DescriptionLipin is a phosphatidate phosphatase enzyme that catalyzes the penultimate step in triacylglycerol synthesis, the conversion of phosphatidic acid to diacylglycerol. By controlling this step, lipin regulates both triacylglycerol and phospholipid synthesis, and it also acts as a transcriptional regulator. It is known that phosphorylation controls the subcellular localization of lipin, and its activity as both a phosphatidate phosphatase and a transcriptional regulator. Multiple phosphorylation sites within lipin have been identified, but the identity of the protein kinases involved is largely unknown. In this work, we sought to identify and characterize kinases that play a role in lipin’s regulation.
Using bioinformatics, we found that lipin 1 beta has a high probability of being phosphorylated by casein kinase II. Using purified lipin 1 beta, we showed that casein kinase II catalyzed the incorporation of the gamma-phosphate of [gamma-32P]ATP into the protein. We then focused on the serine-rich domain of lipin 1 beta, which in lipin 1 beta has been shown to be important in subcellular localization through interaction with 14-3-3 protein. We generated a 22-residue peptide that included 7 putative serine/threonine phosphorylation sites, and found that this peptide was phosphorylated by casein kinase II. Phosphorylation analysis of the peptide with alanine substitutions for the putative sites indicated that Ser-285 and Ser-287 were the targets of the kinase. In full-length lipin 1 beta, the S285A mutation caused a greater degree of phosphorylation, whereas the extent of phosphorylation of the S285A/S287A mutant was about the same as that of the control wild type protein. In phosphopeptide mapping analysis, two phosphopeptides present in the wild type protein were absent in both mutant proteins, but one phosphopeptide present in the mutants was absent in the wild type protein. These observations indicate that the phosphorylation by casein kinase II is complex. This work increases our understanding of the posttranslational modification of lipin 1 beta by phosphorylation.