Gene NomenclatureGene Nomenclature

Budding Yeast:Budding Yeast:CDC28 = wild type geneCDC28 = wild type genecdc28 = recessive mutant allelecdc28 = recessive mutant alleleCdc28 or Cdc28p = CDC28 proteinCdc28 or Cdc28p = CDC28 protein

Fission Yeast:Fission Yeast:cdc2 = the wild type genecdc2 = the wild type genecdc2cdc2-- = recessive mutant allele = recessive mutant allelep34p34cdc2cdc2 = the protein = the protein

Non-genetic systems:Non-genetic systems:not standardnot standarduse either yeast, or use unique name with yeast name superscriptuse either yeast, or use unique name with yeast name superscriptor whateveror whatever

Regulation of G1 to SRegulation of G1 to SRole in G1 for cdc2/cdc28 at START

But cdc2/cdc28 regulators acted at G2 to M ONLYcdc2Dcdc13/B-type Cyclinscdc25 and wee1

cdc2

G2 Mitosis

wee1cdc25

cdc2

G1 S phase

????? ????? G1 CyclinsG1 Cyclins CKIsCKIs

Cyclins NomenclatureCyclins Nomenclature

Cyclin A

Cyclin B’s Vertebrate Cyclin B1, B2 S. pombe cdc13... S. cerevisiae CLB1,2,3,4,5,6

G1’s more similar to each other- PEST domains- required for exit from G1

All contain “cyclin box” domain cdc2 binding domain

A and B’s closely related-destruction box- required for S and M

Differ in expression pattern- “A” accumulates earlier,- degraded earlier

G1 CyclinsS. cerevisiae CLN1, 2, 3Mammalian Cyclin D, E

What do Cyclins do?What do Cyclins do?

1.1. Positive Regulator of CDK catalytic subunitPositive Regulator of CDK catalytic subunit

2.2. Potentially impart CDK substrate specificityPotentially impart CDK substrate specificity

3.3. Potentially impart CKD inhibitor specificityPotentially impart CKD inhibitor specificity

4.4. Potentially determine specific CDK localizationPotentially determine specific CDK localization

Cyclins can be grouped by expression patternsCyclins can be grouped by expression patterns

- CLN1 and CLN2 and CLB5 and CLB6 -

- CLB3 and CLB4 -

- CLB 1 and CLB2

CLN1,2,CLB5,6

G1 Start Metaphase

CLB3,4CLB1,2

CLN3

Figure 13-22

Yeast Cyclin Genes: CLNs and CLBsYeast Cyclin Genes: CLNs and CLBs

G1 Cyclins:CLN1CLN2CLN3 Dominant CLN MutationsDominant CLN MutationsTriple cln1,2,3 mutantTriple cln1,2,3 mutant

G2 CyclinsG2 CyclinsCLB1CLB1CLB2CLB2CLB3CLB3CLB4CLB4

Quadruple clb1,2,3,4 MutantQuadruple clb1,2,3,4 Mutant

S phase CyclinsS phase CyclinsCLB5, CLB6CLB5, CLB6NO PHENOTYPENO PHENOTYPE- double mutants- double mutants-gain of functiongain of function-????????????????????????

Role of Cyclins in G1 to S????Role of Cyclins in G1 to S????

Cdc28/Cln3

“Start-specific” Transcription

Cdc28/Cln1,2 Cdc28/Clb5,6Sic1

BuddingBuddingSPB dupSPB dup

DNADNARepRep

Regulation of G1 to SRegulation of G1 to S

Starting point of this study:Starting point of this study:

- Specific role of CLNs in regulation of G1 to S not clear- Specific role of CLNs in regulation of G1 to S not clear- triple mutant doesn’t bud, dup SPB, or rep DNA- triple mutant doesn’t bud, dup SPB, or rep DNA

- CDC34, CDC53, and CDC4 Required specifically- CDC34, CDC53, and CDC4 Required specificallyfor DNA replicationfor DNA replication

- high Cdc28/Cln activity- high Cdc28/Cln activity- Cdc34 = UBC- Cdc34 = UBC

- CLB1,2,3 and 4 not required for S Phase- CLB1,2,3 and 4 not required for S Phase- What about CLB5 and CLB6, and what’s this to do with- What about CLB5 and CLB6, and what’s this to do withCDC34 et al ?CDC34 et al ?

