A comparative study of the modes of activation of protein kinase C
The key signal transduction enzyme protein kinase C (PKC) is specifically activated by tumor-promoting phorbol esters. In this study, phorbol ester-activation with regards to phorbol ester hydrophobicity, interaction with filamentous actin (F-actin), membrane compositional changes and synergistic activation, was compared between conventional PKCα, novel PKCδ and ϵ, and atypical PKCζ isoforms. The modes of activation by phorbol esters were determined using purified recombinant PKC isoforms. PKC activity was determined in the presence of membrane phospholipids (lipid-dependent activity) or F-actin (lipid-independent activity). The fluorescent phorbol ester, sapintoxin D, was used to measure the binding of phorbol esters to PKC in the presence or absence of membrane phospholipids. The results suggest that PKC contains a hydrophobic binding site for phorbol esters which maintains structural specificity in the absence of phospholipids and Ca2+. Binding of phorbol esters to PKC in the absence of membrane phospholipids, mediates an activating interaction with F-actin. PKCζ and ϵ were specifically activated by phorbol esters and F-actin, while PKCζ was activated by F-actin alone. The results suggest that PKC binds to F-actin via conserved structural features of a single cysteine-rich domain that are exposed upon phorbol ester-binding to the enzyme. PKCaα and δ are shown to be sensitive to subtle changes in the composition of the membrane lipid bilayer that result in an increase in head group spacing. Marginal activator stimulation of PKCϵ, in the presence of membrane phospholipids, suggests that membrane phospholipids play a minor role in the activation of PKCϵ. PKCα contains two distinct phorbol ester binding regions of low and high affinity respectively and PKCδ contains a high affinity phorbol ester binding site. PKCα and δ, but not ϵ, were synergistically activated by diacylglycerols (DAG) and phorbol esters, the level of activity obtained in the presence of both activators, each alone at maximally stimulating concentrations, being greater than that achieved by either activator alone. This shows that the C2 domain is not an absolute requirement for synergistic activation by DAG and phorbol esters. The observed isoform differences in phorbol ester sensitivity and activation suggest that in a cell, phorbol esters specifically activate distinct PKC isoforms via various mechanisms to achieve different biological endpoints.
Taddeo, Frank J, "A comparative study of the modes of activation of protein kinase C" (1998). ETD Collection for Thomas Jefferson University. AAI9915378.