No abstract

Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit A␣ of protein phosphatase 2A. This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate.In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory A␣ subunit could no longer be detected in cell lysates. There was no change in the amount of the catalytic subunit. The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk. These data are consistent with a pathway whereby induction of apoptosis activates caspase-3. This enzyme then cleaves the regulatory A␣ subunit of PP2A, increasing its activity. These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell. These data provide a link between the caspases and signal transduction pathways.

Interfacial catalytic constants for bee venom phospholipase A2 (bvPLA2) have been obtained for its action on vesicles of the anionic phospholipid 1,2-dimyristoylphosphatidylmethanol (DMPM) in the highly processive scooting mode. Spectroscopic measurements which directly measure transbilayer movement of membrane components show that this exchange does not occur in anionic vesicles that have undergone complete bvPLA2-catalyzed hydrolysis of all phospholipids in the outer vesicle monolayer. 3-Hexadecyl-sn-glycero-1-phosphocholine (D-LPC) is an adequate neutral diluent for bvPLA2, which is defined as an amphiphile that forms an aggregate to which enzyme binds but neutral diluent molecules bind weakly in the enzyme's active site. D-LPC has weak affinity for the active site of bvPLA2, and theory and protocols are developed that allow its use to determine equilibrium dissociation constants for competing active site ligands. Some of the properties of bvPLA2 are shared by other 14 kDa PLA2s. (1) Ca2+ is required for binding of ligands to the active site but not for the binding of enzyme to the interface. (2) bvPLA2 does not significantly discriminate between phospholipids with different polar head groups or acyl chains. (3) bvPLA2 does not bind to phosphatidylcholine vesicles, and binding occurs if anionic amphiphiles are present in the vesicle. Novel features of bvPLA2 include the following: (1) Neutral diluents for other 14 kDa phospholipases A2 are not neutral diluents for bvPLA2. (2) Saturation of the active site with a variety of different ligands does not completely prevent histidine alkylation by 2-bromo-4'-nitroacetophenone, and Ca2+ binding does not change the rate of histidine alkylation. Finally, the carbohydrate portion of bvPLA2 does not alter the interfacial catalytic properties of the enzyme. Kinetic analysis of bvPLA2 in the scooting mode together with previous studies with other 14 kDa PLA2s provides a paradigm for the quantitative analysis of interfacial catalysis.