Marian Mosior

Mechanism of Interaction of Protein Kinase C with Phorbol Esters REVERSIBILITY AND NATURE OF MEMBRANE ASSOCIATION

A variety of approaches have been employed to demonstrate that the interaction of protein kinase C βII with phorbol ester-containing membranes is reversible, is not accompanied by significant insertion of the protein into the hydrophobic core of the membrane, and is qualitatively similar to the interaction with diacylglycerol (DG). First, we show that under conditions when protein kinase C is bound with equal affinity to membranes containing either DG or phorbol myristate acetate (PMA), increasing ionic strength causes a similar reduction in membrane binding. The similar sensitivity to ionic strength indicates that the forces mediating the binding of protein kinase C to PMA are not significantly different from those mediating the binding to DG. At sufficiently high concentrations of PMA and relatively low concentrations of phosphatidylserine, the binding of protein kinase C to membranes became markedly less sensitive to ionic strength, suggesting that under these conditions direct non-electrostatic interactions with PMA dominate over electrostatic interactions with the lipid headgroups. Importantly, regardless of the strength of the interaction with PMA, protein kinase C exchanges between vesicle surfaces: protein kinase C bound first to phorbol ester-containing multilamellar vesicles exchanged to large unilamellar vesicles upon addition of an excess surface area of the latter. Lastly, the enzymes intrinsic tryptophan fluorescence was not quenched by bromines located at various positions in the hydrophobic core of the membrane. In contrast, the enzymes tryptophan fluorescence was significantly quenched by probes positioned at the membrane surface. In summary, our results are consistent with protein kinase C binding reversibly to PMA- or DG-containing membranes primarily via interactions at the membrane interface.

Group IV Cytosolic Phospholipase A2 Binds with High Affinity and Specificity to Phosphatidylinositol 4,5-Bisphosphate Resulting in Dramatic Increases in Activity

The group IV cytosolic phospholipase A2 (cPLA2) exhibits a potent and specific increase in affinity for lipid surfaces containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at physiologically relevant concentrations. Specifically, the presence of 1 mol % PtdIns(4,5)P2 in phosphatidylcholine vesicles results in a 20-fold increase in the binding affinity of cPLA2. This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P2 binds to cPLA2 in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P2 on the activity of cPLA2 is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA2 with PtdIns(4,5)P2 and the interaction of the pleckstrin homology domain of phospholipase Cδ1 with PtdIns(4,5)2 combined with sequence analysis of cPLA2 lead us to propose the existence and location of a pleckstrin homology domain in cPLA2. We further show that the very nature of the interaction of proteins such as cPLA2 with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data.

Regional distribution, ontogeny, purification, and characterization of the Ca2+-independent phospholipase A2 from rat brain

Abstract : We purified an 80‐kDa Ca2+‐independent phospholipase A2 (iPLA2) from rat brain using octyl‐Sepharose, ATP‐agarose, and calmodulin‐agarose column chromatography steps. This procedure gave a 30,000‐fold purification and yielded 4 μg of a near‐homogeneous iPLA2 with a specific activity of 4.3 μmol/min/mg. Peptide sequences of the rat brain iPLA2 display considerable homology to sequences of the iPLA2 from P388D1 macrophages, Chinese hamster ovary cells, and human B lymphocytes. Under optimal conditions, the iPLA2 revealed the following substrate preference toward the fatty acid chain in the sn‐2 position of phosphatidylcholine : linoleoyl > palmitoyl > oleoyl > arachidonoyl. The rat brain iPLA2 also showed a head group preference for choline ≥ ethanolamine ≫ inositol. The iPLA2 is inactivated when exposed to pure phospholipid vesicles. The only exception is vesicles composed of phosphatidylcholine and phosphatidylinositol 4,5‐bisphosphate. Studies on the regional distribution and ontogeny of various phospholipase A2 (PLA2) types in rat brain indicate that the iPLA2 is the dominant PLA2 activity in the cytosolic fraction, whereas the group IIA secreted PLA2 is the dominant activity in the particulate fraction. The activities of these two enzymes change during postnatal development.

Activation of caspase-3 alone is insufficient for apoptotic morphological changes in human neuroblastoma cells

Activated caspase‐3 is considered an important enzyme in the cell death pathway. To study the specific role of caspase‐3 activation in neuronal cells, we generated a stable tetracycline‐regulated SK‐N‐MC neuroblastoma cell line, which expressed a highly efficient self‐activating chimeric␣caspase‐3, consisting of the caspase‐1 prodomain fused to the caspase‐3 catalytic domain. Under expression‐inducing conditions, we observed a time‐dependent increase of processed caspase‐3 by immunostaining for the active form of the enzyme, intracellular caspase‐3 enzyme activity, as well as poly(ADP‐ribose) polymerase (PARP) cleavage. Induced expression of the caspase fusion protein showed predominantly caspase‐3 activity without any apoptotic morphological changes. In contrast, staurosporine treatment of the same cells resulted in activation of multiple caspases and profound apoptotic morphology. Our work provides evidence that auto‐activation of caspase‐3 can be efficiently achieved with a longer prodomain and that neuronal cell apoptosis may require another caspase or activation of multiple caspase enzymes.

Role of the Membrane in the Modulation of the Activity of Protein Kinase C

(1999). Role of the Membrane in the Modulation of the Activity of Protein Kinase C. Journal of Liposome Research: Vol. 9, No. 1, pp. 21-41.

The Binding of Peptides and Proteins to Membranes Containing Anionic Lipid

The interaction of proteins with lipids is one of the central problems of membrane biophysics. Of particular interest is that some proteins require a distinct category of lipids for their function. For example, several functionally related groups of proteins that interact preferentially with acidic lipids were discovered in recent years. They include vitamin K-dependent blood coagulation proteins (Jackson and Nemerson, 1980), annexins (Geisow et al., 1987), calpactins (Glenney, 1986), synexins (Creutz et al., 1983), the protein kinase C family (Nishizuka, 1989), and actin-severing or actin-cap-ping proteins (Stossel, 1989). Many other proteins, particularly enzymes, also require acidic lipids for activation (Cornell, 1991; Enyedi et al., 1987; Moritz et al., 1992). There are excellent reviews available on many of the particular groups of proteins that interact with acidic lipids (Geisow et al., 1987; Glenney, 1986; Jackson and Nemerson, 1980; Stossel, 1989). Therefore, we decided to focus this chapter on the roles played by acidic lipids in lipid-protein interactions. For the sake of clarity, we used a limited number of examples to describe the four distinct functions performed by acidic lipids. We also review recent models of the mechanism of acidic lipid-protein interaction.