Inositol pyrophosphates modulate hydrogen peroxide signalling

Abstract

Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis

“…Because of a preference of Vip1 to phosphorylate 5-InsP 7 to 1,5-InsP 8 , vip1D yeast cells accumulate the nonmetabolized substrate 5-InsP 7 (Azevedo et al, 2009;Onnebo and Saiardi, 2009;Padmanabhan et al, 2009) (Figure 2F). Levels of 1-InsP 7 and 1,5-InsP 8 are generally low in wild-type yeast due to the activity of Ddp1 (Safrany et al, 1998;Mulugu et al, 2007).…”

Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

“…The homogenate was centrifuged at 14,000 ϫ g for 20 min at 4°C. Calmodulin-Sepharose 4B (10 l) was added to the supernatant and incubated at 4°C for 2 h. The resin was washed twice with binding buffer containing 500 mM NaCl and then twice with 100 mM NaCl (22). The resin was then used to perform an InsP 6 kinase assay using 50,000 cpm/assay of […”

Importance of Radioactive Labelling to Elucidate Inositol Polyphosphate Signalling

“…These two peaks are absent in extracts from kcs1D (IP6K mutant) yeast. The vip1D mutant instead accumulates InsP 7 [48,49]. Other mutants show increased peaks for inositol polyphosphates: Ins(1,4,5)P 3 accumulates in arg82D multikinase mutants [23], while Ins(1,3,4,5,6)P 5 accumulates and is converted by Kcs1 into PP-IP 4 in an ipk1D mutant.…”

“…The IP6Ks (InsP 6 kinase) are able to pyrophosphorylate position 5 of the inositol ring, generating 5PP-InsP 5 (diphosphoinositol pentakisphosphate, InsP 7 ) from InsP 6 or 5PP-InsP 4 from Ins(1,3,4,5,6)P 5 [25,[38][39][40][41][42][43]. Alternatively, PPIP5K (VIP1 in yeast) enzymes can pyrophosphorylate position 1, generating the 1PP-InsP 5 isomer of InsP 7 from InsP 6 in vitro [44][45][46][47], and InsP 8 ([PP] 2 -InsP 4 ; bisdiphosphoinositol-tetrakisphosphate) from 5PP-InsP 5 in vivo [48,49].…”