Cell Cycle: Start/Restriction Point & S Phase Flashcards
The presence of each cyclin rises and falls at distinct points of the cell cycle. True or false?
True
Which cyclin is highest during S phase?
S-cyclin (Cyclin A)
Which cyclin is highest during G1?
G1/S-cyclin (cyclin E)
Which cyclin is highest during M phase?
M-cyclin (cyclin B) and decreasing S-cyclin (cyclin A)
Which cyclin is highest during G2?
S-cyclin (cyclin A) with M-cycling (cyclin B) rising
Familiarize yourself with this figure.
There are ___ classes of cyclins, each of which binds and activates specific ______..
Four
Cyclin-dependent kinases (Cdks)
There are four classes of cyclins: ____, ______, ______, and _____.
G1
G1/S
S
M
The cyclin and cyclin-dependent kinase complex G1-CDK in yeast is composed of ___________ and _________.
Cyclin 3 (Cln3)
Cyclin-dependent kinase 1 (CDK1)
The G1-CDK complex in mammals is composed of ____________ and _______________.
Cyclin D
Cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6)
The G1/S-CDK complex for yeast is composed of ________ and/or ___________ and ____________.
Cyclin 1 and cyclin 2 (Cln1, Cln2)
CDK1
The G1/S-CDK complex in mammals is composed of ________ and _____________.
Cyclin E
CDK2
The G1-Cdk complex in vertebrates is composed of cyclin ___ and cyclin-dependent kinases ____ and ____. In yeast, this same complex is composed of cyclin (Cln) _____ and cyclin-dependent kinase _____.
Cyclin D
Cdk4 &Cdk6
Cln3
Cdk1
The G1/S-Cdk complex in verebrates is composed of cyclin ____ and cyclin-dependent kinase _____ while in yeast it is composed of cylcin (Cln) _____ and cyclin-dependent kinase ____.
Cyclin E
Cdk2
Cln1, 2
Cdk1
In yeast, G1 cyclin synthesis is stimulated by _________________ and ________________.
Nutrient accumulation and availability
Cell growth (i.e., size)
In mammalian cells, G1 cyclin synthesis is stimulated by __________, ________________, and ___________.
Nutrient accumulation and availability
Cellular growth (size)
Growth factor stimulation
The S-Cdk complex is composed of cyclin _____ and Cdk ____ and ____ in vertebrates and cyclin (Clb) ______ and Cdk1 in yeast.
Cyclin A
Cdk2 & Cdk1
Clb5, 6
Complete the table.
G1-Cdk, Cdk 4 & Cdk6
Cyclin A
Cdk1
Complete the table
G1-Cdk, Cdk4 & Cdk6
Cyclin E
Cyclin A, Cdk2 & Cdk1
M-Cdk, Cdk1
Complete the table.
G1-CDK, Cyclin D, Cdk4 & Cdk6, Cln3, Cdk1
Cyclin E, Cdk1
Cyclin A, Cdk2 & Cdk1, Cdk1
M-Cdk, Cdk1
Complete the table.
G1-Cdk, Cyclin D, Cdk4 & Cdk6, Cln3, Cdk1
G1/S-Cdk, Cyclin E, Cdk2, Cln1, 2, Cdk1
S-Cdk, Cyclin A,Cdk2 & Cdk1, Clb5, 6, Cdk1
M-Cdk, Cyclin B, Cdk1, Clb1, 2, 3, 4, Cdk1
In the M-Cdk complex, cyclin _____ and cdk _____ are present in vertebrates and cyclin ________ and cdk ____ in yeast.
Cyclin B
Cdk1
Clb 1, 2, 3, 4
Cdk1
The G1/S boundary is the “start” or _______________ point.
Restriction
What do cyclin dependent kinases do?
Regulate progression of the cell cycle at distinct stages
Cyclin-dependent kinases regulate progression at distinct stages of the _______ cycle.
Cell
Cyclin-dependent kinases can be regulated by _________ binding, ____________________, and ________________ binding.
Cyclin
Phosphorylation
CDK inhibitory proteins (CKIs)
What are three ways in which cyclin-dependent kinases (CDKs) are regulated?
