CDK Inhibitor p27 Degradation and Cell Proliferation

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CDK Inhibitor p27 Degradation and Cell
Proliferation

• Presented by Layne Norton
• 05/11/2003

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Cell Replication

• G1 to S transition
• (G1) is the interval between mitosis and DNA
  replication
• G1 phase is the growth phase
• G1 phase is the interval between M phase and S
  phase
• Transition commits cell to replication
• If proper signals are not provided cell enters G0
  phase

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Cell Replication
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Cyclin Dependent Kinases

• Transition from G1 to S requires CDK activity
• Phosphorylated CDK complexes phosphorylate Rb
  Protein
• This allows cell to proceed from G1 to S phase
• Activity of CDKs is controlled by CDK inhibitors

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CDK inhibitors

• Two classes of CDK inhibitors
• INK4 family
• Cip family
• One of the most important inhibitors is p27
• Suppression/degradation of p27 is required for G1
  to advance to S phase
• P27 inhibits (via cAMP) complexes formed by
  cyclin E-CDK2, cyclin A-CDK2, cyclin D1-CDK4, and
  -CDK6
• Inhibited complexes can no longer phosphorylate
  Rb protein
• This prevents cell from proceeding to S phase

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CDK inhibition
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P27 degradation

• Degradation of p27 via ubiquitination
• Ubiquitin attachment marks proteins for
  proteasome degradation
• Attachment to substrate follows a mechanism
  involving the sequential action of 3 enzymes.
• E1-Ubiquitin-activating enzyme (usually only 1)
• E2-Ubiquitin-conjugating enzyme (several E2s)
• E3-Ubiquitin-protein ligase enzyme (many E3s)

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p27 Degradation (contd)

• E1 (ubiquitin-activating enzyme)
• Forms thiolester linkage with C-terminus of
  ubiquitin
• Then transfers ubiquitin to an E2 enzyme
• E2 enzyme involved in p27 degradation is
  CDC34/UBC3
• Transfer of ubiquitin from E2 to substrate is
  mediated by E3 enzyme

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p27 Degradation (contd)
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p27 Degradation (contd)

• E3 enzyme involved in p27 degradation is an SCF
  ligase
• SCF ligase consists of Skp1, Cul1 protein, Rbx1,
  and an F-box protein (Skp2)
• F-box protein is largely responsible for
  recognition of substrate
• F-box protein is variable and depends on the
  substrate. The F-box protein for p27 is Skp2

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SCF-Ligase

• Skp2 binds Skp1 through their F-Box motif
• C-terminal region interacts with substrate
• Skp2 also binds Cyclin A-CDK2 (not shown)
• Skp2 also interacts with Cks1 (not shown)
• Skp2 may also position p27 optimally for
  ubiquitination
• P27 binding domain is composed of leucine rich
  repeats (LRRs)

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p27 Degradation (SCF ligase)

• So how does this whole sha-bang work?
• Cul1 interacts with E2 enzyme (UBC3)
• Skp2 recruits only phosphorylated p27
• P27 is phosphorylated on Thr187 by Cyclin A-CDK2
• If p27 is not phosphorylated it will NOT be bound
  by Skp2
• Cks1 is association is required for binding of
  p27 to Skp2
• Cks1 confers and allosteric change in Skp2 that
  increases its affinity for phosphorylated p27
• The Cks1-Skp2 interaction enables the transfer of
  ubiquitin to p27 by the SCF ligase
• There is no known reason why Cyclin A-CDK2
  associates with Skp2 (but I have a semi-theory)

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p27 Degradation

• Proteasome
• 30 subunit complex that degrades proteins
• Subunits are arranged in a self-compartmentalized
  structure
• Active sites of proteasome are inaccessible to
  p27
• p27 must be ubiquitinated (usually
  poly-ubiquitinated) in order for them to be
  translocated to its hydrolytic chamber
• Proteasome then degrades ubiquitinated p27

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p27 Degradation

• Consequences of p27 degradation
• Less p27 is available to inhibit CDKs
• CDKs increase phosphorylation of Rb proteins
• If Rb is phosphorylated, it can no longer block
  transcription of genes involved in DNA synthesis
• Cell proceeds from G1 to S phase
• Cell proliferation!

