Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts.

Z Szallasi,C.B Smith,G.R Pettit,P.M Blumberg

doi:10.1016/s0021-9258(17)42143-0

Abstract

Bryostatin 1 and phorbol 12-myristate 13-acetate (PMA) are both potent activators of protein kinase C (PKC), although in many systems bryostatin 1 induces only a subset of the responses to PMA and blocks those which it does not induce. We report here that in NIH 3T3 fibroblasts PMA showed similar potencies for translocating PKC isozymes alpha, delta, and epsilon to the Triton X-100-soluble and -insoluble fractions and for the down-regulation of the three isozymes. Bryostatin 1 was slightly was more potent than PMA for down-regulating it. Bryostatin 1 was markedly more potent than PMA for translocating PKC delta but showed a biphasic dose-response curve for down-regulating this isozyme. 1-10 nM bryostatin 1 down-regulated PKC delta to a similar extent as PMA; lower (10-100 pM) or, unexpectedly, higher (100 nM to 1 microM) doses of bryostatin 1 caused either no or reduced down-regulation. Moreover, these high (100 nM to 1 microM) doses of bryostatin 1 inhibited the down-regulation of PKC delta by 1 microM PMA when coapplied. Bryostatin 1 caused translocation of PKC epsilon with slightly higher potency than PKC delta, but there was no protection of this isozyme at any of the doses examined. Bryostatin 1 induced a long-term increase in c-Jun level. The dose-response curve for bryostatin 1 was biphasic, with maximal induction at 1-10 nM bryostatin 1, coincident with the maximal down-regulation of PKC delta. We conclude that bryostatin 1 showed substantially different regulation for PKC alpha, PKC delta, and PKC epsilon, whereas PMA distinguished only weakly between these isozymes.

Highlights

From the $Laboratory of Cellular Carcinogenesis and lbmorPromotion, National Cancer Znstitute, Bethesda, Maryland 20892 and the gCancer Research Znstitute and Department of Chemistry, Arizona State University, Ikmpe, Arizona 85287
Fraction contains about 45-50% of the total protein.) The dis- bryostatin 1
Our results show that in NIH 3T3 fibroblasts bryostatin 1 was more potent than phorbol 12-myristate13-acetate (PMA) for translocating and down-regulating PKCa, -6, and -e (Table I)

Summary

EXPERIMENTAL PROCEDURES

Between the biological responses induced by typical phorbol Cells and Materia1s“NIH 3T3 cells were grown in Dulbecco’s modiesters,such as phorbol 12-myristate13-acetate (PMA) and fied Eagle’s medium supplemented with nw glutamine and 10% fetal bryostatin 1, the mechanistic basis for these differencesre- calf serum (complete Dulbecco’s modified Eagle’s medium). Amnity purified polyclonal antibody against the C terminus (PKCS amino acids 662-673)of PKCG was purchased from Research& Diagnostics Antibodies (Berkeley, CA) and applied at a 1:50,000 dilu-. Amnity purifiedpolyclonal antibody against a polypeptide corresponding to amino acids 313-326 of PKCc was purchased from Life Technologies Inc. and applied a t a concentrationof 2 pg/ml. Polyclonal antibody against the C terminuosf PKCC (PKCC amino acids 480-492). Awas purchased from Research& Diagnostics Antibodies and applieda t a dilution 15,000.Polyclonal antibody was raised against the 18-amino acid C terminus of PKCq in our laboratory and applied a t a dilution. The blots were incubated overnight at 4 "C with the indicated amounts of the primary antibody dissolved in 4% milk in phosphate-buffered saline.

RESULTS

C Isozymes by Bryostatin 1

DISCUSSION