21

Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

Chemical structures of small molecular cdk inhibitors (none approved so far)

Summary

- The cell cycle is controlled by Cdks, activated by cyclins and CAKs, and inhibited by CKIs

- Cyclins are positively and negatively regulated by cyclin-Cdks complexes

- Any process in the cell cycle is dependent on the previous one

- The cell cycle progresses in the right order

Cyclins regulate other cyclins, both negatively and positively

But, it cannot be just an intrinsic cell cycle clock…

Mitogens stimulate the onset of the cell cycleIn this case, we are very different from yeasts

Mitogens control cyclin D expression

- Mitogens act by activating the D-Cdk4/6 complexes

- Mitogens act by inhibiting CKIs

- Mitogen signaling is correlated with growth, answering the question: “have I grown enough?”

Mitogens control cyclin D expression

We actually have:

3 D-type cyclins 2 E-type cyclins2 A-type cyclins3 B-type cyclins

START/Restriction point

w/o growth signals, sub-threshold levels of enzymes will lead to quiescence (G0)

cyclin D and growth

- Activated D-Cdk4/6 initiates transcription of cyclin E and activation of E-Cdk2

- Activated E-Cdk2 allows progression through START

- From here on, it’s a cell cycle clock game

In the next lectures we will focus on the molecular machinery that acts at the START point in normal

and cancer cells