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Linking Skp2 and p27 to cancer

• Skp2
• Skp2 is a rate limiting component of the
  machinery that degrades p27
• Skp2 is over expressed in tumor cells
• Found to have oncoprotein properties
• Aggressive carcinomas have high p27 specific
  degradation activity
• Skp2 levels and p27 levels are inversely
  correlated
• Over expression of Skp2 may allow cancer cells to
  escape growth control mediated by p27

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Linking Skp2 and p27 to cancer

• p27
• Reports show that p27 levels are severely
  diminished in various human cancers
• Low p27 levels correlate with tumor
  aggressiveness
• Low p27 levels correlate with poor survival rates
  in cancer patients
• Low p27 levels are strong predictors of disease
  relapse
• Most normal epithelial tissues express high
  levels of p27

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Possible Treatments

• Protein Tyrosine Phosphatase (PTP) inhibitor-
  bpV(phen)
• Increased binding of p27 to CDK2/cyclin E
• Increased p27 availability
• Proteasome inhibitors
• Lactacystin blocks p27 degradation
• Blocks transition from G1 to S
• p27mt
• A mutant of p27
• changes amino-acids Thr187 / Pro188 to Met187 /
  Ile188
• prevents the phosphorylation of Thr187
• Thus, it prevents it from being recognized by
  Skp2 and it is not ubiquitinated

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Possible Treatments

• A few treatments I have cooked up
• Prevent phosphorylation of Serine 10 on p27
• This should prevent p27 from being degraded by
  keeping it in the nucleus
• Prevent Cks1 from associating with the SCF ligase
• This will prevent degradation by making it
  difficult for the substrate to reach the
  substrate binding domain of Skp2
• Skp2 inhibitors
• An inhibitor that mimics p27
• p120 is a possible Skp2 inhibitor (more tests are
  needed)

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Conclusion

• Skp2 is a possible oncoprotein which may
  contribute to cancer growth if over expressed
• p27 is a anti-cancer protein which prevents cell
  cycle progression from G1 to S
• High levels of Skp2 are associated with low
  levels of p27
• Low levels of p27 are excellent markers of tumor
  aggressiveness
• Agents that raise p27 levels or lower Skp2 levels
  may have a place as anti-cancer treatments
• Much more research into the intricacies of this
  pathway is needed

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• THATS ALL FOLKS!

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References

• 1. Schulman, et al. Insights into SCF
  ubiquitin ligases from the structure of the
  Skp1-Skp2 complex (2000) Nature, 408,
  6810-6816
• 2. McIntyre, et al. Differential expression of
  the cyclin-dependent kinase inhibitor p27 in
  primary hepatocytes in early-mid G1 and G1/S
  transitions. (1999) Oncogene, 18, 4577-4585
• 3. Carrano, et al. Skp2 is required for the
  ubiquitin-mediated degradation of the CDK
  inhibitor p27. (1999) Nature Cell Biology, 1,
  193-200.
• 4. Bartek, Jiri and Lukas, Jiri. p27
  destruction Cks1 pulls the trigger. (2001)
  Nature Cell biology, 4, 95-97.
• 5. Caron et al. Cytostatic effect of PTP
  inhibition on ovarian cancer cells A first
  glance at cell cycle regulatory components and
  demonstration of the presence of a pool of
  SHP-1/P27Kip1. (2000) http//www.med.miami.edu/
  mnbws/Caron87.html 4/24/03.

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References (contd)

• 6. Amati, Bruno, and Vlach, Jaromir. Kip1
  meets Skp2 new links in cell-cycle control.
  (1999) Nature Cell Biology, 1, August, E91-E93.
• 7. Chiarle, et al. The cyclin dependent kinase
  inhibitor p27 and its prognostic role in breast
  cancer. (2001) Breast Cancer Res, 3(2) 91-94.
• 8. Lu, et al. The F-box protein Skp2 mediates
  androgen control of p27 stability in LNCaP
  human prostate cells. (2002) BMC Cell Biology,
  3, 22
• 9. Kamura, et al. Degradation of p27(Kip1) at
  the G(0)-G(1) transition mediated by a
  Skp2-independent ubiquitination pathway. (2001)
  J Biol Chem, 276, 52, 48937-48943.