Diacylglycerol kinase ζ interacts with sphingomyelin synthase 1 and sphingomyelin synthase-related protein via different regions.

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG-generating sphingomyelin synthase (SMS)-related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N-terminal cytosolic region of SMSr binds to the N-terminal half catalytic domain of DGKζ. However, the C-terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.

Phosphatidylinositol 4,5-bisphosphate-specific phospholipase C ß1 selectively binds dipalmitoyl and distearoyl phosphatidic acids via Lys946 and Lys951.

Phospholipase C (PLC) ß1 hydrolyzes 1-stearoyl-2-arachidonoyl (18:0/20:4)-phosphatidylinositol (PtdIns) 4,5-bisphosphate to produce diacylglycerol, which is converted to phosphatidic acid (PtdOH), in the PtdIns cycle and plays pivotal roles in intracellular signal transduction. The present study identified PLCß1 as a PtdOH-binding protein using PtdOH-containing liposomes. Moreover, the comparison of the binding of PLCß1 to various PtdOH species, including 14:0/14:0-PtdOH, 16:0/16:0-PtdOH, 16:0/18:1-PtdOH, 18:0/18:1-PtdOH, 18:0/18:0-PtdOH, 18:1/18:1-PtdOH, 18:0/20:4-PtdOH, and 18:0/22:6-PtdOH, indicated that the interaction of PLCß1 with 16:0/16:0-PtdOH was the strongest. The PLCß1-binding activity of 18:0/18:0-PtdOH was almost the same as the binding activity of 16:0/16:0-PtdOH. Furthermore, the binding of PLCß1 to 16:0/16:0-PtdOH was substantially stronger than 16:0/16:0-phosphatidylserine, 16:0/16:0/16:0/16:0-cardiolipin, 16:0/16:0-PtdIns, and 18:0/20:4-PtdIns. We revealed that a PLCß1 mutant whose Lys946 and Lys951 residues were replaced with Glu (PLCß1-KE) did not interact with 16:0/16:0-PtdOH and failed to localize to the plasma membrane in Neuro-2a cells. Retinoic acid-dependent increase in neurite length and numbers was significantly inhibited in PLCß1-expressing cells; however, this considerable attenuation was not detected in the cells expressing PLCß1-KE. Overall, these results strongly suggest that PtdOHs containing only saturated fatty acids, including 16:0/16:0-PtdOH, which are not derived from the PtdIns cycle, selectively bind to PLCß1 and regulate its function.

Relative intensities of prompt γ-rays from the 35Cl(n,γ)36Cl reaction with thermal neutrons as secondary γ-ray intensity standards.

The relative intensities of 16 prompt γ-rays from the (35)Cl(n,γ)(36)Cl reaction with a thermal neutron were precisely determined as secondary γ-ray intensity standards with HPGe detectors. The detection efficiencies were calibrated from 0.2 to 10.8 MeV using the standard sources (152)Eu and (56)Co and the (14)N(n,γ)(15)N reaction. We performed appropriate analyses for the evaluation of doublet peaks, subtraction of mixing with escape γ-rays and other corrections; consequently, the values were determined within 1% accuracy. Relative intensities in the range of 0.7 to 8.6 MeV are proposed as reliable secondary standards for 16 γ-rays.