Phosphorylation of CDK Targets Changes Their Activity Now performs a cell cycle function

Yeasts have one CDK and several cyclins Humans have 4 CDKs and 4 cyclins

CDK/Cyclin complex regulation controls the cell cycle - Cyclin-Cdk complexes function in different phases - G1/S-Cdk complexes commit the cell to a new cell cycle - S-Cdk complexes promote S phase (and block it in G2!) - M-Cdk complexes trigger entry into mitosis - M-Cdk complexes are inactivated before anaphase

Example: cycB-Cdk1 appears in mitosis, phosphorylates lamin and leads to nuclear envelope breakdown during early mitosis cycB-Cdk1 will be destroyed during mitosis to allow formation of a new nuclear envelop breakdown during telophase

How are CDK’s Regulated? By cyclin synthesis and destruction By phosphorylation By binding to CDK inhibitory proteins (CKIs)

Generation of a “Cycling” Frog Egg Extract Inject females with hormones so that they lay eggs 2. Pack eggs into a centrifuge tube and spin 4. Add sperm chromatin and away you go! 3. Remove Cytoplasmic Extract

Cyclin Synthesis and Destruction Is Essential for Cell Cycle Progression sea urchin! sea urchin!

But what is ubiquitylation??!! Cyclin Destruction is Controlled by Ubiquitylation But what is ubiquitylation??!!

Ubiquitylation: a post-translational protein modification E3 ubiquitin ligase is a protein complex that confers specificity: i.e. which protein to target Proteasome: the cellular garbage can E1, E2, and E3 enzyme cascade

An E3 ubiquitin ligase called the Anaphase Promoting Complex (APC) destroys mitotic cyclins (and other things) Not to be confused with: APC (adenomatous polyposis coli) APC (antigen-presenting cell)

How does the APC function? M APC M Cyclin and securin must be destroyed in order for anaphase to take place

APC Activity changes during the cell cycle Negative feedback generates a repeating oscillator

The Cell Cycle According to Oscillating Cyclin/CDK and APC Activity A cyclin promotes synthesis of the next cyclin that in turn, promotes destruction of the previous one

Once and Only Once S phase is Controlled by CDKs

NO! The Cell Cycle According to Oscillating Cyclin/CDK and APC Activity But is cyclin abundance the only way to control CDK activity? NO!

How else are CDKs Regulated? Genetic studies in fission yeast.

CDKs are Regulated by Phosphorylation is a kinase is a phosphatase CAK Activating Kinase) is a kinase is a phosphatase

Conformational Changes Associated with CDK Phosphorylation Free CDK CDK + Cyclin T161 phosphorylation The T-loop blocks substrate access Binding of cyclin moves the T-loop Phosporylation moves the T-loop more

How else are CDKs Regulated? Biochemical studies in mammalian cells. Figure 8.8 The Biology of Cancer (© Garland Science 2007)

Cyclin Dependent Kinase Inhibitors (CKIs) CDKs are regulated by Cyclin Dependent Kinase Inhibitors (CKIs) p21 Cyclin CDK CDK p21 Cyclin p16 Cyclin CDK4 CDK4 p16

The Discovery of p21 and p16: what binds to CDKs? Transformed Transformed Cultured cells Cell Line Normal Normal Competing - + - + Adding 35S[Met] peptide Cyclin CDK4 p21 Metabolic labeling Lysis cells midly Add anti-CDK4 antibody Add protein A-agarose beads Immunoprecipitate Cyclin D SDS-PAGE CDK4 Autoradiography p21 p16 Xiong et al. (1993) Genes & Dev. 7:1572

The p21 Family of CDK inhibitors (p21CIP1/WAF1, p27KIP1, p57KIP2) CDK Cyclin active p21 + inactive CDK Cyclin

Jeffrey et al. (1995) Nature 376:313 Russo et al. (1996) Nature 382:325 Figure 8.13b The Biology of Cancer (© Garland Science 2007)

+ + The INK4 Family of CDK inhibitors (p16INK4a, p15INK4b, p18INK4c, p19INK4d) CDK4/6 Cyclin D active CDK4/6 Cyclin D INK4 + inactive INK4 + Russo et al. (1998) Nature 395:237 Brotherton et al. (1998) Nature 395:244

