Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity

The Journal of Biological Chemistry
Nileeka BalasuriyaPatrick O'Donoghue

Abstract

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only part...Continue Reading

References

Nov 10, 1995·The Journal of Biological Chemistry·S E TsutakawaD E Koshland
Sep 6, 1996·The Journal of Biological Chemistry·A D KohnR A Roth
Feb 12, 1998·The Journal of Biological Chemistry·M AndjelkovićB A Hemmings
Sep 25, 1999·Protein Expression and Purification·D FabbroS Ferrari
Mar 18, 2000·The Journal of Biological Chemistry·A Toker, A C Newton
Aug 7, 2002·Biochemistry·Hadas ReuveniAlexander Levitzki
Aug 9, 2002·Molecular and Cellular Biology·Michael P ScheidJames R Woodgett
Mar 17, 2004·Biochimica Et Biophysica Acta·Masahito HanadaBrian A Hemmings
Dec 8, 2004·The Journal of Biological Chemistry·Maya T KunkelAlexandra C Newton
Feb 19, 2005·Science·D D SarbassovDavid M Sabatini
Mar 1, 2005·Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology·Derek L DaiGang Li
Apr 2, 2005·Protein Expression and Purification·Shoshana KleinAlexander Levitzki
Sep 9, 2005·American Journal of Clinical Pathology·Ana SlipicevicVivi Ann Flørenes
Nov 17, 2005·Oncogene·Deborah A Altomare, Joseph R Testa
Mar 17, 2006·The Journal of Biological Chemistry·Xiaoling ZhangRichard L Kendall
Jul 3, 2007·Cell·Brendan D Manning, Lewis C Cantley
May 6, 2008·Bioorganic & Medicinal Chemistry Letters·Blaise LippaJoel Morris
Dec 17, 2009·Current Opinion in Genetics & Development·Kwok-Kin WongLewis C Cantley
Jun 15, 2011·Oncotarget·Jonathan R Hart, Peter K Vogt
Dec 1, 2009·Current Protocols in Chemical Biology·Maya T Kunkel, Alexandra C Newton
Aug 27, 2011·Science·Hee-Sung ParkDieter Söll
Nov 15, 2011·Cell·Samuel M PearlmanJames E Ferrell
Feb 16, 2012·The Journal of Biological Chemistry·Jon M SteichenSusan S Taylor
Aug 15, 2012·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Tony Hunter
Mar 28, 2013·Angewandte Chemie·Sangsik LeeHee-Sung Park
Jun 22, 2013·Molecular & Cellular Proteomics : MCP·Corina E Antal, Alexandra C Newton
Aug 10, 2013·Molecular & Cellular Proteomics : MCP·Demetris C IacovidesJoe W Gray
Sep 10, 2013·Surgical Oncology Clinics of North America·Burhan HassanFunda Meric-Bernstam
Jan 8, 2014·Annual Review of Pharmacology and Toxicology·Alexandra C Newton, Lloyd C Trotman
Jun 6, 2014·Annals of Medicine·Miriam MartiniEmilio Hirsch

❮ Previous
Next ❯

Citations

May 7, 2019·RNA Biology·Christina Z ChungIlka U Heinemann
May 1, 2020·The Journal of Biological Chemistry·Nileeka BalasuriyaPatrick O'Donoghue
Sep 13, 2018·Genes·Nileeka BalasuriyaPatrick O'Donoghue
Jul 11, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Hao ChenChenguang Fan
Sep 25, 2019·The Journal of Biological Chemistry·Alison L KearneyDavid E James
Feb 23, 2021·Frontiers in Bioengineering and Biotechnology·McShane McKennaPatrick O'Donoghue
Mar 23, 2019·Current Opinion in Structural Biology·Philip A ColeHwan Bae
Apr 15, 2021·Science Signaling·Timothy R BaffiAlexandra C Newton
Jul 22, 2021·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Li MengXiao-Yu Hou
Aug 14, 2021·Proceedings of the National Academy of Sciences of the United States of America·Linda TruebesteinThomas A Leonard
Dec 18, 2021·Proceedings of the National Academy of Sciences of the United States of America·Bo GuoHai-Lan Chen

❮ Previous
Next ❯

Related Concepts

Related Feeds

AKT Pathway

This feed focuses on the AKT serine/threonine kinase, which is an important signaling pathway involved in processes such as glucose metabolism and cell survival.

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.