Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity

Bernard P.C Kok,Tamara D Skene-Arnold,Ji Ling,Matthew G.K Benesch,Jay Dewald,Thurl E Harris,Charles F.B Holmes,David N Brindley

doi:10.1074/jbc.m114.552612

Abstract

Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg(2+) or Mn(2+) and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ.

Highlights

Lipin-1 functions as a phosphatidate phosphatase in glycerolipid synthesis and as a co-transcriptional regulator
The function of the N terminus domain of lipins (NLIP) domain is less clear, a point mutation (G84R) in mice produces the same phenotype as lipin-1-deficient fld mice [34]; this mutation abrogates Phosphatidate Phosphatase (PAP) activity [5] and blocks lipin-1 nuclear localization [34]
Val-Arg-Phe, HVRF) in the NLIP domain, which is conserved in the yeast lipin, Pah1p (Fig. 1)

Summary

Background

Lipin-1 functions as a phosphatidate phosphatase in glycerolipid synthesis and as a co-transcriptional regulator. Lipins are predominantly cytosolic proteins that translocate to their sites of action in the endoplasmic reticulum and nucleus [5,6,7,8,9,10,11,12,13] These changes are dictated by a polybasic nuclear localization motif [6, 9, 14], which promotes an electrostatic interaction with negatively charged phosphatidate, fatty acids, and acyl-CoA esters on the membrane surface (9, 14 –17). Increasing the negative charge on the lipins through phosphorylation decreases their interactions with negative charges on the surfaces of membranes to control subcellular distribution and function [5, 6, 10, 19] This is demonstrated by the cytosolic localization of hyperphosphorylated forms of lipins, whereas hypophosphorylated lipins translocate to the nucleus and endoplasmic reticulum [5, 6, 11, 19]. The conserved N-terminal domain of lipin-1 is important for the actions of lipin-1 as a phosphatidate phosphatase and for its nuclear localization

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION