Understanding Postranslational Modifications Involved in Adi3 Programmed Cell Death Signaling
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Programmed cell death (PCD) is an active process by which organisms coordinate the controlled destruction of cells. In tomato, the protein kinase Adi3 (AvrPto-dependent Pto-interacting kinase 3), acts as a negative regulator of PCD and shares important functional homologies with the mammalian anti-apoptotic AGC kinase PBK/Akt. Adi3 was originally identified as an interactor of the complex formed by the tomato resistance protein Pto and the Pseudomonas syringae pv. tomato (Pst) effector protein AvrPto. The complex formed by AvrPto and Pto causes a resistance response characterized by a rapid form of PCD that limits the spread of Pst and prevents the onset of the tomato bacterial speck disease. In an effort to characterize the mechanisms by which Adi3 regulates PCD, we identified Adi3 interacting partners in a Y2H screen. Here, I describe the interaction of Adi3 with two interacting partners identified: the Sucrose Non-fermenting (SNF1) kinase complex (SnRK) which is a eukaryotic master regulator of energy homeostasis and the E3 RING Ubiquitin ligase AdBiL. Using a combination of in vitro and in vivo approaches I found that AdBiL is an active ubiquitin ligase that ubiquitinates Adi3. Interestingly, Adi3 was found to be degraded in a proteasome-dependent manner suggesting ubiquitination could play a role in its degradation. On the other hand, Adi3 was found to inhibit the SnRK complex by directly interacting with its catalytic subunit as well as by phosphorylating the regulatory subunit SlGal83 at Ser26. SlGal83 is phosphorylated at multiple sites in vivo, and this phosphorylation state, as well as its intracellular localization was found to depend on a myristoylation signal present at its N-terminus. Phosphorylation at Ser26 by Adi3 was found to alter the localization of this subunit in a myristoylation-dependent manner. The interactions studied in this dissertation provide additional evidence on the functional homologies shared by Adi3 and PKB. In addition, the regulatory control of SnRK activity and cellular localization offers a novel connection between pathways involved in energy homeostasis and pathogen-mediated PCD.
Avila Pacheco, Julian Ricardo, 1983- (2012). Understanding Postranslational Modifications Involved in Adi3 Programmed Cell Death Signaling. Doctoral dissertation, Texas A&M University. Available electronically from