title: Structure-activity relationship of diacylglycerol kinase theta.
abstract: Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid (PA). Among the nine mammalian isotypes identified, DGKtheta is the only one with three cysteine-rich domains (CRDs) (instead of two) in its N-terminal regulatory region. We previously reported that DGKtheta binds to and is negatively regulated by active RhoA. We now report that RhoA strongly binds to the C-terminal catalytic domain, which would explain its inhibition of DGK activity. To help finding a physiological function of DGKtheta, we further determined its activity in vitro as a function of 15 different truncations and point mutations in the primary structure. Most of these alterations, located throughout the protein, inactivated the enzyme, suggesting that catalytic activity depends on all of its conserved domains. The most C-terminal CRD is elongated with a stretch of 15 amino acids that is highly conserved among DGK isotypes. Mutation analysis revealed a number of residues in this region that were essential for enzyme activity. We suggest that this CRD extension plays an essential role in the correct folding of the protein and/or in substrate presentation to the catalytic region of the protein.
authors.affiliation: Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsterdam, The Netherlands.
authors.forename: Alrik P
authors.initials: AP
authors.lastname: Los
authors.forename: Jürgen
authors.initials: J
authors.lastname: van Baal
authors.forename: John
authors.initials: J
authors.lastname: de Widt
authors.forename: Nullin
authors.initials: N
authors.lastname: Divecha
authors.forename: Wim J
authors.initials: WJ
authors.lastname: van Blitterswijk
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D017931:DNA Primers; D019852:Diacylglycerol Kinase; D020742:rhoA GTP-Binding Protein