(1) Binding of their respective cyclins,
(2) phosphorylation, which can activate or deactive them, and
(3) binding of CDK inhibitory proteins (CKIs)
What does CKI stand for?
Cyclin-dependent kinase (Cdk) inhibitory proteins
In yeast, G1 cyclin synthesis is stimulated by _________________ and ________________________.
Nutrient accumulation and availability
Cellular growth (increase in size)
In mammals, G1 cyclin (cyclin D) synthesis is stimulated by __________________, ____________________, and ________________.
Nutrient accumulation and availability
Cellular growth
Growth factor stimulation
What is the primary difference between yeast and mammalian cells regarding G1 cyclin synthesis?
Mammalian cells require growth factor stimulation
For all other cyclins, each active _______ initiaties a wave of ____________ activity, which produces a new _______.
Cdk
Transcriptional
Cyclin
How are cyclins degraded in the cell?
Ubiquitin-dependent proteolysis
What is required for ubiquitin-dependent proteolysis during the cell cycle?
Two E3 ubiquitin-ligases:
- SCF complex
- APC/C complex (anaphase promoting complex or cyclosome)
What does APC/C complex stand for?
Anaphase promoting complex or cyclosome
The SCF complex is active throughout the cell cycle but only recognizes _______________ targets.
Phosphorylated
What are two targets of the SCF complex?
G1 cyclins and CKIs (cyclin-dependent kinase inhibitors)
How does the SCF complex recognize its targets G1 cyclins and CKIs (cyclin-dependent kinase inhibitors)?
Phosphorylation
When is APC/C (anaphase promoting complex or cyclosome) active?
When phosphorylated by the M-cyclin/Cdk complex at the metaphase/anaphase transition
What phosphorylates APC/C (anaphase promoting complex or cyclosome)?
The M-cyclin/Cdk complex (cyclin B/Cdk1)
What does APC/C target?
Proteins that possess destruction box motifs (recognition sites for APC/C)
What proteins have destruction boxes?
S-cyclin (cyclin A), M-cyclin (cyclin B), and proteins that connect sister chromatids
What are the three targets of APC/C?
- S-cyclin (cyclin A)
- M-cyclin (cyclin B)
- Proteins that connect sister chromatids
In the absence of phosphorylation, ____________________________ (Cdks) still have activity but are much lower.
Cyclin-dependent kinases
Cyclin-dependent kinases (Cdks) are more active when ___________________.
Phosphorylated
CDKs are phosphorylated by ____________.
CDK activating kinases (CAKs)
CAKs (CDK activating kinases) are active throughout the cell cycle and _________________ the amino acid ____________ at the active site.
Phosphorylate
Threonine
Phosphorylation of the amino acid threonine by CAKs (CDK activating kinases) at the active site of CDKs increases their activity but is not the ______________ step.
Rate-limiting
What kinase phosphorylate and inhibit mitotic CDKs?
Wee1 kinases
What do Wee1 kinases phosphorylate on CDKs?
Tyrosine and threonine residues in the ATP binding site of CDK
Wee1 kinases phosphorylate a tyrosine and threonine residue in the _______________ site of CDK, which potently inhibits CDK activity.
ATP-binding site
How are phosphorylations completed by Wee1 kinases removed?
Cdc25 phosphatases
________ phosphatases dephosphorylate and therefore ___________ mitotic CDKs inhibitied by Wee1 kinase phosphorylation.
Cdc25
Activate
How many classes of CKIs are present in mammalian cells?
4
What does CKI stand for?
CDK inhibitors
What are the four classes of CKIs (CDK inhibitors) in mammalian cells?
- Wee1 kinases
- INK4 (G1 CDK inhibitors)
- p21CIP (G1/S and S CDK inhibitors)
- p27<strong>KIP1</strong> (G1/S and S CDK inhibitors)
What does INK4 stand for?
Inhibitors of kinase 4
What inhibits G1 CDKs (CDKs 4 & 6)?
INK4 (inhibitors of kinase 4)
How does INK4 (inhibitors of kinase 4) (a CKI) inhibit CDK4 and CDK6 in the G1 phase?
INK4 binds them, preventing cyclin binding
When is INK4 most likely to be expressed?
