Structural basis of protein kinase C activation by tumor promoters.

Abstract

Protein kinase C (PKC) is an important enzyme that helps govern cell metabolism and growth. The enzyme is physiologically activated when an (S)-diglyceride binds to its own regulatory domain. The saturable binding site of the regulatory domain can also be bound by any of a group of structurally diverse tumor promoters, including debromoaplysiatoxins (DATs), phorbol esters, ingenols, teleocidins, and bryostatins. The question of how the same binding site can be the target of these structurally diverse molecules is of considerable importance and is addressed in this article. The relatively rigid structure of DAT and the fact that it possesses a diglyceride moiety renders it an ideal starting template. Structure-activity studies with PKC reveal that the C29 but not the C30 stereocenter of DAT is critical for activity. Furthermore, 3-deoxy-DAT and DAT are equipotent as PKC activators, hence the C3 hydroxyl group of DAT is not critical for activity. Straightforward structural considerations show that the C30 hydroxyl group ofDAT matches the C3 hydroxyl group of diglyceride, the C29 stereocenter of DAT matches the C2 stereocenter of (S)-diglyceride, and the C1 ester moiety of DAT matches the C2 ester moiety of diglyceride. Based on these studies and on published structure-activity observations on other tumor promoters, a structural hypothesis is developed to account for the chemical mechanism of tumor promoter action. Experimentally testable predictions are made concerning the interactions with PKC of several classes of tumor PKC activators.A central question in the mechanism of protein kinase C (PKC) action is concerned with the nature of the activation process. This physiologically important enzyme is activated when a diglyceride binds to an effector site on its regulatory domain (1, 2). The diglyceride itself is produced as a consequence of the regulated hydrolysis of phosphatidylinositol phosphates by a specific phospholipase (3). The interaction of PKC with diglyceride is stoichiometric (4) and stereospecific, with the (S)-enantiomer being active (5, 6). The specificity of this process has been further probed with diglyceride analogs (7-9). These studies have shown an unusual degree of specificity directed toward the glycerol backbone and the hydrophilic moieties of the diglycerides. The strict specificity shown toward the diglycerides must be contrasted with the behavior of PKC toward the tumor promoters, including phorbol esters, ingenols, aplysiatoxins, and teleocidins (see Fig. 7). All these structurally diverse molecules share PKC as their common target and appear to function by binding to and activating the enzyme permanently, whereas the diglycerides bind and activate temporarily (10)(11)(12).Given that it is highly likely that the various tumor promoters and active diglycerides bind to the same site on the regulatory domain ofPKC, it must be possible to demonstrate structural commonalities in these various agents. Several models, generated by various computer-driven modeling protocols, have alread...

The stereochemical requirement for protein kinase C activation by 3‐methyldiglycerides matches that found in naturally occurring tumor promoters aplysiatoxins

“…Furthermore, the corresponding diolein analogs 8 and 9 exhibit biological behavior parallel to that of 6 and 7, demonstrating that the stereochemical requirement at the second asymmetric center of diglycerides is identical with that of aplysiatoxins. This finding lends strong support to the structural hypothesis that we have proposed which links structural elements of the diglycerides to the pharmacophore of the tumor promoters [10].…”

“…It was thus important to probe the stereochemical requirement in the synthetic 3-methyldiglyceride series, to determine if it matches that found in the aplysiatoxin series. If this turned out to be the case, it would further strengthen the structural link forged between diglycerides and aplysiatoxins [10]. In this connection, it is interesting to note that methylated diglycerides 6 and 7 were studied previously, but their behavior towards PKC appeared to be inconsistent with our hypothesis [10].…”

“…If this turned out to be the case, it would further strengthen the structural link forged between diglycerides and aplysiatoxins [10]. In this connection, it is interesting to note that methylated diglycerides 6 and 7 were studied previously, but their behavior towards PKC appeared to be inconsistent with our hypothesis [10]. For this reason, we have independently synthesized methylated diglycerides 6 and 7, established their stereochemistry unambiguously, and proved their biological behaviors to be consistent with our hypothesis.…”

“…For example, 1,3-diglycerides are inactive, and the addition of a single methyl group at the sn-1 position of an otherwise active diglyceride almost completely abolishes activity [8,9]. PKC's great specificity with respect to diglycerides must be contrasted, however, to its apparent lack of specificity with regard to the structurally diverse tumor promoters [10]. The tumor promoters capable of potently activating PKC include phorbol esters, ingenols, teleocidins, and aplysiatoxins [1,[11][12][13].…”

“…Recently, we have presented a stereochemical model which addresses this issue and explains how the structurally diverse tumor promoters and the diglycerides can all be accommodated by the same binding-site [I0]. Debromoaplysiatoxin (DAT; 1) and 3-deoxydebromoaplysiatoxin play a central role in this model [10]. The aplysiatoxins provide synthetically manipulable templates which are crucial in the development of the structural hypothesis.…”