brucei, TbPRMT1 [27]. Of particular interest to us are proteins whose functions might be affected by arginine methylation. Here, we report that TbPRMT1 directly interacts in both Far Western and co-immunoprecipitation assays with a novel protein. We termed this protein TbLpn, based on the presence of two URMC-099 mw conserved (N-LIP and C-LIP) domains

found in a family of proteins called lipins. We further demonstrate that, like TbPRMT1, TbLpn is cytoplasmic in PF T. brucei, consistent with a function in TbLpn methylation. Together, these data point to TbLpn as a candidate protein whose post-transcriptional NSC 683864 price gene regulatory functions are affected by arginine methylation. We demonstrated that, as predicted from the amino GSK458 nmr acid sequence, recombinant TbLpn, as other members of the lipin family, exhibits phosphatidic acid phosphatase enzymatic activity. Mutation of the conserved aspartic acid residues (Asp-445 and Asp- 447) to alanines results in a significant reduction in the enzymatic activity of TbLpn. These two aspartic acid residues are part

of the conserved DxDxT motif found in lipin proteins and other members of the haloacid dehalogenase (HAD)-like superfamily [53, 54]. Based on the crystal structure of L-2-haloacid dehalogenase from Pseudomonas, it is likely that Asp-445 in TbLpn acts as a nucleophile in the phosphoryl transfer reaction. Compared to the recombinant yeast PAH1 (3000 nmol/min/mg) and human Lipin-1 (1,600 nmol/min/mg), His ~ TbLpn displays a lower but still significant specific activity [43]. One possible explanation for this lower specific activity

is the fact that the recombinant protein may not contain the same post-translational modifications as those found in the native protein. It is of interest that several lipin Selleck Pazopanib homologues are highly modified at the post translational level. In rat and in mouse adipocytes, Lipin 1 contains at least 19 and as many as 23 sites that are phosphorylated in response to insulin [49, 55, 56]. Although it does not affect its intrinsic phosphatidic acid phosphatase activity, phosphorylation of Lipin-1 decreases the association with intracellular membranes, thus the active lipin fraction [49]. In addition, the lipin homologue SMP2 is phosphorylated by the cyclin-dependent kinase Cdc28/Cdk1 in budding yeast [57]. The authors have shown that phosphorylation of SMP2 by Cdc28/Cdk1 enhances its association with promoters of lipid biosynthetic genes, which leads to their transcriptional down-regulation. Careful analysis of TbLpn amino acid sequence revealed the presence of 5 conserved amino acid residues shown to be phosphorylated in either mouse (Mm) Lipin-1 or yeast (Sc) Smp2. These residues are Ser-102 (Ser-110 in Sc), Thr-239 (Thr-282 in Mm), Thr-255 (Thr-298 in Mm), Ser-282 (Ser-328 in Mm), and Ser-343 (Ser-392 in Mm). In addition, a previous analysis of the cytosolic phosphoproteome of BF T.