In response to excessive growth stimulation because it inhibits CDKs 4 & 6 from binding cyclin D
What does the “p” stand for in p21CIP and p27KIP1?
Protein
What are the G1/S (CDK2) and S (CDK2 & 1) CDK inhibitors (CKIs)?
p21CIP and p27KIP1
How do p21CIP and p27KIP1 act as CKIs (CDK inhibitors)?
Binding G1/S and S cyclin/CDK complexes, inhibiting CDK activity
As the activity of a particular CDK begins to increase, one of its targets is the ___________ that is trying to inhibit it.
CKI (CDK inhibitor)
How do CDKs inactivate CKIs?
CDKs bind CKIs when CDK levels increase, and CDK binding to CKIs inhibits CKIs
What happens after a cyclin-dependent kinase (CDK) binds its CDK inhibitory protein (CKI) target?
SCF complex recognizes it via its substrate recognition partner, an F-box protein, and ubiquinates it
How does a CDK inhibitor (CKI) become degraded?
Binding by CDK, ubiquinated by SCF complex via help of its substrate recgonition partner, an F-box protein
What helps the SCF complex recognize CKIs (CDK inhibitors)?
A substrate recognition partner, an F-box protein
What is an F-box protein?
A substrate recognition partner for the SCF complex
How do cells commit to moving through the cell cycle?
Moving past the G1/S boundary
What is the rate-limiting step for the cell cycle?
The G1/S boundary
In yeast, the G1/S boundary is known as the “start” point whereas it is known as the “________________” point in mammalian cells.
Restriction point
What do we mean by a “start” or “restriction point” in the cell cycle?
The cell is committed to moving through the cycle unless some significant problem occurs
What is required for start in yeast?
- Sufficient nutrients and growth
- Sufficient level of Cln3 (cyclin 3), the G1 cyclin
- Synthesis of Cln1 and Cln2 (cyclin 1, cyclin 2), the G1/S cyclins
What three things are required for start in yeast?
- Synthesis of Cln1 & 2
- Synthesis of enough Cln3 (G1 cyclin)
- Nutrient availability and appropriate cell size
Cln3/CDK1 phosphorylate the transcriptional co-repressor of Cln1 and Cln2, ________, thereby enabling transcription of Cln 1 & 2 required for start.
Whi5
What does Whi5 do?
It represses transcription of Cln 1 & Cln 2 and thereby regulates start
What deactivates Whi5?
Phosphorylation by Cln3/CDK1, the G1 cyclin/CDK complex
Regulation of Cln3 occurs at what level?
The level of translation
The regulation of Cln 1 and Cln 2 occurs at what level?
The level of transcription
How is Cln3 regulated?
When nutrients are scarce, translation of all proteins is inhibited, and nutrient available is detected and relayed through the Target of Rapamycin (TOR) kinase
What is TOR kinase stand for?
Target of Rapamycin kinase
What does TOR kinase do?
Target of Rapamycin kinase detects nutrient levels in yeast cells, ultimately regulating whether the cell will translate Cln3
In mammalian cells, passing the _______________ point requires both growth factor signaling and sufficient nutrients.
Restriction
Nutrient availability and binding by growth factors are relayed through a kinase called _____________________ in mammalian cells.
Mammalian Target of Rapamycin (mTOR)
Which specific mTOR is involved in identifying nutrient availability and growth factoring binding?
mTOR complex 1 (mTORc1)
Active mTOR phosphorylates three key substrates. What are they?
- S6 kinase
- 4E binding protein
- Transcription factors
mTOR phosphorylates ______ and activates it, which then phosphorylates its target ___________.
S6 kinase
Ribosomal protein S6
mTOR phosphorylates S6 kinase, which then phosphorylates ribosomal protein S6 because ribosomal protein S6 must be phosphorylated for the _______________ to engage translation.
Ribosome
mTOR phosphorylates 4E binding protein, which normally binds eIF4E and ______________ eIF4E.
Inhibits
How does 4E binding protein inhibit eIF4E?
By binding and sequesting eIF4E
When 4E binding protein is phosphorylated by _______, it releases _________, allowing it to bind other eIFs and the 5’ cap of mRNA.
mTOR
eIF4E
mTOR phosphorylates other transcription factors, too, which increase the _________________ synthesis.
Ribosomal subunit
What regulates mTOR?
- Low ATP levels and environmental stress inhibit mTOR
- High amino acid levels and growth factor stimulation activate mTOR
What two things inhibit mTOR?
- Low ATP levels, specifically ATP/AMP rato
- Environmental stress, like osmotic stress, oxidative stress, and high temperatures
_____ ATP levels, and specifically low ATP:AMP ratios, ___________ mTOR as do ______________ stressors.
Low
Inhibit
Environmental
What two things stimulate mTOR?
- High amino acids levels, especially leucine
- Growth factor stimulation
What amino acid is especially significant in activating mTOR?
Leucine
What G-protein activates mTOR?
Rheb
What protein acts as the GAP (GTPase activating protein) for Rheb, which functions as the G-protein for mTOR?
Tuberous sclerosis 1/2 (TSC1/TSC2)
What do TSC1/TSC2 stand for?
Tuberous sclerosis 1/2
When TSC1/TSC2 is active, it binds and activates the G-protein _____, stimulating the hydrolysis of GTP to GDP. This _______ the G-protein and thereby decreases its ability to activate ______. Thus, for cell growth to occur, we want TSC1/TSC2 to be __________.
Rheb
Deactivates
mTORc1
Inactive
What do tuberous sclerosis 1/2 (TSC1/TSC2) do regaring mTOR activation?
TSC1/TSC2 act as the GAP for Rheb, which is the G-protein that activates mTOR
What phosphorylates and inactivates TSC2 of the TSC1/2 complex?
Protein kinase B (PKB)
Under favorable conditions, Akt/PKB is activated. PKB then phosphorylates and _________ TSC2 of the _________ complex. Phosphorylation likely leads to the ___________ of the complex. As such, the complex can no longer function as the ______ for Rheb, so Rheb remains in its _____-bound state and active, thereby activating mTORc1 and increasing _________.
Inactivates
TSC1/TSC2
Dissociation
GAP (GTP-ase activating protein)
GTP-bound
Cell growth
Under unfavorable conditions, specifically low ATP/AMP ratios, AMP kinase is activated, which in turn phosphorylates and activates _______, which increaseTSC1/2 complex levels. This allows for Rheb to hydrolyze its GTP for ____, thereby inhibiting its ability to activate _______.
TSC2
GDP
mTORc1
______ ATP/AMP ratios activate ______, which in turn phosphorylates and activates TSC2, which causes the association of the TSC1/2 complex. The TSC1/2 complex can now act as the GAP for ____, thereby hydrolyzing its GTP for GDP. This causes its deactivation and decreases mTOR activity.
Low
AMP kinase
Rheb
Which pathway mediates mTOR activation via growth factors?
PI3K via G-protein Rheb and its GAP TSC1/TSC2
Which pathway mediates mTORc1 activation via amino acid levels?
The Rag complex of G-proteins
When amino acid levels are high, mTORc1 is activated by ___________ G-proteins.
Rag complex
Which pair of the Rag complex is GTP bound under high amino acid levels?
A&B
Which pair of the Rag complex is GDP bound under high amino acid levels?
C&D
What acts as the GEF (guanine nucleotide exchange factor) for RagA&B?
Ragulator
Under low amino acid conditions, RagA&B are bound to ______________.
GDP
Under low amino acid conditions, RagC&D are bound to ____________.
GTP
What acts as the GAP (GTP-ase activating protein) for RagD?
Leucyl-tRNA synthetase
When can leucyl-tRNA synthetase act as the GAP for Rag D?
Upon leucine binding
Be able to describe the pathway that leads to mTORc1 activation via growth factor binding.
In the presence of amino acids, mTORc1 is activated by what pathway?
The Rag complex
How is mTORc1 activated by the Rag complex of G-proteins? In other words, what must be present in the cell for the pathway’s activation?
Amino acids, especially leucine
How is mTORc1 activation different from other G-proteins we’ve encountered throughout the semester?
