Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells.

Abstract

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 40C as well as at 30'C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase I, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase U by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.Phorbol esters influence cellular functions at various levels (1, 2), presumably by activating protein kinase C (3, 4). Some cellular effects can be detected rapidly after the addition of phorbol esters. These effects include the regulation of ionic transport, release of bioactive substances, and receptor down-regulation (1,2,5). Other effects of phorbol esters target the genome, resulting in the regulation of DNA replication or in the modulation of gene expression. Examples of genes that appear to be induced by phorbol esters include ornithine decarboxylase in epidermal cells (6,7), interleukin 2 and IL-2 receptor in mouse T cells and Tlymphoma cells (8-10), c-fos in U937 monocytes and in HL-60 cells (11), actin and vimentin in K562 erythroleukemia cells (12), and calcitonin in thyroid medullary carcinoma cells (13). Genes that seem to be repressed by phorbol esters include globin in Friend erythroleukemia cells (14), glycophorin in K562 erythroleukemia cells (15), and c-myc in thyroid medullary carcinoma cells (13). Moreover, phorbol esters increase the frequency of initiation of DNA replication for bacteriophage X injected into Xenopus eggs (16).The effects of phorbol esters on gene transcription generally require more time to become manifest than the effects on ionic fluxes or receptors. Thus, the regulation of c-fos mRNA in U937 monocytes is maximal after 30 min (11) and that of glycophorin and actin genes becomes evident after about 1 hr (12, 15), whereas the effect on c-myc mRNA is observed 4 hr after exposure of the cells to the tumor promoter (13). The relative delay in these responses may reflect an indirect effect ofphorbol esters on gene transcription, requiring one or more intermediary steps or a cascade of reactions; alternatively, phorbol esters may alter gene transcription more directly by stimulating protein kinase C, which then may phosphorylate a polypeptide(s) involved in transcriptional regu...

Altered stability of etoposide-induced topoisomerase II-DNA complexes in resistant human leukaemia K562 cells

“…Topoisomerase II has been shown to be phosphorylated in intact cells at serine and threonine residues (Saijo et al, 1990;Kroll & Rowe, 1991;Cardenas et al, 1992) and in vitro serves as a substrate for casein kinase II, protein kinase C and p34cdc2 kinase (Ackerman et al, 1985;1988;Sahyoun et al, 1986;Cardenas et al, 1992;Devore et al, 1992;Corbett et al, 1993). In addition, the activity and degree of phosphorylation of topoisomerase II has been found to increase during cell cycle progression from GI to G2-M phase (Heck et al, 1989;Woessner et al, 1991;Saijo & Enomoto, 1992).…”

Terminal deoxynucleotidil transferase is a nuclear PKC substrate

“…DNA polymerase ~ has also been reported to be phosphorylated in vitro by PKC [35] and in vivo, the enzyme undergoes phosphorylation dependent on phosphatidyl inositol mono and diphosphate [36]. Other nuclear enzymes affecting DNA structure and function, such as DNA topoisomerase II, could be substrates for PKC [37] as well as the nucleoside phosphatase that mediates mRNA transport out of the nucleus [38]. The role of PKC in T cell activation has recently been described and this contributes to understanding the involvement of PKC in specific biological processes, including regulation of the immune responses [39].…”

The role of protein phosphorylation in the control of cell growth and differentiation

“…Alterations in DNA topology have also been implicated in the control of differential gene expression (Sahyoun et al, 1986). Protein phosphorylation may be a key factor in this process.…”

“…Gene expression may be modulated by several routes. These include: changes in the activity of DNA replication and transcription enzymes (Krauss et al, 1987;, alteration of DNA topology (Sahyoun et al, 1986) and regulation of the association of transcriptional control proteins with specific DNA sequences (Montminy et al, 1987). Protein phosphorylation may be the regulatory mechanism operating in each of these cases.…”

“…Protein phosphorylation may be the regulatory mechanism operating in each of these cases. DNA polymerase alpha (Krauss et al, 1987) RNA polymerases I and II (Rose et al, 1981; and topoisomerases I and II (Durban et al, 1983;Ackerman et al, 1985;Sahyoun et al, 1986) are all phosphoproteins whose activity is related to their state of phosphorylation.…”