[Diagram of Phospholipases and Lipid Kinases]

Phospholipases and Signal Transduction. A variety of phospholipid-derived molecules have been implicated as signalling molecules, see rounded boxes in the Figure. These include the protein kinase C regulator diacylglycerol, the calcium signal inositol 1,4,5-trisphosphate, probably phosphatidic acid, and phosphorylated forms of phosphatidylinositol. The phospholipase C system which generates diacylglycerol and IP3 from phosphatidylinositol 4,5-bisphosphate has been extensively studied in recent years. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid and released choline headgroup. The phosphatidic acid may itself act as a signal molecule (e.g., by activating a PA-activated kinase), or can be hydrolyzed to form diacylglycerol by the enzyme PA phosphohydrolase. One of the focus areas in the Lambeth lab is to investigate phospholipase D and its relation to signal transduction.

Kinetics of Signal Generation. Early studies from our lab implicated protein kinase C in signalling in the neutrophil (e.g., activation of the respiratory burst) and we subsequently showed that in cells which were activated with a chemoattractant, there were two "waves" of diacylglycerol generation, one corresponding to a calcium flux and IP3 release, and a second larger wave which was independent of PIP2 breakdown. Subsequent studies used the chemical linkage and fatty acid composition of the diradylglycerol as a "fingerprint" which identified the parent phospholipid as phosphatidylcholine and not PIP2. Diacylglycerol generation was also activated by a protein kinase C activator, PMA. Studies from Billah's and from our laboratory pointed to phospholipase D as the source of the second wave of diacylglycerol (formed from phosphatidic acid hydrolysis). [Diagram of the Kinetics of Antagonist-Activated Signal Generation]

A Cell-Free System for Reconstitution of Phospholipase D Activity. Olson in our laboratory developed a cell-free system for reconstitution of phospholipase D activity. Activity was calcium dependent and required protein factors in both the plasma membrane and cytosol. Activation stimuli included both GTPgS and PMA, implying the participation of both a GTP-binding/regulatory protein and protein kinase C. Because phosphatidic acid is metabolically unstable, phospholipase D activity is assayed by transphosphatidylation, a reaction in which the phosphatidate is preferentially transferred from phosphatidylcholine to a primary alcohol e.g. to form phosphatidylethanol. This metabolically stable product is easily detected by thin layer chromatography due to its unique migration.

[Diagram of a PLD Activation Model]
The model depicts the involvement of the various regulatory factors in synergistically activating PLD.

RhoA. Using biochemical separation and reconstitution approaches, the cytosol and membrane from neutrophils were resolved into a minimum of five required protein factors. The GTP-binding component was shown to be the small GTPase RhoA. The related small GTPase Rac1 was only modestly effective. RhoA also participates in regulation of the cytoskeleton (actin stress fibers) and regulation of transcription.

50 kDa Factor. An as-yet unidentified 50 kDa cytosolic factor was found to be present in cytosol, and was required along with RhoA for phospholipase D activity. Despite repeated attempts and many person-years of effort, this factor has resisted purification.

Phospholipase D. The phospholipase D itself is located in the plasma membrane. It can be assayed in the plasma membrane itself using endogenous substrate or in a detergent-solubilized extract using exogenous substrate. In the latter case, activity requires the presence of PIP2, which is presumed to be present also in the plasma membrane. It has not been successfully purified.

Protein Kinase C. Using protein kinase C-depleted cytosol, PMA-dependent activation was lost, and added protein kinase C reconstituted PMA-dependent phospholipase D activity. Unlike phospholipase D in some other tissues which show ATP-independent activation by protein kinase C, activation using neutrophil subcellular fractions required ATP and was inhibited by staurosporine, implying that protein kinase C is functioning by classical phosphorylation-dependent mechanisms. The phosphorylation target was shown to be in the plasma membrane, and may be the phospholipase D itself. Calcium-dependent PKC isoforms, particularly the beta isoform, reconstituted activity, whereas calcium-independent and atypical isoforms were not capable of reconstituting activity.

Arf. Arf, while not essential for activity, synergized with the 50 kDa factor when the latter was present at sub-optimal concentrations. In the presence of higher concentrations of the 50 kDa factor, Arf caused no further activation.

Cloning of Phospholipase D Isoforms. Two mammalian isoforms of phospholipase D (PLD1 and PLD2) have been cloned, based on their homology with yeast and plant phospholipase D. hPLD1 is an Arf-activated isoform which can be activated synergistically by Rho and the regulatory domain of protein kinase C (calcium-dependent isoforms). It does not have a requirement for a 50 kDa factor, and differs in other respects from the neutrophil phospholipase D. PLD2 has recently been cloned (the mouse enzyme by the Frohman lab and the human enzyme by the Lambeth lab). PLD2 is consituitively active when expressed in insect cells, but remains a candidate for the neutrophil enzyme. Other isoforms of PLD apparently exist, but have not yet been identified by cDNA cloning.