The yeast phosphatidic acid (PA) phosphatase, Pah1, regulates the synthesis of triacylglycerol (TAG) and phospholipids by controlling the relative amounts of its substrate PA and product diacylglycerol. Pah1 phosphorylation, which is mediated by multiple protein kinases, has been shown to protect the protein against proteasomal degradation and regulates its localization and catalytic activity. Phosphorylated Pah1 is cytoplasmic, but it translocates to the nuclear/endoplasmic reticulum membrane for catalytic function through its dephosphorylation. In this study, we show that Rim11, a yeast homolog of glycogen synthase kinase phosphorylates Pah1. The rim11Delta mutant shows alterations in lipid composition with elevated TAG levels during exponential growth, the phase in which Rim11 is the most abundant. Rim11 purified from yeast phosphorylated Escherichia coli-expressed Pah1, which lacks endogenous phosphorylation, in a manner that is dependent on reaction time and the amounts of Rim11, Pah1, and ATP. Phosphoamino acid analysis and phosphopeptide mapping showed that Pah1 phosphorylation by Rim11 occurs at multiple serine and threonine residues, which was supported by mass spectrometry analysis which indicated that Rim11 has a major phosphorylation site that is shared with multiple kinases (Ser-602) and two unique minor phosphorylation sites (Thr-163 and Thr-164). Furthermore, in vitro PA phosphatase assays show that Pah1 catalytic activity is inhibited following phosphorylation by Rim11.

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Journal Article

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Khondker SP, Han GS, Carman GM

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