Role of substrate in determining the phospholipid specificity of protein kinase C activation

Mohammad D. Bazzi, Gary L. Nelsestuen

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43 Scopus citations

Abstract

The phospholipid selectivity of protein kinase C (PKC) activation was examined by using two substrates, histone and a random copolymer of lysine and serine [poly(lysine, serine)] (PLS), plus phospholipids provided as vesicles or as Triton-mixed micelle preparations. The results indicated that substrate-phospholipid interaction was an essential component of PKC activation and that many in vitro properties of PKC activation are attributable to this interaction. The substrate histone interacted with phospholipid-Triton mixed micelles containing phosphatidylserine (PS), but not with those containing phosphatidylinositol (PI) or phosphatidylglycerol (PG). In direct correlation, only PS-Triton mixed micelles were effective in supporting PKC activity. Also, the minimum PS composition (4 mol % in Triton) required to induce significant histone-PS interaction coincided with the minimum composition required for phosphorylation of histones. Moreover, the PS composition required for maximum activity varied with the histone concentration of the reaction. In contrast to histone, PLS interacted with phospholipid-Triton mixed micelles containing either PS, PI, or PG, and all these mixed micelles supported the phosphorylation of PLS. In fact, by selection of appropriate experimental conditions (e.g., concentration of substrate and phospholipid), any of the three mixed micelles could appear the most effective in supporting PKC activity. Phospholipid vesicles containing PS, PG, or PI were found to interact with both histone and PLS and to support the activity of PKC. Physical properties of the solution and conditions used for preparation of phospholipid vesicles had considerable influence on PKC activation. At high phospholipid concentrations, vesicles containing PS, PI, or PG supported the activity of PKC to essentially the same level, provided that the physical differences among the phospholipid vesicles were minimized. In agreement with this general property, maximum binding of phorbol dibutyrate to PKC was not influenced by the head group of the phospholipids. Three major implications for the activation of PKC were apparent: (1) the phospholipid selectivity of PKC activation was related to properties of substrate-phospholipid interaction; (2) the best PKC substrates are membrane-associated proteins; and (3) selection of experimental conditions could render opposing relationships and conclusions for the influence of the same component(s) measured under different conditions.

Original languageEnglish (US)
Pages (from-to)5002-5008
Number of pages7
JournalBiochemistry
Volume26
Issue number16
StatePublished - 1987

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