Avram Hershko

1. Avram Hershko Aaron Ciechanover Irwin Rose The Nobel Prize in Chemistry 2004 JBC 1983 "for the discovery of ubiquitin-mediated protein degradation"

2. SCF catalyzes ubiquitination of phosphorylated substrates using interchangeable substrate-targeting subunits. There are a number of different, interchangeable, F-box proteins, each of which recruits a specific subset of target proteins to SCF for ubiquitination. The rate of ubiquitination is regulated by changing the affinity of the substrate for its corresponding F-box protein usually by phosphorylation.

3. The APC initiates anaphase and mitotic exit. APC catalyzes ubiquitination of many substrates, two of the most important in terms of the cell cycle are: Securin and S and M cyclins. Targets contain aminoacids motifs instead of the phosphorylation recognition of the F box. One of them is the destruction (D) box present in cyclins.

4. APC is another E3 complex. The activator in the case of cyclin is Cdc20 Scf is an E3 ligase complex involved in degradation of phospho proteins

5. Resumption of Meiosis II Knowing that Cdc20 activation is essential for cyclin degradation and resumption of meiosis. What could be the mechanism to arrest the egg in metaphase II?

6. Activation of separase and initiation of sister chromatid separation. Binding of securin to the protease separase has both positive and negative effects on the separase. First, it is thought to promote the proper folding and localization of separase. Second, it binds tightly to both termini of separase thereby blocks separase activation. Separase activity is also suppressed by Cdk-dependent phosphorylation. Activation of Cdc20 triggers destruction of securin and mitotic cyclins, thereby reversing both inhibitory mechanisms that restrain separase activity. Calcium CSF: Activity that maintains Cdc20 inactive and inhibits degradation of cyclin and securin Meiosis resumption after fertilization and calcium increase: Activity that release inactivation of Cdc20 and stimulate degradation of cyclin and securin Fertilization

7. Criteria for CSF 1) It should appear in the oocyte cytoplasm after Progesterone treatment 2) It should cause nuclei to arrest in metaphase 3) It should be maintained until the oocyte is activated 4) It should become inactivated during fertilization or egg activation

8. Assays used to investigate CSF A and B are gain of function experiments. C is a loss of function assay.

9. MPF and CSF levels during oocyte maturation and fertilization How calcium regulates cyclin degradation? MAPK inactivation is delayed with respect to Cyclin degradation and MPF inactivation.

10. Nature 1993 Effect of adding constitutively active CaMKII to a CSF extract in the presence of the MLCK inhibitory peptide. As control WT CamKII was used. - Ca2+ Addition of MLCK peptide that binds and inhibit calmodulin blocks degradation of 35S-labeled cyclin B1 Parallel H1 kinase experiments Ca2+ Autoradiography of exogenously added cyclin B1 to a CSF extract

11. H1 kinase activity after microinjection of constitutively active CaMKII or WT CaMKII. The difference with the previous experiment is that this is done with real eggs and not with the CSF extract. Metabolic labeling of Xenopus oocytes; the Mos protein is immunoprecipitated in eggs that have been microinjected with constitutively active CaMKII or the WT control. Ability of CSF extract to arrest the cell cycle in the presence of constitutively active CaMKII or the WT control. A and B represents two ways of conducting the experiment. A: Oocyte extracts with or without additions. B: Microinjected eggs and then cytosol to donor.

12. Kinase activity assay using syntide a CaMKII consensus sequence substrate. Controls were done with inhibitors of CaMKII (the MLCK peptide used before and another even more specific peptide inhibitor derived from the regulatory portion of the same CaMKII); as a negative control a peptide inhibitor for PKC was used. CSF extract time curve after Ca2+ addition H1 kinase activity Syntide phosphorylation by extracts from eggs activated parthenogentically.

