Phosphorylation regulates myo‐inositol 3‐phosphate synthase: A novel regulatory mechanism of inositol biosynthesis (LB190)

Abstract

Myo‐inositol phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate (VPA), suggesting that the enzyme is post‐translationally regulated. Using 32Pi ‐ labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation‐deficient and phosphomimetic site‐mutants indicated that the three conserved sites in yeast (S184, S296, and S374) and human (S177, S279, and S357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phospho‐deficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374 caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to VPA. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPSGrant Funding Source: Supported by National Institutes of Health Grant DK 081367, and Wayne State University Graduate Research Fellowship and Graduate Research Enhancement Funds