Phosphorylation of CDK Targets Changes Their Activity

Now performsa cell cycle function

How are CDK’s Regulated?

1. By cyclin synthesis and destruction

2. By phosphorylation

3. By binding to CDK inhibitory proteins (CKIs)

Generation of a “Cycling” Frog Egg Extract

1. Inject females with hormonesso that they lay eggs

2. Pack eggs into acentrifuge tube and spin

3. RemoveCytoplasmic Extract

4. Add sperm chromatinand away you go!

Cyclin Synthesis and DestructionDrives the Early Embryonic Cell Cycle

sea urchin!sea urchin!

Cyclin Destruction is Controlled by Ubiquitination

But is cyclin abundance the only

way to control CDK activity?

The Cell Cycle According to Cyclin Abundance

How are CDKs Regulated?

Isolate mutants that phenocopy cdc2- alleles

CDKs are Regulated by Phosphorylation

is a kinase is a phosphatase

CAK(CDKActivatingKinase)

Conformational Changes Associated with CDK Phosphorylation

The T-loop blockssubstrate access

Free CDK CDK + Cyclin T161 phosphorylation

Binding of cyclinmoves the T-loop

Phosporylation movesthe T-loop more

How does the G1-S Transition Work?

but…S. pombe cdc2+ can subsitute

for S. cerevisiae cdc28 and vice versa

cdc2 mutants arrest in G2

cdc28 mutants arrest in G1

Why?

and, cln1 and cln2 !!

The Identification of G1 Cyclins in S. cerevisiae

How Does High Copy Suppression Work?

The S. cerevisiae Cell Cycle

The G1-S Transition in S. cerevisiae

Growth Signal

Cln3/Cdc28

Cln1/Cdc28Cln2/Cdc28

make a bud

SIC1

G1

S

Clb5/Cdc28

Clb6/Cdc28

(cyclin/cdk)

Sic1 is Destroyed by Ubiquitination

(Cdc4,Cdc53)

Once and Only Once Replication is Controlled by CDKs

Human cdc2 (cdk1) Rescues cdc2 Mutant

cdk1cdk1

cdc2 mutantcells at 25C

Transformation with humancDNA library expressedwith SV40 viral promoter cdc2 transformed

mutant cells at 35C

Human cyclin E Rescues cln1,2,3 Mutant

cyc Ecyc E

cln1,2,3 triple mutantcells at 25C

Transformation with humancDNA library expressed

with yeast promoter cln1,2,3 transformed mutant cells at 35C

The Human Cell Cycle

Cyclin Dependent Kinase Inhibitors (CKIs)

Cyclin

CDKp21

Cyclin

CDK4

Cyclin

CDK

CDK4p16

p16

p21

The Discovery of p21 and p16

Cultured cells

Adding 35S[Met]

Metabolic labeling

Lysis cells midly

Immunoprecipitate

Add anti-CDK4 antibody

Add protein A-agarose beads

SDS-PAGE

Autoradiography

CDK4p16

Xiong et al. (1993) Genes & Dev. 7:1572

Cyclin

CDK4p21

CDK4

Cyclin D

p21

p16

Cell Line

No

rma

l

Tra

ns

form

ed

Tra

ns

form

ed

No

rma

l

Competing peptide - -+ +

-CDK4

The p21 Family of CDK inhibitors(p21CIP1/WAF1, p27KIP1, p57KIP2)

CDK

Cyclin

active

p21+

inactive

CDK

Cyclinp21

p27Kip1

Cyclin ACDK2

Russo et al. (1996) Nature 382:325

Cyclin A

CDK2

Jeffrey et al. (1995) Nature 376:313

The INK4 Family of CDK inhibitors(p16INK4a, p15INK4b, p18INK4c, p19INK4d)

INK4+CDK4/6

Cyclin D

active

CDK4/6Cyclin D

INK4+

inactive

Russo et al. (1998) Nature 395:237Brotherton et al. (1998) Nature 395:244

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