Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters.

U Kikkawa,Y Takai,Y Tanaka,R Miyake,Y Nishizuka

doi:10.1016/s0021-9258(17)44245-1

Abstract

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.

Highlights

A tumor-promoting phorbol ester, [3H]phorbol- by Niedel et al [13], who have shown that a phorbol esterlZ,lS-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated,phospholipid-dependent protein kinase(protein kinase C) in the simultaneous presence of Ca2+ and phospholipid
Using a homogeneous preparation of protein kinase C, the present studies were undertaken toexplore the kinetics and propertieosf the binding of tmor-promoting phorbol ester to theenzyme
The binding of the phorbol ester is prevented by the addition of diacylgly- Materials and Chemicals-A homogeneous preparation of protein cerol, which serves as activator of protein kinase C kinase C was obtained from the soluble fraction of rat brain by the under physiological conditions

Summary

Protein Kinase C As a Possible Receptor Protein of Tumorpromoting PhorbolEsters*

A tumor-promoting phorbol ester, [3H]phorbol- by Niedel et al [13], who have shown that a phorbol esterlZ,lS-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated,phospholipid-dependent protein kinase(protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. 13-acetate are well known as potent tumor promoters, anda Binding Assay-The complete assay mixture (0.2 ml) contained 4 number of kinetic studieswith various cell types suggest that the primary site of action of the tumor-promoting phorbol esters is located on cell surface membranes (for review, see pmol of Tris/malate at pH 6.8, 20 pmol of KCI, 30 nmol of CaCl,, 20 pg of phosphatidylserine suspended in an aqueous buffer solution as described (ll), 10 pmol of [3H]PDBu (1.51 X lo4cpm/pmol), 100 ng of protein kinase C (this preparation contained 10 nmol of EGTA), and 0.5% (final concentration) of Me2S0. Protein was determined as described previously [24]

RESULTS AND DISCUSSION

Protein Kinase C and Phorbol Ester Receptor

Phorbol derivative added