The document summarizes key aspects of cell cycle regulation. It describes the various phases of the cell cycle and factors that affect its regulation. Checkpoints ensure conditions are suitable before progression, and cyclin-CDK complexes drive phase transitions when activated by cyclins. The Rb and p53 tumor suppressor proteins also play important roles by integrating signals to arrest the cycle or induce apoptosis in response to DNA damage. Ubiquitination targets proteins like cyclins for degradation by the proteasome, controlling complex activity and moving the cycle forward.

1. Regulation of Cell
Cycle
By – Mohit Kumar

2. overview
• Described by Howard and Pelc in 1953.
• From single cell, zygote to adult cell requires a
series of cell division known as ‘cell cycle’.

3. Division cycles
• Somatic division cycle: used to fulfill specific requirements.
• Embryonic cycle: having lack of G1 and G2 phase.
• Meiotic cell cycle: allow formation of haploid gametes.
• Endoreduplication: S phase is not followed by mitosis.

4. Human cell cycle
• Length of cell cycle is highly variable, even within cells of a
single organism.
• In human, frequency of cell turnover range few hours in early
embryonic development to an average of two to five days for
epithelial cells.
• In human, rapidly cell cycle length is about 24 hours.
 G1 phase lasts approximately nine hours.
 S phase lasts ten hours.
 G2 phase lasts about four and one-half hours.
 M phase lasts approximately one-half hour.

5. Factors that affects cell cycle regulation
• The death of nearby cells.
• As sweeping as the release of growth promoting hormones.
• Crowding of cells.
• Size of the cell during division (decreasing surface to volume
ratio).
• Chromosomal mutation

6. Checkpoints
• A checkpoint is one of several points in the eukaryotic cell
cycle at which the development of a cell to the next stage in
the cycle can be halted until conditions are favorable.
• These checkpoints occur near the end of G1, at the G2/M
transition, and during the metaphase.

7. G1 checkpoint
• Also known as restriction point in yeast.
• Check for genomic DNA damage.
• Plays crucial role at this check post due to proliferation of the
cell.
• The cell can halt the cycle and waits, whether to moves G°
phase or signal to improves condition.

8. G2 checkpoint
• Entry into mitotic phase.
• Whether chromosome have been replicated properly.
• No DNA damage occurs, if occurs than repair
mechanism repair the mutation and completes proper
replication.

9. M checkpoint
• Known as spindle checkpoint.
• At the end of metaphase in interphase.
• Sister chromatids are correctly attached to the
microtubule or not.
• Cycle get arrested if spindle fiber attached chromatids
doesn’t moves at poles.

10. cyclins and cyclin-dependent kinases (CDKs)
CDKs
• Master regulator of cell cycle.
• Role is to phosphorylate protien on ‘Ser’ and ‘Thr’ amino
acids.
• Yeast having 1 CDK while animals having 9 CDKs.
Cyclins
• Regulatory subunits of complexes with kinase activity (with
Cdks).
• Their presence “oscillates” in the cell cycle.
• There are G-1cyclin, G1-S cyclin, S cyclin and M cyclin.

12. Cyclins and CDKs in vertebrates

14. Activation of CDKs-cyclin complex

15. Regulation of activity of cyclin-CDKs complex's
• Cyclins undergo a cycle of synthesis and degradation
in each cell cycle, while CDKs level remains constant
during the cell cycle.
• Cyclins-CDKs complex inactivated by regulated
proteolysis of cyclin at specific cell cycle stage.
• Cyclin level in cells are controlled not only by
changes in cyclin destructions but also by changes in
cyclin synthesis.

16. M-CDKs
• They are mitosis promoting factor or maturation
promoting factor (MPF).
• Synthesized during S and G-2 phase
• Include chromosomal condensation.
• Breakdown of nuclear envelop
• Assembly of mitotic spindle apparatus.
• Alignment of condensed chromosome at metaphase
plate.
• It phosphorylate number of protein such as
condensin, lamin etc.

17. Regulation of CDKs-cyclin in S. pombe

18. • After the proper association of chromosomes
with spindle microtubules, the M-CDKs
complexes activates the anaphase promoting
complex/cyclosome (APC/C).
• APC/C acts on different substrate at different
time. It bears to different co activators i.e.
cdc20 and cdh1.