13. Mad: mitotic arrest defective. Bub: budding uninhibited by benzimidazole. These proteins localized on kinetochores until the chromosomes are aligned in Metaphase II plate. Displacement of the proteins from the kinetochores signaled the end of the spindle assembly checkpoint. CaM Kinase II Ca2+ APC/C: Anaphase-promoting Complex or Cyclosome

14. Cdc20

15. UO126 Nature 2002 Emi1 is sufficient to prevent release from CSF block in the presence of Ca2+. Addition of Ca2+. WB with: All panels are Western blots with the respective antibodies. The experiments are done in CSF extracts counting the time from addition of Ca2+. Constitutively active CaMK in the absence of calcium

16. Specificity of Emi1 immunodepletion (dpn). Emi1 accumulates in the maturing oocyte. Emi1 depletion causes cyclin B destruction and mitotic exit in the absence of Ca2+ Quantification of:

17. Also known as Emi2 ?

18. Model

19. Destruction of Polo Box domain Dominant negative N172A W408F The Polo box domains target the kinase to specific substrates and subcellular locations. The polo box domain has a high affinity for proteins that are phosphorylated at serine or threonine residues within specific sequence contexts. Plx1NA: a kinase dead Plx1 whose kinase activity is abolished by a N172A point mutationD Plx1WF: a kinase active Plx1 with a W408F point mutation, which has been demonstrated to dramatically abolish the localization capability of the Polo Box domain (PBD). This mutation also blocks the effects of Plx1 or Plx1NA on mltiple mitotic steps, including CSF release.

20. 2005 Plx1 Induces CSF Release in the Absence of Calcium in a Polo-Box-Dependent Manner Plx1NA: a kinase dead Plx1 whose kinase activity is abolished by a N172A point mutationD Plx1WF: a kinase active Plx1 with a W408F point mutation, which has been demonstrated to dramatically abolish the localization capability of the Polo Box domain (PBD).

21. Figure 2. Depletion of Plx1 Blocks Calcium-Induced CSF Release Figure 3. Plx1 and CamCat Require Each Other to Induce CSF Release CamCat-induced release was monitored with or without Plx1 depletion, and the corresponding nuclear morphology is shown in the lower panel.

22. Figure 3. Plx1 and CamCat Require Each Other to Induce CSF Release CSF release by CamCat was monitored in the presence of Plx1NA (Dominant negative), as indicated. The corresponding nuclear morphology is shown in the lower panel at the indicated times. Effect of CamK inhibitor +281-309 on CSF release and cyclin B1 degradation.

23. Combined action of PLx1 and Cam-Cat overcomes inhibition by Emi2. +Ca2+ -Ca2+ +Ca2+ -Ca2+ -Ca2+ +Ca2+ -Ca2+ CSF release was monitored by cyclin WB in the presence of Emi2 and or different kinases Phosphorylation of Emi2 by CaMKII, by Plx1 and by both together CSF release and Emi2 degradation by the combination of both kinases

24. Model SCF catalyzes ubiquitination of phosphorylated substrates APC catalyzes ubiquitination of many substrates, two of the most important in terms of the cell cycle are: Securin and S and M cyclins. Targets contain aminoacids motifs instead of the phosphorylation recognition of the F box. One of them is the destruction (D) box present in cyclins.

25. Which is the relationship between the Mos Pathway and Emi2? Nature 2007

26. Nature 1991 Two approaches to analyze the sequence important for cyclin degradation: 1) cyclin deletion mutants; 2) addition of sequence to another protein. In this case Protein A.

27. Autoradiography 125I proteins are added to Xenopus mitotic or interphase extracts. Degradation is evaluated after different time periods. Cyclin deletion mutants Protein A-cyclin fusion Interphase extract Mitotic extract

28. 125I ubiquitin form high Mr complexes with cyclin-Protein A fusion proteins. The autoradiography is developed after immunoprecipitation of Protein A with Ig-Sepaharose. 13-91cyclin-protein A fusion is degraded and it is ubiquitinated. However, 13-91prA-R42C mutant is not degraded and not ubiquitinated. Kinetic analysis of degradation of 13-91prA. A) extracts; B) Immunoprecipitated with Ig Sepharose. Experiment was done using mitotic extracts.