RagA/B must be GTP bound
Rag C/D must be GDP bound
mTORc1 is activated by the ____________ complex of G-proteins. Amino acids activate ______________, the GEF for RagA/B, and leucine binds and activates _________________, the GAP for RagC/D. This allows for RagA/B to become ____-bound and RagC/D to become ____-bound, which is required for mTORc1 activation.
Rag complex
Ragulator
leucyl tRNA synthetase
GTP-bound
GDP-bound
mTORc1 can be regulated by ATP levels when _____ kinase phosphorylates and activates TSC2.
AMP kinase
ATP levels can also regulate mTORc1 by __________.
Inhibition
____ATP:AMP ratios activate AMP kinase to either phosphorylate and activate ________, thereby inhibiting mTORc1, or directly phosphorylate and inhibit _______ because ____ levels signal to the cell that there is not enough energy for cell growth.
Low
TSC2
mTORc1
Low
There are four cyclin/CDK pairs in ___________, two in ___________ yeast, and three in budding yeast.
Mammals
Fission yeast
Be able to explain the major differences between cyclin/CDK complexes in fission yeast cells and mammalian cells.
The order of cyclin/CDK complexes from G1 to M phase in mammalian cells: D, E, A, B; 4&6, 2, 2, 1.
Cyclin D/CDK4&6
Cyclin E/CDK2
Cyclin A/CDK2
Cyclin B/CDK1
Growth factor binding its receptors activates the MAPK pathway. This results in transcription and translation of the G1 cyclin/CDK complex: _________. The G1 cyclin/CDK complex phosphorylates ______, which releases E2F, a ______________ that upregulates expression of S cyclin/CDK complex: _____________.
Cyclin D/CDK 4&6
Rb (Retinoblastoma)
Transcription factor
Cyclin E/CDK2
_____ upregulates expression of cyclin E/CDK2, which then phosphorylates ____, releasing E2F and increasing its own expression.
E2F
Rb
When enough cyclin __/CDK__ is accumulated in the yeast cell, the cell moves past the ____________ point or ______ boundary.
Cyclin 3/CDK2
start point
G1/S boundary
At the beginning of G1 phase, Rb or _____________ binds and sequesters the transcription factor _______________.
Retinoblastoma
E2F
What does Rb stand for?
Retinoblastoma
What happens when cyclin D/CDK4&6 phosphorylate Rb?
Rb releases transcription factor E2F
E2F then upregulates transcription of cyclin E and CDK2
Cyclin E/CDK2 are required for transition from G1 phase to S phase
What does Rb do in its unphosphorylated form?
Sequesters the transcription factor E2F
In mammalian cells, cyclin ___/CDK4&6 promote progression from G1 to ____ phase while cyclin ___/CDK2 initiate ________ in late G1 phase. Cyclin ___/CDK2 initiate ________ in S phase, again, and cyclin B/CDK1 promotes mitosis in late ____ phase.
Cyclin D/CDK4&6
S phase
Cyclin E/CDK2
Replication
Cyclin A/CDK2
Replication
G2
The transcription factor E2F, active G1/S cyclinE/CDK2 complex, and active S cyclinA/CDK2 complex all have a _______ feedback loop with E2F.
Positive
E2F increases transcription of cyclin ____. Cyclin E/CDK2 in turn phosphorylate ____, which releases E2F.
Cyclin E
Rb
Cyclin E/CDK2 or __________ phase complex increases the synthesis of the S-phase cyclin, cyclin ____.
G1/S phase
Cyclin A
As protein synthesis increases, cyclin D and CDK4&6 levels increase; cyclin D/CDK4&6 phosphorylate ____ (retinoblastoma), which then releases the transcription factor E2F in the cytosol.
Rb
The S phase is regulated in cells by regulating the S phase cyclin, which in yeast is ______ and in animals is ______. Initial S cyclin synthesis is stimulated by G1/S cyclin/CDK activity (i.e., cyclin E/CDK2). Upon its synthesis, the S chase cyclin is inhibited by _____ in yeast and _____ in animals. This keeps the cyclin inactivae until the correct time.
