Association of GCN1-GCN20 regulatory complex with the N-terminus of eIF2alpha kinase GCN2 is required for GCN2 activation

Stimulation of GCN4 mRNA translation due to phosphorylation of the alpha-subunit of initiation factor 2 (eIF2) by its specific kinase, GCN2, requires binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like domain in GCN2. GCN2 function in vivo also requires GCN1 and GCN20, but it was unknown whether these latter proteins act directly to promote the stimulation of GCN2 by uncharged tRNA. We found that the GCN1-GCN20 complex physically interacts with GCN2, binding to the N-terminus of the protein. Overexpression of N-terminal GCN2 segments had a dominant-negative phenotype that correlated with their ability to interact with GCN1-GCN20 and impede association between GCN1 and native GCN2. Consistently, this Gcn(-) phenotype was suppressed by overexpressing GCN2, GCN1-GCN20 or tRNA(His). The requirement for GCN1 was also reduced by overexpressing tRNA(His) in a gcn1Delta strain. We conclude that binding of GCN1-GCN20 to GCN2 is required for its activation by uncharged tRNA. The homologous N-terminus of Drosophila GCN2 interacted with yeast GCN1-GCN20 and had a dominant Gcn(-) phenotype, suggesting evolutionary conservation of this interaction.     

c-Cbl is not required for ERK1/2-dependent degradation of BimEL

Bim_EL the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim_EL. It has been proposed that the RING finger protein Cbl binds to Bim_EL and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim_EL is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim_EL turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim_EL is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim_EL does not involve its tyrosine phosphorylation; indeed, Bim_EL is not a tyrosine phosphoprotein. Furthermore, Bim_EL fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim_EL turnover proceeds normally in Cbl-containing or Cbl−/− fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim_EL turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.     

ABA is required for Leptosphaeria maculans resistance via ABI1- and ABI4-dependent signaling

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLMcol pathway. ABA-deficient and -insensitive mutants in Ler-0 background (abal-3 and abil-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abil-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abil-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. beta-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1co1)pad3 and rlmlLerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1col. ABII, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1co, expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABIJ activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.     

Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells

GCN2 stimulates GCN4 translation in amino acid-starved cells by phosphorylating the alpha-subunit of translation initiation factor 2. GCN2 function in vivo requires the GCN1/GCN20 complex, which binds to the N-terminal domain of GCN2. A C-terminal segment of GCN1 (residues 2052-2428) was found to be necessary and sufficient for binding GCN2 in vivo and in vitro. Overexpression of this fragment in wild-type cells impaired association of GCN2 with native GCN1 and had a dominant Gcn(-) phenotype, dependent on Arg2259 in the GCN1 fragment. Substitution of Arg2259 with Ala in full-length GCN1 abolished complex formation with native GCN2 and destroyed GCN1 regulatory function. Consistently, the Gcn(-) phenotype of gcn1-R2259A, but not that of gcn1Delta, was suppressed by overexpressing GCN2. These findings prove that GCN2 binding to the C-terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo. GCN1 expression conferred sensitivity to paromomycin in a manner dependent on its ribosome binding domain, supporting the idea that GCN1 binds near the ribosomal acceptor site to promote GCN2 activation by uncharged tRNA.