Casein kinase II–mediated phosphorylation of lipin 1β phosphatidate phosphatase at Ser-285 and Ser-287 regulates its interaction with 14-3-3β protein

Meagan Hennessy,Mitchell E Granade,Azam Hassaninasab,Dana Wang,Joanna M Kwiatek,Gil-Soo Han,Thurl E Harris,George M Carman

doi:10.1074/jbc.ra118.007246

Meagan Hennessy, Mitchell E Granade + Show 6 more

Open Access

https://doi.org/10.1074/jbc.ra118.007246

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Abstract

The mammalian lipin 1 phosphatidate phosphatase is a key regulatory enzyme in lipid metabolism. By catalyzing phosphatidate dephosphorylation, which produces diacylglycerol, the enzyme plays a major role in the synthesis of triacylglycerol and membrane phospholipids. The importance of lipin 1 to lipid metabolism is exemplified by cellular defects and lipid-based diseases associated with its loss or overexpression. Phosphorylation of lipin 1 governs whether it is associated with the cytoplasm apart from its substrate or with the endoplasmic reticulum membrane where its enzyme reaction occurs. Lipin 1β is phosphorylated on multiple sites, but less than 10% of them are ascribed to a specific protein kinase. Here, we demonstrate that lipin 1β is a bona fide substrate for casein kinase II (CKII), a protein kinase that is essential to viability and cell cycle progression. Phosphoamino acid analysis and phosphopeptide mapping revealed that lipin 1β is phosphorylated by CKII on multiple serine and threonine residues, with the former being major sites. Mutational analysis of lipin 1β and its peptides indicated that Ser-285 and Ser-287 are both phosphorylated by CKII. Substitutions of Ser-285 and Ser-287 with nonphosphorylatable alanine attenuated the interaction of lipin 1β with 14-3-3β protein, a regulatory hub that facilitates the cytoplasmic localization of phosphorylated lipin 1. These findings advance our understanding of how phosphorylation of lipin 1β phosphatidate phosphatase regulates its interaction with 14-3-3β protein and intracellular localization and uncover a mechanism by which CKII regulates cellular physiology.

Highlights

The mammalian lipin 1 phosphatidate phosphatase is a key regulatory enzyme in lipid metabolism
We demonstrate that lipin 1␤ is phosphorylated by casein kinase II (CKII), a conserved serine/threonine protein kinase that is essential for viability and cell cycle progression in mammalian cells (54 –56)
When lipin 1␤ was phosphorylated by CKII after pretreatment with ␭-phosphatase [58] to remove phosphates from its endogenous phosphorylation, it did not show a difference in the extent of phosphorylation

Summary

Results

Lipin 1␤ PA phosphatase is the predominant isoform in most tissues, and it is known to be phosphorylated on multiple residues [42, 47]. CKII activity followed Michaelis–Menten kinetics with apparent Vmax and Km values, respectively, for the WT peptide of 45 Ϯ 5 nmol/min/mg and 146 Ϯ 40 ␮M and for ATP of 10.7 Ϯ 0.8 nmol/min/mg and 6.2 Ϯ 1.5 ␮M These results indicated that the lipin 1␤ peptide (i.e. serine-rich region) is phosphorylated by CKII. After determining the phosphorylation of the serine-rich region by CKII, we examined which of the seven serine/threonine residues is a target phosphorylation site For this purpose, CKII activity was measured on the derivatives of the lipin 1␤ peptide in which the alanine residue was substituted for the Ser or Thr residue (Fig. 7). The CKIIphosphorylated WT lipin 1␤ was digested with TPCK-treated trypsin, and the resulting peptides were separated by electrophoresis and TLC This analysis showed multiple phosphopeptides from the CKII-phosphorylated lipin 1␤, indicating that the protein is phosphorylated on many sites (Fig. 8). Whereas these reductions caused by the individual and combined mutations were significant, the mutational effects were not significantly different from each other (Fig. 9D)

Discussion

Experimental procedures

Analysis of phosphopeptides and phosphoamino acids

PA phosphatase assay

Analyses of data