Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes

Abstract

Lipin-1 is a bifunctional protein involved in lipid metabolism and adipogenesis. Lipin-1 plays a role in the biosynthesis of triacylglycerol through its phosphatidate phosphatase activity and also acts as a transcriptional co-activator of genes involved in oxidative metabolism. Lipin-1 resides in the cytoplasm and translocates to the endoplasmic reticulum membrane to catalyze the phosphatidate phosphatase reaction. It also possesses a nuclear localization signal, which is required for its translocation to the nucleus and may therefore be important for lipin-1 co-activator function. Thus, subcellular localization may be an important factor in the regulation of this protein. Here, we show that the nuclear localization signal alone is not sufficient for lipin-1 nuclear localization, and identify lipin-1 interaction with 14-3-3 as a determinant of its subcellular localization. We demonstrate that lipin-1 interacts with 14-3-3 proteins and that overexpression of 14-3-3 promotes the cytoplasmic localization of lipin-1 in 3T3-L1 adipocytes. The effect of 14-3-3 is mediated through a serine-rich domain in lipin-1. Functional mapping of the 14-3-3-interacting region within the serine-rich domain indicates redundancy and cooperativity among several sites, including five phosphorylated serine and threonine residues. Insulin stimulation of 3T3-L1 adipocytes results in increased lipin-1 phosphorylation, enhanced interaction with 14-3-3, and predominantly cytoplasmic localization. In summary, our studies suggest that insulin may modulate the cellular function of lipin-1 by regulating its subcellular localization through interactions with 14-3-3 proteins.

FIGURE 1.

Subcellular localization and the nuclear localization signal of lipin-1α. A, confocal immunolocalization of wild-type lipin-1α in transfected 3T3-L1 adipocytes. Representative images of cells with predominantly nuclear (NUC), cytoplasmic (CYTO), or simultaneous cytoplasmic and nuclear (CYTO + NUC) lipin-1 staining are shown. B, schematic representation of the domain structure of wild-type lipin-1 and the ΔNLS mutant lacking the putative NLS (black box and amino acid sequence). The N- and C-terminal evolutionarily conserved regions (NLIP and CLIP, respectively) are indicated. C, quantitative assessment of the subcellular localization of lipin-1α and the ΔNLS mutant in 3T3-L1 adipocytes. The fraction of cells exhibiting lipin-1α distribution patterns illustrated in A are shown.

FIGURE 2.

Lipin-1 interacts with 14-3-3. A, GST pulldown experiment. Cell lysates were prepared from HEK293 cells transfected with lipin-1α-V5 and incubated with GST alone, wild-type, or mutant (K49E) GST-14-3-3β fusion proteins attached to glutathione-agarose. Eluted material was analyzed by Western blotting using anti-V5 antibody (top panel) or Coomassie staining (bottom panel). B, co-immunoprecipitation of transfected wild-type lipin-1α-V5 (top panel) or ΔNLS (bottom panel) with endogenous 14-3-3 from HEK293 cells.

FIGURE 3.

14-3-3 affects the nucleocytoplasmic localization of lipin-1α. A, quantitation of the subcellular localization of lipin-1α by confocal immunofluorescence microscopy. HEK293 cells were co-transfected with lipin-1α-V5 and empty (−) or 14-3-3-expressing vector (+). B, subcellular localization of lipin-1α in 3T3-L1 adipocytes analyzed as in panel A. Wild-type 14-3-3β and 14-3-3θ isoforms, or a mutant of 14-3-3θ with the R56A/R60A (A^56/60) double mutation, were co-transfected with lipin-1α-V5. * and + indicate statistically significant (p < 0.05) differences compared with no or wild-type 14-3-3 transfections, respectively.

FIGURE 4.

14-3-3 interaction motif in the SRD of lipin-1. A, schematic representation of lipin-1 showing the serine-rich domain (SRD). Pinheads indicate the locations of consensus 14-3-3 interaction motifs (S-X-P). The amino acid positions of the respective Ser residues are 228, 245, 248, 286, 294, and 569. The solid pinhead for Ser²⁴⁸ indicates that this residue is known to be phosphorylated in adipocytes (7). The nuclear localization signal is represented as a black bar. B, lipin-1 contains a phosphorylated 14-3-3 interaction motif. Lipin-1α-V5 immunoprecipitated from transfected HEK293 cells was treated with or without λ-phosphatase and detected by Western blotting using a phosphorylation-specific antibody recognizing the 14-3-3 interaction motif (top panel) or anti-V5 (bottom panel). C, the phosphorylated 14-3-3 interaction motif is located in the SRD. HEK293 cells were transfected with wild-type or mutant lipin-1α lacking the SRD and analyzed as in panel B.

FIGURE 5.

Structural determinants of lipin-1 nucleocytoplasmic localization and 14-3-3 interaction. A, mutant lipin-1α constructs used in the experiments of panel B. Gray boxes and lines represent sequences present or deleted in various mutants, respectively. The diagram above wild-type lipin-1 shows the ΔSRD mutant, in which the entire SRD has been deleted. H1–2 and Q1–4 represent constructs with subdeletions within the SRD, and pinheads indicate the locations of 14-3-3 interaction motifs, as described in Fig. 4A. The Q4 mutant was used to generate the following mutations of known (7) phosphorylated residues: Ser²⁴⁸ → Ala (A²⁴⁸), Thr²⁴⁹ → Ala (A²⁴⁹), Ser²⁴⁸ → Ala/Thr²⁴⁹ → Ala (AA), Ser²⁵² → Ala (A²⁵²), Ser²⁵⁴ → Ala (A²⁵⁴), and Ser²⁶⁰ → Ala (A²⁶⁰). B, quantitative assessment of the nucleocytoplasmic distribution of mutant lipin-1α proteins in transfected 3T3-L1 adipocytes using confocal immunofluorescence microscopy. Gray and open bars represent co-transfection with or without 14-3-3θ. * and + indicate statistically significant (p < 0.05) differences compared with wild-type lipin-1α or co-transfection without 14-3-3 construct, respectively.

FIGURE 6.

Insulin stimulates phosphorylation, interaction with 14-3-3, and cytoplasmic localization of lipin-1. A, insulin (I, 10 milliunits/ml) stimulates, whereas rapamycin (R, 20 nm) and wortmannin (W, 100 nm) inhibit phosphorylation of endogenous lipin-1 in 3T3-L1 adipocytes. Lipin-1 was detected with anti-lipin-1 antibody (top panel), and phosphorylation of S6K was confirmed with a phosphorylation-specific antibody recognizing Thr³⁸⁹ (bottom panel). B, GST pulldown experiment. Lysates prepared from 3T3-L1 adipocytes treated as described above were incubated with GST-14-3-3β or control GST-FKBP12 fusion proteins attached to GSH-Sepharose. Eluates were analyzed by Western blotting using anti-lipin-1 (top panel) or anti-GST antibody (bottom panel). C, the effects of insulin (I) and rapamycin (R) treatments were analyzed in serum-starved 3T3-L1 adipocytes cotransfected with lipin-1α-V5 and 14-3-3θ using confocal immunofluorescence microscopy. * and + indicate statistically significant (p < 0.05) differences compared with control (C) or insulin only, respectively. D, 3T3-L1 adipocytes were serum-starved and treated with insulin (I), rapamycin, and insulin, or not treated (C). After subcellular fractionation, the localization of endogenous lipin-1 to the cytosolic (CYTO) and nuclear (NUC) fractions was analyzed by Western blotting using anti-lipin-1 antibody.