Clb5&6
Cyclin A
Sic1
p27<span>KIP</span>
The G1/S cyclin/CDK complex ultimately overcomes the inhibition of the S phase cyclin via ____ in yeast and ____ in animals. The G1/S cyclin/CDK complex phosphorylates these CDK inhibitory proteins, leading to their ubiquination and degradation by the _____ complex.
Sic1
p27KIP1
SCF complex
In yeast, Sic1 inhibits the S phase cyclin: _________. Once start occurs, the G1 cyclin/CDK complex (Cln3/CDK1) phosphorylates Sic1. This results in ubiquination and degradation of Sic1 by the SCF complex, which requires the help of _______ in identifying targets. Sic 1 must be phosphorylated ___ times in order for degradation to occur.
Clb5&6/CDK1
F-box protein
6 times
How many times must Sic1 be phosphorylated in __________ yeast before it can be recognized by _______ and ubiquinated by SCF complex?
Budding
F-BOX protein
6 times
Why must Sic1 be phosphorylated 6 times before it can be recognized by FBOX?
Poor affinity at some binding sites; requires high levels of G1/S cyclin/CDK (cyclin E/CDK2)
What is the significance of Sic1 requiring phosphorylation 6 times?
Provides a sharp transition between G1 and S phases
During _____ phase, replication of DNA occurs via multiple ________ of replication, but the pre-replication complex is assembled during _____ phase.
S phase
Origins of replication
G1
What makes up the pre-recognition complex?
Origin recognition complex
Helicase
Loading factors, Cdc6 and Cdt1
What three things are required for assembly of the pre-replication complex?
- Origin recognition complex
- Helicase
- Loading factors, Cdc6 and Cdt1
Initiation of replication requires phosphorylation of the replication complex by two kinases: (1) _________, which phosphorylate initiator proteins that in turn recruit helicase activating factors and the DNA polymerase complex, and (2) __________, which phosphoryates MCM helicase.
CyclinA/CDK2
DDK
When do the kinases that phosphorylate the replication complex become active?
S phase; they assembly during G1 and wait
How does the cell ensure replication occurs only once?
- Loading factor Cdc6 is phosphorylated by cyclin A/CDK2 and targeted for ubiquination and degradation by SCF complex
- Loading factor Cdt detaches from pre-replication complex and bound by its inhibitor, geminin, which is later targeted by APC/C complex in M phase
- Helicase only loads when DNA is unphosphorylated; following replication, phosphorylated helicase is exported from the nucleus and only reenters the nucleus following dephosphorylation by phosphatases active during G1
The loading factor ______ is phosphorylated by cyclin A/CDK2 and then targeted by ______ for degradation by the proteosome.
Cdc6
SCF complex
After it helps the pre-replication complex attach, loading factor _____ is bound by its inhibitor ________, which is later targeted for degradation by the ________ complex in M phase.
Cdt1
Geminin
APC/C
Helicase only loads onto DNA when it is phosphorylated or non-phosphorylated?
Non-phosphorylated
What links sister chromatids together?
Cohesins
Cohesins are made up of four subunits: two _____________ and two ___________.
Coiled-coils
Globulars
Why are sufficient levels of Cln3 required in yeast for start to occur?
Cln3/CDK1 phosphorylate the transcriptional co-repressor Whi5, releasing its inhibition of Cln 1 and Cln2 transcription
In yeast, Cln3/CDK1 complex phosphorylates the transcriptional co-repressor _______, which releases from DNA and allows for transcription of the next round of cyclins: _____ and _____.
Whi5
Cln1
Cln2
For mTORc1 activation, RagA&B must be bound to _____ and RagC&D must be bound to ____.
GTP
GDP
For activation of mTORc1, RagA&B must be in its GTP form. This requires that its GDP be exchanged for GTP by its GEF ______________. Simultaneously, RagC&D must be in its GDP form. This requires that its GTP be hydrolyzed to GTP by its GAP _______________.
Ragulator
Leucyl-tRNA synthetase
For mTORc1 activation to occur under high amino acid conditions, __________ must bind leucyl-tRNA synthetase in the ___________ membrane. This activates the GAP function of leucyl-tRNA synthetase and results in RagC&D becoming _____ bound.
Leucine
Lysosomal
GDP-bound
