Crosstalk between Ras and inositol phosphate signaling revealed by lithium action on inositol monophosphatase in Schizophyllum commune

Abstract

Mushroom forming basidiomycete Schizophyllum commune has been used as a tractable model organism to study fungal sexual development. Ras signaling activation via G-protein-coupled receptors (GPCRs) has been postulated to play a significant role in the mating and development of S. commune. In this study, a crosstalk between Ras signaling and inositol phosphate signaling by inositol monophosphatase (IMPase) is revealed. Constitutively active Ras1 leads to the repression of IMPase transcription and lithium action on IMPase activity is compensated by the induction of IMPase at transcriptome level. Astonishingly, in S. commune lithium induces a considerable shift to inositol phosphate metabolism leading to a massive increase in the level of higher phosphorylated inositol species up to the inositol pyrophosphates. The lithium induced metabolic changes are not observable in a constitutively active Ras1 mutant. In addition to that, proteome profile helps us to elucidate an overview of lithium action to the broad aspect of fungal metabolism and cellular signaling. Taken together, these findings imply a crosstalk between Ras and inositol phosphate signaling.

Keywords: IMPA; Inositol monophosphatase; Inositol phosphates; Ras; Schizophyllum commune; Signaling; basidiomycete fungi; lithium.

Fig. 1

Inositol phosphate (IP) signaling pathway in S. commune. The binding of extracellular stimuli to G-alpha subunit (G_α) activates subsequently phospholipase C (PLC). PLC cleaves phosphatidylinositol (PI)-4,5-bisphosphate (PIP₂) generating 2 s messengers, inositol 1,4,5 triphosphate (IP₃), shuttling between higher phosphorylated inositol phosphate (IP₄ to IP₈) and regenerating inositol through IP₂ and IP₁, and diacylglycerol (DAG) activating protein kinase C (PKC). The lithium sensitive and Ras-regulated inositol monophosphatase (IMPase) generates inositol from inositol monophosphate (IP₁). The Ras module with its guanosin exchange factor (GEF) and its GTPas activating protein (GAP) is shown. Inositol is used to build up phosphoinositides (PI; PIP; PIP₂) and inositol phosphates using inositol phosphate kinases (black arrow) and phosphatases (dashed orange arrow).

Fig. 2

Expression of imp1. Expression levels on transcript (A) and IMPase enzyme activity (B) were tested for the wild type S. commune 4–39 and constitutively active Ras1 mutant S. commune ras1^G12V. Lithium was tested for imp1 gene induction, counter-acting enzyme repression; tef1 was used as reference gene.

Fig. 3

Influence of lithium on colony and hyphal morphology, and on growth rates.S. commune 4–39 (A–F) and constitutively active Ras1 mutant S. commune ras1^G12V (G–L) were screened after 7 days for the effects of 5 mM LiCl (C, F, I, L) or KCl (B, E, H, K); red arrows show irregular hyphae; black arrow shows bulbous or swollen hyphae. Growth rates of S. commune 4–39 (M) and S. commune ras1^G12V (N) were measured; n = 3.

Fig. 4

Effect of lithium on the inositol phosphates profile of wildtype S. commune 4–39 and constitutively active S. commune Ras1^G12Vmutant. Chromatographic elution profile of soluble inositol phosphates of S. commune wild type 4–39 (A) and constitutive active Ras1^G12V mutant (B). Mycelia were labeled 24 h in absence (black trace) or presence of lithium (red trace) as described in material and methods, before extraction and SAX-HPLC analysis. To reveal changes in the inositol pyrophosphate IP₇ and IP₈, the chromatogram was presented in a different scale (inset). Elution time of genuine standard (IP₃, [³H]I(1,4,5)P₃; IP₆, [³H]IP₆; IP₇, [³H]PP-IP5) was used for peak identification. Of the three IP₅ species identified in S. commune, IP5_a was identified as [³H]I(1,3,4,5,6)P₅. The elution profile is representative of experiments independently repeated three times.

Fig. 5

KOG annotation for proteins regulated by lithium.S. commune 4–39 and S. commune ras1^G12V induced and repressed proteome changes are presented.