The document summarizes research findings about the role of Protein Kinase C ζ (PKCζ) in the regulation of translation. The key findings are: 1. PKCζ affects translation independently of the mTOR pathway and through its interaction with the scaffold protein RACK1 on ribosomes. 2. PKCζ associates with ribosomes upon activation and binds specifically to the 40S ribosomal subunit. 3. Disrupting the interaction between PKCζ and RACK1 or knocking down RACK1 impairs translation, suggesting their interaction on ribosomes is required for PKCζ to modulate translation.

1. SAN RAFFAELE SCIENTIFIC INSTITUTE & UNIVERSITA’ DEGLI STUDI DEL PIEMONTE ORIENTALE “AMEDEO AVOGADRO” PKC  II Modulates Translation independently from mTOR and through RACK1 Viviana Volta FISV 2008, September 24-27, Riva del Garda

2. Translation Control Roux et al. Journal of Biological Chemistry 2007 MAPK and PI3K-Akt signaling pathways activate translation Signaling pathways converge on mTOR (mammalian target of rapamycin) The phorbol ester PMA , a strong PKC agonist, is also able to increase translation

3. RACK1 is both a scaffold and a ribosomal protein Sengupta et al., Nature Structural and Molecular Biology, 2004 RACK1 (Receptor for Activated C Kinase 1) resides on the 40S next to the mRNA exit channel RACK1 belongs to the WD40 family and interacts with several signaling molecules Macahill et al, Molecular Pharmacology, 2002 RACK1 binds eIF6 makes it to be release from 60S subunit and allows 40S and 60S joining RACK1 mRNA exit 40S subunit 60S subunit PTPμ Fyn GABA A insulin-like growth factor receptor NMDA receptor

4. PKC  II binds RACK1 Ron et al, Proceedings of the National Academy of Science, 1994 RACK1 binds active PKC  II and enhances its activity PKC  II (Protein Kinase C  II) is a conventional PKC isoform, activated both by DAG and Ca 2+ Mackay et al, Nature Review Cancer, 2007

5. RACK1 is a scaffold protein for PKC  II is a structural component of 40S PMA (PKC stimulus) increases translation Can RACK1 be a link between PKC  II and translation

6. PKC  inhibition decreases translation BMI (bisindolylmaleimide I) is a broad PKC inhibitor Hispidin is an inhibitor specific for PKC  isoforms BMI Volta, Grosso et al, Biochem. J., 2008

7. mTOR pathway is not affected by PKC  inhibition LY294002 Hispidin decreases translation without affecting 4E-BP1 and rpS6 phosphorylation Volta, Grosso et al, Biochem. J., 2008

8. Conclusion I: PKC  affects translation independently from mTOR pathway

9. PKC  II cosediments with ribosomes upon PMA stimulation Volta, Grosso et al, Biochem. J., 2008

10. PKC  II binds 40S subunit in vitro PKC  II selectively binds 40S ribosomal subunit in vitro 60S ribosomal subunit does not recruit PKC  II Volta, Grosso et al, Biochem. J., 2008

11. PKC  II copurifies with ribosomes Volta, Grosso et al, Biochem. J., 2008

12. Conclusion II: PKC  II associates with ribosomes

13. R7 peptide inhibits PKC  II-RACK1 binding R7 peptide inhibits the binding of PKC  II with RACK1 R9 is a control peptide Volta, Grosso et al, Biochem. J., 2008

14. R7 peptide impairs PKC  II association to ribosomes R9 treated cells R7 treated cells Volta, Grosso et al, Biochem. J., 2008

15. R7 decreases translation in an mTOR-independent fashion R7 peptide inhibits translation without affecting 4E-BP1 and rpS6 phosphorylation (as hispidin) Volta, Grosso et al, Biochem. J., 2008

16. RACK1 downregulation impairs translation A 70% downregulation of RACK1 was obtained by RNAi Translational rate is decreased in RACK1 depleted cells Volta, Grosso et al, Biochem. J., 2008

17. Conclusion III: PKC  II interaction with RACK1 on the ribosome is required for modulation of translation

18. Ribosomes host PKC activity ribosomes purified by sucrose gradient ribosomes immunopurified by GFP-S18 MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) is a specific PKC substrate Ribosomes purified by two different techniques are able to phosphorylate MARCKS Volta, Grosso et al, Biophys. Biochem Res. Comm., 2008

19. The model PI3K Insulin receptor mTOR Akt eIF4E 4E-BP1 P P P P extracellular environment S6 P S6K intracellular environment insulin growth factors, mitogens (PMA), ecc… eIF4F RACK1 AAAAAAAAAAAA PABP 40S PKC ERK PKC  II MEK 4E-BP1 eIF4E 60S eIF6 P eIF4B P

20. Acknowledgements Molecular Histology and Cell Growth Stefano Biffo Pier Carlo Marchisio Stefano Grosso Marina Vietri Anne Beugnet Annarita Miluzio Valentina Ruggeri External collaborator Dorit Ron (Ernest Gallo Research Center, Emeryville, California ) Thanks for your attention!