Clb5/Cdc28 kinase accumulates near/after StartClb5/Cdc28 kinase accumulates near/after Start

Protein Levels

Clb/Cdc28 Activityby Histone H1 Kinase Assay

Clb5,6/Cdc28 Necessary and Sufficient forClb5,6/Cdc28 Necessary and Sufficient forDNA ReplicationDNA Replication

clb1,clb3,clb4,clb5,clb6clb2(ts)GAL::CLB5

Grows at 23 deg on Grows at 23 deg on Galactose mediumGalactose medium

Dies at 37 degrees on Dies at 37 degrees on Glucose mediumGlucose medium

Cdc34 involved in Cdc34 involved in degradation of a degradation of a Cdc28/Clb inhibitor??Cdc28/Clb inhibitor??

CLBs redundant for S CLBs redundant for S Phase, with 5 and 6 Phase, with 5 and 6 most importantmost important

Inhibition of Clb/Cdc28 by Sic1 in vitroInhibition of Clb/Cdc28 by Sic1 in vitro

Cdc28/Clb specific Cdc28/Clb specific inhibitor accumulates inhibitor accumulates in cdc34 et al mutantsin cdc34 et al mutants

Inhibitor depends on Inhibitor depends on SIC1 functionSIC1 function

Sic1 is a Cdc28 Sic1 is a Cdc28 inhibitorinhibitor

In vitro In vitro demonstration of Clb-demonstration of Clb-specific inhibition and specific inhibition and Sic1/Cdc28/Clb Sic1/Cdc28/Clb complex formationcomplex formation

Implications and Predictions...Implications and Predictions...

Clb5,6 expressed in late G1

Clb5,6 major role in S phase induction after Start

Clb5,6/Cdc28 and other Clb/Cdc28s held inactive by Sic1

Sic1 function is opposed by cdc34,4,53 after Start…

Sic1 should be regulated - on before start, off after start

Sic1 regulation dependent on cdc34,4,53

Phenotype of cdc34 mutants should be dependent on Sic1

sic1 mutants should be sensitive to CLB5 over expression

Cdc34 dependent Regulation of Sic1 AccumulationCdc34 dependent Regulation of Sic1 Accumulation

Sic1 phosphorylated by Sic1 phosphorylated by Cdc28/Cln in vitroCdc28/Cln in vitro

Sic1 destroyed in a CDC34-Sic1 destroyed in a CDC34-dependent mannerdependent manner

Phosphorylation correlates Phosphorylation correlates with time of destructionwith time of destruction

Sic1 is required to prevent S phase in cdc34Sic1 is required to prevent S phase in cdc34

G2/M arrest?G2/M arrest?

Regulation of G1/SRegulation of G1/S

Cdc28/Cln3

“Start-specific” Transcription

Cdc28/Cln1,2 Cdc28/Clb5,6Sic1

BuddingBuddingSPB dupSPB dup

DNADNARepRep

only essentialonly essentialfunction of CLNs!function of CLNs!

cln1,cln2,cln3 deadcln1,cln2,cln3 deadcln1,cln2,cln3,sic1 alivecln1,cln2,cln3,sic1 alive

Mammalian regulation of G1 to SMammalian regulation of G1 to S

Compare and Contrast RolesCompare and Contrast Rolesof G1 CDKs and CKIs in G1 to S inof G1 CDKs and CKIs in G1 to S inbudding yeast and mammalian cellsbudding yeast and mammalian cells

PICK 1PICK 1CDK/CKICDK/CKIPairPair

Ubiquitin Dependent Proteolysis in Cell Ubiquitin Dependent Proteolysis in Cell Cycle RegulationCycle Regulation

Cell Free Cell Cycling ExtractsCell Free Cell Cycling Extracts

Visualizing Mitotic Spindle Formation and Visualizing Mitotic Spindle Formation and Chromosome Segregation in vitroChromosome Segregation in vitro

Cyclin is destroyed at the time of sister chromatid Cyclin is destroyed at the time of sister chromatid separation by ubiquitin-dependent proteolysisseparation by ubiquitin-dependent proteolysis

Ubiquitin-dependent proteolysis of other mitotic Ubiquitin-dependent proteolysis of other mitotic proteins control separation of sister chromatidsproteins control separation of sister chromatids

Identification of the Anaphase Promoting ComplexIdentification of the Anaphase Promoting Complex(aka Cyclosome or APC/C)(aka Cyclosome or APC/C)

Cell Free Cell Cycling ExtractsCell Free Cell Cycling Extracts

Cyclin mRNA isCyclin mRNA isnecessary and sufficientnecessary and sufficientto drive cell oscillationsto drive cell oscillations