CKIs Regulate the G1-S Transition (p16) (p21, p27)

p16 is Frequently Mutated in Human Tumors

Therapeutic Targeting of the Hallmarks of Cancer Hanahan and Weinberg, Cell 144:646 (2011)

Chemical structures of small molecule cdk inhibitors Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

http://www.clinicaltrials.gov/ 141 studies for CDK inhibitors

Growth Factors Induce Cell Proliferation act Prior to the Restriction Point Modified from The Biology of Cancer (© Garland Science 2007)

Cell Division + Growth = Let’s think about the Difference Between Growth and Cell Division Growth with No Cell Division Cell Division No Growth Cell Division + Growth = Proliferation!

A Differentiated Neuron Growth with No Cell Division: A Differentiated Neuron

Cell Division with No Growth: Early Development OOCYTE GROWS WITHOUT DIVIDING (MONTHS) FERTILIZED EGG DIVIDES WITHOUT GROWING (HOURS) FERTILIZATION 1 mm sperm tadpole feeds, grows and bcecomes an adult frog

Unregulated Proliferation: Cancer! A Typical Solid Tumor…Needs to Grow The size of a tumor first detectable by X-ray: 108 cells The size of a tumor first palpable: 109 cells The size of tumor at death of patient: 1012 cells Unregulated Proliferation: Cancer!

Budding Yeast Saccharomyces cerevisiae

Cdc Mutants Affect the Cell Cycle, Not Growth Permissive (low) temperature Restrictive (high) temperature

Mammalian Cells Growing in Cell Culture Amino Acids Vitamins Salts Miscellaneous Arginine Cystine Glutamine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine Biotin Choline Folate Nicotinamide Pantothenate Pyridoxal Thiamine Riboflavin NaCl KCl NaH2PO4 NaHCO3 CaCl2 MgCl2 Glucose Penicillin Streptomycin Phenol red Whole serum Specific growth factors 1961Hayflick and Moorhead Showed that human fibroblasts die after a finite number of divisions in culture. This is called “The Hayflick Limit”

Some Commonly Used Cell Lines Cell Type and Origin 3T 3 fibroblast (mouse) BHK 21 fibroblast (Syrian hamster) MDCK epithelial cell (dog) HeLa epithelial cell (human) PtK 1 epithelial cell (rat kangaroo) L 6 myoblast (rat) PC 12 chromaffin cell (rat) SP 2 plasma cell (mouse) *Many of these cell lines were derived from tumors. All of them are capable of indefinite replication in culture and express at least some of the differentiated properties of their cell of origin. BHK 21 cells, HeLa cells, and SP 2 cells are capable of growth in suspension; the other cell lines require a solid culture substratum in order to multiply.

Growth Factors Induce Cell Proliferation Growth + Cell Cycle Progression: But How? Growth Factors act Prior to the Restriction Point Modified from The Biology of Cancer (© Garland Science 2007)

Growth Factors Induce Gene Expression

Growth Factors Induce Oncogene Expression As Early Response Genes

Growth Factors Induce Cyclin D Expression They also act by inhibiting CKIs We actually have: 3 D-type cyclins 2 E-type cyclins 2 A-type cyclins 3 B-type cyclins And the D-type cyclins respond to different cues

Growth Factors Get the Cell Cycle Going

ONCOGENES!!!! Growth Factors Induce Cyclin D Expression So, is Cyclin D an oncogene?

YES! Cyclin D is also an Oncogene Disease: INVOLVED IN B-LYMPHOCYTIC MALIGNANCY (PARTICULARLY MANTLE-CELL LYMPHOMA) BY A CHROMOSOMAL TRANSLOCATION T(11;14)(Q13;Q32) THAT INVOLVES CCND1 AND IMMUNOGLOBULIN GENE REGIONS (BCL1 ONCOGENE). Disease: INVOLVED IN A SUBSET OF PARATHYROID ADENOMAS BY A CHROMOSOMAL TRANSLOCATION T(11;11)(Q13;P15) THAT INVOLVES CCND1 AND THE PARATHYROID HORMONE (PTH) ENHANCER (PRAD1 ONCOGENE).

Translocations Cause One Gene to be Controlled by Another