the regulation of protein kinase C by anionicphospholipids - PNAS

5 downloads 0 Views 1MB Size Report
Protamine sulfate, ATP sodium salt, EGTA, and histone III-S were supplied by Sigma. The peptide VRKRTLRRL was obtained from Bachem. The radiolabeled ...
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1907-1912, March 1996 Biophysics

Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: Evidence for the lack of a specific binding site for phosphatidylserine (protein-lipid interactions/phospholipid inverted-phase propensity/membrane lateral-phase separation)

MARIAN MOSIOR*, ERIN S. GOLINI, AND RICHARD M. EPANDt Department of Biochemistry, McMaster University, Hamilton, ON, L8N 3Z5, Canada

Communicated by Bruce Merrifield, The Rockefeller University, New York, NY, November 9, 1995

independently assess the chemical and physical properties of the membrane that modulate the activity of PKC. The nonspecific alteration of PKC activity by a variety of amphiphiles was commonly interpreted as caused by changes in the local lipid environment of the membrane-bound enzyme (9-12). However, the extent of the actual association of the enzyme with the lipid bilayer was not tested. In this work we study how changes in the chemical composition of the membrane modulates the enzymatic activity of PKC and if this modulation occurs through changes in the association of the enzyme with the lipid bilayer. We also identified an additional factor, the miscibility of lipid components, which has a major influence on the binding of PKC to membranes. We demonstrate that it is not possible to simply compare the relative activity or lipid binding of the enzyme at one particular lipid composition. For several pairs of lipids, one pair can promote more or less binding of PKC, depending on the mole fraction of PS.

ABSTRACT The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidylL-serine (PS). In particular, a synthetic phospholipid, dansylphosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansylphosphatidylethanolamine was also able to activate the enzyme in a Ca2l-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

EXPERIMENTAL PROCEDURES Materials. Phospholipids and 1,2-sn-dioleoylglycerol (DG) were purchased from Avanti Polar Lipids. The N-dansyl-PE (DNS-PE) (dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) was a commercial product from Avanti Polar Lipids, prepared by transphosphatidylation of egg PC. Only DG displayed minor contamination (TLC; 19:1 chloroform/acetone) that was identified as 1,3-dioleoylglycerol. Due to its minor character, DG was used without further purification. Protamine sulfate, ATP sodium salt, EGTA, and histone III-S were supplied by Sigma. The peptide VRKRTLRRL was obtained from Bachem. The radiolabeled compounds were supplied by NEN. Bovine serum albumin (BSA), fraction V, fatty acid free, was obtained from Boehringer Mannheim. BSA, Tris (Life Technologies, Grand Island, NY), and all salts, analytical grade, supplied by Fisher, were provided with the actual lot analysis, indicating the amount of contaminating calcium. Lipid Vesicles. Mixtures of lipids in chloroform were dried under a stream of nitrogen and subsequently evacuated under high vacuum. The mixture also contained tracer amounts of 3H-labeled PC at 1 ppm (mol/mol) to monitor subsequent lipid

The protein kinase C (PKC) family, defined through homology in primary structure, is regulated by diacylglycerol, Ca2+, and anionic lipids (1, 2). Enzymatic assays employing Triton X-100 micelles demonstrated strong specificity of Ca2+-dependent isotypes of PKC for phosphatidylserine (PS) over other anionic lipids (3-6). However, there have been some reports (7, 8) indicating that other phospholipids can replace PS with various degrees of effectiveness. In addition to the role of negative charge, there are several other chemical and physical properties of the lipid that affect the binding of PKC to the membrane and its subsequent activation. Among these factors are the composition of acyl chains (9-11) and substitution of phosphatidylcholine (PC) with phosphatidylethanolamine (PE) (11, 12). In this work we consider the various properties of membrane lipids that regulate the association of PKC with the bilayer and subsequent activation of the enzyme. Different headgroups of phospholipids have the ability to alter physical properties of lipid bilayers, such as miscibility of their components, local curvature, molecular packing, or hydration, just to name a few. Therefore, we have investigated how the alterations in some of these properties, evoked by the change in the acyl chain composition but with the PS headgroup left intact, affect the association of PKC with a lipid bilayer. This has allowed us to

Abbreviations: PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PA, phosphatidic acid; DG, 1,2-sn-dioleoylglycerol; DM-, dimyristoyl-; DP-, 1,2dipalmitoyl-; dansyl-, 5-dimethylaminonaphthalene-1-sulfonyl-; DSC, differential scanning calorimetry; PO-, 1-palmitoyl-2-oleoyl-; DNSPE, N-dansyl-PE; PKC, protein kinase C; BSA, bovine serum albumin; LUV, large unilamellar vesicle. *Present address: Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0640. tTo whom reprint requests should be addressed at: Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, ON, L8N 3Z5, Canada.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1907

1908

Proc. Natl. Acad. Sci. USA 93 (1996)

Biophysics: Mosior et al.

recovery. Lipids were then suspended in either 170 mM sucrose or 100 mM KCl, both buffered with 20 mM Tris HCl to pH 7.0. Aliquots of 0.5 ml of up to 50 mM lipid were subjected to five freeze-thaw cycles, followed by 21-51 rounds of extrusion through two stacked 0.1-,tm (pore size) polycarbonate filters in a Lipofast microextruder (Avestin, Ottawa, ON, Canada) or an extruder manufactured by Lipex Biomembranes (Vancouver, Canada). Vesicles containing either 1,2dipalmitoyl-PC (DPPC) or dimyristoyl-PS (DMPS) were extruded at 50°C under nitrogen; however, they were subsequently used like all other vesicles at 25°C. PKC Purification. Rat brain PKC was purified to apparent homogeneity by a modification of the procedure by Huang et al. (13) as described (14). Purified protein displayed a single band on a silver-stained electrophoresis gel. The specific activity of the enzyme for the histone in a micellar assay (15) was 1 ,umol per mg per min. The phospholipid-independent kinase activity in this assay was

Suggest Documents