90 = 90 = nondegradablenondegradable

NH2-terminus or D-box sufficient for NH2-terminus or D-box sufficient for mitotic-specific, ubiquitin-dependent, proteolysismitotic-specific, ubiquitin-dependent, proteolysis

Role of Cyclin B destruction in progression Role of Cyclin B destruction in progression through Mitosisthrough Mitosis

Cdc2/Cyclin B (MPF) activity peaks at metaphaseCdc2/Cyclin B (MPF) activity peaks at metaphase

Cyclin destruction correlates with Cdc2/Cyclin B inactivationCyclin destruction correlates with Cdc2/Cyclin B inactivation

Regulation of metaphase to anaphase by Cyclin B ProteoysisRegulation of metaphase to anaphase by Cyclin B Proteoysis

half right...half right...

““Cell Free Mitosis”Cell Free Mitosis”

Reversible Arrest at MetaphaseReversible Arrest at MetaphaseVisualize MTs and DNAVisualize MTs and DNASynchronous release from Metaphase-Anaphase-G1Synchronous release from Metaphase-Anaphase-G1

+ EGTA (minus calcium) + EGTA (minus calcium) extract arrests in “mitosis” = “CSF extractextract arrests in “mitosis” = “CSF extract

analogous to mature egganalogous to mature egghaploid genomehaploid genome

+ sperm nuclei, + + sperm nuclei, + rhodamine-labelled tubulinrhodamine-labelled tubulin+ calcium, enter G1, S + calcium, enter G1, S

-sister chromatids-sister chromatids

+ “CSF extract”, enter and arrest at metaphase+ “CSF extract”, enter and arrest at metaphase

+ initiate anaphase by adding calcium again+ initiate anaphase by adding calcium again

Metaphase to Anaphase in vitroMetaphase to Anaphase in vitro

Extract dependent on exogenous Cyclin B mRNA - WT Cyclin BExtract dependent on exogenous Cyclin B mRNA - WT Cyclin B

TIME AFTER SECOND CALCIUM ADDITIONTIME AFTER SECOND CALCIUM ADDITION

Extract dependent on exogenous Cyclin B mRNA = Extract dependent on exogenous Cyclin B mRNA = 90 Cyclin B90 Cyclin B

TIME AFTER SECOND CALCIUM ADDITIONTIME AFTER SECOND CALCIUM ADDITION

Extract contains endogenous Cyclin B mRNA PLUS D-box peptideExtract contains endogenous Cyclin B mRNA PLUS D-box peptide

Interpretation...Interpretation...

Data:Data:

Preventing Cyclin B destruction blocks after anaphasePreventing Cyclin B destruction blocks after anaphase

Adding competitive inhibitor of Cyclin B destruction Adding competitive inhibitor of Cyclin B destruction blocks at metaphaseblocks at metaphase

Interpretation...Interpretation...

Cell Cycle Regulation of Ubiquitin Cell Cycle Regulation of Ubiquitin Dependent ProteolysisDependent Proteolysis

SCF SCF - Skp1, Cdc53, F-box (Cdc4)- Skp1, Cdc53, F-box (Cdc4)- Sic1, Cln’s...- Sic1, Cln’s...

APC/C - Anaphase Promoting Complex or Cyclosome APC/C - Anaphase Promoting Complex or Cyclosome - Securing (anaphase inhibitor), mitotic Cyclins,...- Securing (anaphase inhibitor), mitotic Cyclins,...

Exam 1 - will write in after Tuesday’s lectureExam 1 - will write in after Tuesday’s lecturequestions on cell cycle analysis using mutants, questions on cell cycle analysis using mutants, synchronous culturessynchronous cultures

- interpret data- interpret data- design experiment- design experiment

your comparison contrast of G1 to S regulation by your comparison contrast of G1 to S regulation by Cdk/Cyclin/CKI in mammalian cells and budding yeastCdk/Cyclin/CKI in mammalian cells and budding yeast

review article is sufficient for mammalian cdk inforeview article is sufficient for mammalian cdk infono wrong answersno wrong answersI don’t care how long/short - just thoughtfulI don’t care how long/short - just thoughtfulcan bring the answer to class with youcan bring the answer to class with you

questions on cell cycle regulated, ubiquitin-dependent questions on cell cycle regulated, ubiquitin-dependent proteolysisproteolysis

- interpret data from in vivo (genetic) and in vitro- interpret data from in vivo (genetic) and in vitroexperimentsexperiments