Module 2- Cell Cycle Flashcards
What needs to be regulated in the cell cycle
Each chromosome only duplicated once, chromosomes separates and distributed- coordinated with cell growth and only when required
Damaged cells dont replicate
G1 checkpoint/ restriction
G2 checkpoint
M checkpoint
how many cells in body and how many of these replaced daily
10^13 in body with 10^10 replaced daily
Detail on each cell cycle checkpoint- what is checked in each
G1= are cells ready to divide- is it big enough, is there enough energy and other resources, is DNA damaged
G2= how is the DNA- did chromosomes get correctly replicated, is DNA damaged
M checkpoint= how is chromosome alignment- did all chromosomes line up in centre of the cell ready to be pulled apart
Where do different CDKs operate
4/6 with cyclinD in G1
2 with cyclinE before S
2 with cyclinA in S and start of G2
1 with cyclinB for M
7/MAT1 with cyclinH for activation of CDKs
Overview of CDKs
Kinases which drive cell cycle, core enzymes of machinery
Ser/Thr protein kinases and constitutively active- activity is regulated
P >1000 proteins
CDK structure
N and C lobes (B sheets and a helices respectively)
Get P for P from ATP
Helix L12 site for binding
Change from active to inactive through T loop (thr160)
Signals feed in to control activity of these enzymes
Roles of geminin and Cdt1 in cell cycle- how was it seen
See with levels of them in different parts of the cell cycle
Geminin= inhibits early parts of DNA replication, switched off for G1 and on for S- also inhibits cdt1
Cdt1- helps organise replication
Visualising the cell cycle
Tags on certain proteins which are expressed at different levels in different stages of the cell cycle with different colours- see cells in different stages, can look at effect of drugs or mutation also or if things arent working
Cdt1 for G1, SLBP for S, geminin for everything except G1, H1 for all DNA
CDK regulation
Cyclin binding
Phosphorylation= activating or inhibitory
Dephosphorylation= activating
Binding of regulatory proteins
What are cyclins
Proteins which bind to CDKs and regulate their activity- CDKs only function when cyclin bound
Expression is cyclical/ regulated- can work out which ones are needed when based on when expressed with western blot
Cyclin structure and interaction with CDKs
Core helical structure= cyclin box bundles of a-helices
Helices 3 and 5 make contact with CDKs (hydrophobic interactions)
N-term is reg region- can have extra seq to control activity= more variable
6 important in humans- D1,2,3, E, A, B
In CDKs, T-loop and PSTAIRE helix move with interaction so opens up S binding site. Thr160 then P by CAK for activation
What is CAK
Complex of CDK7, cyclin H and mat1
P CDK T loop at Thr160 for activation
Optimises active site conf via H bond network
Helps support ATP binding site structure
Not usually a rate-limiting step
Regulatory phosphorylation of CDKs= limit activity to correct phase
Thr14 and Tyr15 regulatory P sites in roof of ATP binding site
Myt1 P both sites, Wee1 P Tyr15 and both cause inactivation as prevents peptide binding
CDC25 phsophatase family A/B/C remove inhibitory P so cause activation
What happens when wee1 and cdc25 mutated in cells
Wee1 not there= get cell division but no growth as inhibits mitosis until cells are big enough
Cdc25 not there= keeps growing but no division occurs as there is no mitosis occurring
Protein-protein interactions to control CDKs (CKIs)
CKI- cyclin kinase inhibitors
INK family and CIP/KIP family
INK family of CKI
Inhibit CDK4/6 through binding to kinase monomer
p15, p16, p18 and p19
INK4 binds on opposite CDK side to cyclin binding, shifts N lobe of CDK so cyclin cant bind and ATP binding site also changed so ATP cant bind
CIP/KIP family of CKI (CDK interacting protein/ kinase inhibitory protein)
Interact with CDK-cyclin complex
p21, p27 and p57
Eg when p27 binds, sits over top of complex and dostorts ATP binding, blocking substrate for the kinase binding. Can be P and this changes conf so it no longer inhibits
Over view of ubiquitin mediated degradation
E1 uses ATP to get ub, transferred to E2 and then E3 catalyses transfer of ub onto protein
E3 provides specificity- two main types being SCF and APC/C (anaphase promoting complex)
SCF complex relevance to G1/S phase
Complex with Skp1 adapter, Cullin scaffold, Rbx1 catalyst and F-box
F-box protein provides specificity eg SCFskp2 degrades CKIs and SCFfbox4 degrades cyclin D
Constitutively active, rate determined by f-box affinity for substrate and regulated by S phosphorylation- needs P for recognition by f-box
Signals controlling entry into the cell cycle
Growth factors and hormones (mitogens) external factors which signal for expression of cyclin D through NGF pathway
Nutritional sufficiency internal factor which signals for expression of cyclin E and inhibition of anti-mitogenic signals through mTOR
Anti-mitogens which inhibit progression into S phase
Regulation of transcription at G1/S phase
Regulates expression of proteins required for S phase eg histone unwinding, nucleotide synthesis and DNA synthesis
E2F 1-3 activators bind to DNA and E2F4-8 with p107 and p130 inhibitors bind to DNA
Rb protein binds to E2F proteins, when phosphorylated by CDK4/6 and CDK2 is inactive so releases E2F proteins
Depends where they are localised in cells too leads to regulation also
events which occur entering the cell cycle
Cyclin D is expressed through ERK from external signal which activated CDK4/6
CDK4/6 P many proteins including Rb, allowing E2F1-3 to be active and bind to target genes, allowing cyclinE expression
CyclinE activates CDK2 which P proteins and hyperP Rb so it really doesnt inhibit at all
CKIs also P and degraded so they dont inhibit the process
Cross-talk and checks involved= complex
How do anti-mitogens work eg TGF-beta
Inhibit progression through restriction checkpoint (G1 checkpoint)
TGF-beta acts through SMAD family of TFs
Causes expression of cyclin kinase inhibitors such as p27
What does cancer need to do in relation to the cell cycle- how do therapeutics relate
Overcome G1-S checkpoint with inhibitors (Rb, CKIs) or cause inappropriate active mitogen signalling
Therapeutics aim to inhibit G1-s transition