20. Inactivation of cyclin-CDKs complex's

21. Inactivation of CDKs –cyclin complex
• Occurs by ubiquitin dependent proteolysis.
• Proteolysis of cyclin is catalyzed by ubiquitin lygase.
• Enzyme SCF(SKp1, cullin, F-box) & E3 ubiquitin
lygase responsible for destruction of G1 phase cyclin.
• E-3 ubiquitin ligase- anaphase promoting
complex/cyclosome (APC/C) is responsible for S&M
phase cyclin inactivation.

22. Role of Rb protein in cell cycle regulation
• RB1(retinoblastoma) gene encodes Rb protein
of 110 KDa.
• It is a tumor suppressor protein and loss of
function mutation in Rb1 gene leads tumor.
• It is present on 13q14.2 chromosome location.

24. Rb protein
• Its active, un or hypophosporylated form
binds with E2-F transcription factor and
suppress the gene transcription which is
required for transition from G1 to S phase.
• The product of these genes are required for S
phase of the cell cycle.
• Cyclin-D – CDKs 4/6 phosphorylate the Rb
protein results losses its affinity with EF-2.

25. Rb protein
• Restriction point: This site ensure that the transition
is responsive to extracellular signals.
• Then cyclin E level is increase, cyclin-E +CDK-2
complex drives fully Phosphorylated form of Rb
protein due to that E2F is released.
• E2F binds with DP(DNA binding protein) &
transcription translation could occurs.

26. p53 protein
• Tp53 gene encodes for p53 protein of molecular mass
of 53KDa.
• It is also a tumor suppressor gene encodes a
polypeptide chain of 393 amino acid residue.
• It is described as Guardian of the genome &
cellular gatekeeper because it involves in the
complex network of cellular events
• It regulates the expression of several genes involves
in DNA repair, apoptosis, cell growth, antioxidant
defense etc.

27. p53 protein
• p53 is inactivated by its –ve regulator MDM2 (mouse
double minute 2).
• MDM2 functions as E3 ubiquitin ligase & cause
proteosomal degradation of p53 protein.
• Upon DNA damage or other stress leads to
dissociation of p53 and MDM2 complex, activated
p53 will induce a cell cycle arrest to either repair and
survival of the cell or apoptosis.

30. pP53 protein
• In double stranded DNA break, protein kinase ATM is
activated which in turn activates CHK 2 kinase.
• Both ATM &CHK2 phosphorylate p53 at distinct sites. At the
same time ATM phosphorylate MDM2 in way that cause its
functional inactivation.
• Phosphorylated p53 acts as transcription factor for p21 gene.
• p21 protein binds with G1/S-CDK and S CDK complexes and
inhibit their activity, thereby helping to block entry into the
cell cycle.
• Hence, p53 level is increase and arrest the cell in the G1 phase
of the cell cycle.

31. Ubiquitin mediated protein degradation
• Mainly two type of proteolytic system are there:
1. Lysosome mediated- membrane bound + acid
hydrolysis including many proteolytic enzyme.
2. Proteasome mediated- regulated breakdown of
protein by energy dependent manner.

32. Proteasome
• Found in both prokaryotes and eukaryotes.
• In eukaryotes they present in both cytosol and
nucleus.
• In eukaryotes they are of two types:
 20S Proteasome- cylindrical barrel shaped structure
arranged as four rings in heptamer form upon one
another.
 26S Proteasome- it catalysis the ATP-dependent
degradation of polyubiquitinated proteins.

34. Ubiquitination
• Most well established means of targeting protein to
proteasome is by addition of ubiquitin.
• Ubiquitin is highly conserved protein of 76 amino acid residue
and covalently attached to a lysine residue on a target protein.
• Protein can be monoubiquitinated, multiubiquitinated or
polyubiquitinated.

35. Activation of ubiquitin
• Activated by ubiquitin E-1 (ubiquitin activating
enzyme), in energy dependent manner E-1 attach to
ubiquitin at C- terminal.
• Then, E-1 transfer to a ‘cysteine residue’ present on
an E-2 (ubiquitin conjugating enzyme).
• Finally E-3 (ubiquitin ligase)transferred the activated
ubiquitin from E-2 to a lysine amino acid residue of
its target protein, forming an isopeptide bond.

37. Attractions:
• Cell cycle
• Checkpoints
• Activation/inactivation of cyclin-cdks complex
• Rb protein
• P53 protein
• Ubiquitin mediated proteasomal degradation