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. 2002 Jan 22;99(2):1047-52.
doi: 10.1073/pnas.022634399. Epub 2002 Jan 15.

Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin

Todd A Huffman  1 , Isabelle Mothe-Satney, John C Lawrence Jr
Affiliations

Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin

Todd A Huffman et al. Proc Natl Acad Sci U S A. .

Abstract

The phosphorylation of a previously uncharacterized protein of apparent M(r) approximately 140,000 was found to be increased when rat adipocytes were incubated with insulin. The sequences of peptides generated by digesting the protein with trypsin matched perfectly with sequences in mouse lipin. Lipin is the product of the gene that is mutated in fatty liver dystrophy (fld) mice [Peterfy, M., Phan, J., Xu, P. & Reue, K (2001) Nat. Genet. 27, 121-124], which exhibit several phenotypic abnormalities including hyperlipidemia, defects in adipocyte differentiation, impaired glucose tolerance, and slow growth. When immunoblots were prepared with lipin antibodies, both endogenous adipocyte lipin and recombinant lipin overexpressed in HEK293 cells appeared as bands ranging in apparent M(r) from 120,000 to 140,000. Incubating adipocytes with insulin decreased the electrophoretic mobility and stimulated the phosphorylation of both Ser and Thr residues in lipin. The effects of insulin were abolished by inhibitors of phosphatidylinositol 3-OH kinase, and by rapamycin, a specific inhibitor of the mammalian target of rapamcyin (mTOR). The inhibition by rapamycin was blocked by FK506, which competitively inhibits those effects of rapamycin that are mediated by inhibition of mTOR. Moreover, amino acids, which activate mTOR, mimicked insulin by increasing lipin phosphorylation in a rapamycin-sensitive manner. Thus, lipin represents a target of the mTOR pathway, and potentially links this nutrient-sensing pathway to adipocyte development.

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Figures

Figure 1
Figure 1
Identification of lipin. Rat adipocytes were incubated in medium containing 32Pi for 90 min, and then incubated without or with 1 milliunit/ml insulin for an additional 20 min before extracts were prepared. Immunoprecipitations were conducted by using nonimmune IgG (NI IgG) or sheep PKB-β antibody (PKB-β sAb). Samples of extracts and immunoprecipitated proteins were subjected to SDS/PAGE, and an autoradiogram of the dried gel was prepared (A). The bands of 32P corresponding to ATP-citrate lyase (ACL, appMr = 130,000), isp62 (appMr = 62,000), ribosomal protein S6 (S6, appMr = 32,000), and PHAS-I (appMr = 22,000) are indicated by the arrows. The major 32P-labeled proteins immunoprecipitated correspond to PKB-β and an unknown protein (filled arrow), which we later identified as lipin. In a separate experiment, immunoprecipitations from extracts were performed with nonimmune IgG and two lipin antibodies, LAb-1 and LAb-2 (B). The filled arrow points to lipin.
Figure 2
Figure 2
Amino acid sequence of mouse lipin. The lipin sequence (25) with underlined residues denoting peptides sequenced by mass spectrometry are shown (A). Lipin cDNA having a 99-bp insert was cloned from a mouse liver cDNA library. The extra bases encode 33 aa, which would be inserted between amino acids 240 and 241 in lipin (B). The sequence of the insert has a region homologous to that of the peptide used to generate the PKB-β antibody (C).
Figure 3
Figure 3
Lipin levels in wild-type and fld mice. Extracts (75 μg protein) of different tissues from wild-type (+/+) mice or mice homozygous (fld/fld) for the fld allele were subjected to SDS/PAGE, and immunoblots were prepared with LAb-1. Pictures of regions of the blot surrounding lipin, denoted by the filled arrows, are shown.
Figure 4
Figure 4
Sheep PKB-β antibodies crossreact with recombinant lipin. HEK293 cells were transfected with pKH3 or with pKH3-lipin. After preparation of extracts, immunoprecipitations were conducted with the sheep PKB-β Ab (PKB-sAb) or with the rabbit PKB-β antibody (PKB-β rAb), which we generated for this study. Samples of the immunoprecipitated proteins were subjected to SDS/PAGE, and immunoblots were prepared with the HA antibody, 12CA5, or with the sheep PKB-β antibody (A). Immunoprecipitations were also conducted with the two PKB-β antibodies and with 12CA5, before an immunoblot was prepared with the sheep PKB-β antibody (B). The filled and open arrows point to lipin and PKB-β, respectively.
Figure 5
Figure 5
mTOR-dependent phosphorylation of lipin. 32P-Labeled rat adipocytes were incubated with 25 nM rapamycin, 200 nM FK506, or the combination of agents for 30 min. After adding insulin (1 milliunit/ml) as indicted, the incubations were continued for 20 min before extracts were prepared. [32P]Lipin was immunoprecipitated with LAb-1 and subjected to SDS/PAGE. An autoradiogram of the dried gel in the region surrounding lipin, and a lipin immunoblot of cell extracts, are shown (A). In a separate experiment, 32P-labeled rat adipocytes were incubated without or with 25 nM rapamycin for 30 min, and then incubated for an additional 10 min after insulin (1 milliunit/ml final concentration) and/or MEM amino acids (2.5× final concentration) were added. 32P-labeled lipin and PKB-β were immunoprecipitated with LAb-1 and the rabbit PKB-β antibody, respectively. Autoradiograms of [32P]lipin and [32P]PKB-β, and an immunoblot showing lipin from the cell extracts, are shown (B).
Figure 6
Figure 6
Phosphoamino acid analyses of 32P-labeled lipin. Samples of 32P-labeled lipin from control adipocytes (CON) or cells that had been incubated for 20 min with either 1 milliunit/ml insulin (INS) or 25 nM rapamycin plus insulin (INS + RAP) were subjected to SDS/PAGE. The protein was transferred to an Immobilon P membrane, then hydrolyzed in 5.7 M HCl for 1.5 h at 110°C. After adding standards of P-Ser (P-S), P-Tyr (P-Y), and P-Thr (P-T) to allow visualization with ninhydrin, the phosphoamino acids were resolved by two-dimensional high voltage electrophoresis at pH 1.9 and pH 3.5. Pictures of autoradiograms in the region surrounding the phosphoamino acids are shown.
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