Dataset Information

Diacylglycerol kinase ? and sphingomyelin synthase-related protein functionally interact via their sterile ? motif domains.

ABSTRACT: The ? isozyme of diacylglycerol kinase (DGK?) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGK? preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGK? and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile ? motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGK?-SAMD. Full-length DGK? co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGK? interacted only weakly with full-length DGK? and SMSr, respectively. These results strongly suggested that DGK? interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGK? and SMSr, we used LC-MS/MS to investigate whether overexpression of DGK? and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGK?-overexpressing COS-7 cells. Moreover, SMSr enhanced DGK? activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGK? and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

SUBMITTER: Murakami C

PROVIDER: S-EPMC7062193 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Diacylglycerol kinase δ and sphingomyelin synthase-related protein functionally interact via their sterile α motif domains.

Murakami Chiaki C Hoshino Fumi F Sakai Hiromichi H Hayashi Yasuhiro Y Yamashita Atsushi A Sakane Fumio F

The Journal of biological chemistry 20200124 10

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known abo ...[more]

PMID: 31980461

Similar Datasets

Project description:Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.

Dataset Information

Diacylglycerol kinase ? and sphingomyelin synthase-related protein functionally interact via their sterile ? motif domains.

Publications

Diacylglycerol kinase δ and sphingomyelin synthase-related protein functionally interact via their sterile α motif domains.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure