Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1.beta. gene expression

Abstract

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Bryostatin-1 and 1α,25-dihydroxyvitamin D3 synergistically stimulate the differentiation of NB4 acute promyelocytic leukemia cells

“…2 The reason for this was postulated to be a differential competition between bryostatin and PMA on activation of different PKC isozymes 41,46,47 or that PMA is not a native compound and cannot be hydrolyzed by in vivo enzyme system. 48 ATRA has been successfully used for the treatment of patients with APL disease and can lead to clinical remission.…”

Immunocharacterization of β‐ and ζ‐subspecies of protein kinase C in bovine neutrophils

“…In the case of bovine brain PKC, 6 peptides were synthesized, which corresponded to the following residues: 318-331 (ISPSEDRRQPSNNL) for o~-PKC [9], 319-332 (EKTTNTISKFDNNG) for ~-PKC [9], 316-332 (VRTGPSSSPIPSPSPT) for 7-PKC [9], 318-334 (KTAVSGNDIPDNNGTYG) for ~-PKC [10,11], 836-853 (NQEEFKGFSYFGEDLMP)for e-PKC [10,11] and 128-142 (SEDLKPVIDGDDGIK)for ~'-PKC [10,11]. Each antipeptide antibody reacted effi- ciently with its own antigen [9][10][11].…”

“…Each antipeptide antibody reacted effi- ciently with its own antigen [9][10][11]. Concerning the o~-, ~-and ,y-isoforms of rat brain PKC, the peptides used corresponded to the following residues: 320-335 (PSEDRKQPSLNLDR) for c~-PKC, 319-335 (EKTALTISKFPNNGNR) for /3-PKC and the Cterminus peptide 683-697 (PDARSPTSPVPVPVM) for -y-PKC (Pelosin et al, unpublished).…”

“…Non-specific sites were blocked by incubating the membrane with 5 % instant milk (Carnation, USA) and 0.5% Tween 20 for 1 h at 41°C. The immunoreaction complex was detected with [lzSI]protein A (Amersham) (0.1 #Ci/ml) exactly as described [9,10]. The nitrocellulose membrane was exposed to Kodak XA5 film with intensifying screens at -70°C overnight for/3-PKC and two days for all other isoenzymes.…”

“…They were coupled either to keyhole limpet hemocyanin [10] or to tuberculin PPD [11], and injected into rabbits. The specificity of the antisera was previously documented [9,11]. Another set of peptides corresponding to selected sequences of c~-,/3-and 7-PKC isoform from rat brain was prepared and coupled to BSA before injection into rabbits (Pelosin et al, unpublished).…”