A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.

We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.     

Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Epidermal growth factor increases c-myc mRNA without eliciting phosphoinositide turnover, protein kinase C activation, or calcium ion mobilization in Swiss 3T3 fibroblasts

Incubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, bis-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reaction.     

Enhancement of growth factor-induced DNA synthesis by colon tumor-promoting bile acids in Swiss 3T3 cells. Their different mode of action from that of phorbol esters

In Swiss 3T3 cells, colon tumor-promoting deoxycholate (DOC) enhanced DNA synthesis which was induced by fibroblast growth factor (FGF) in the presence of insulin. This effect was observed only when DOC was added within 10 h after the addition of FGF. DOC by itself did not induce DNA synthesis irrespective of the presence or absence of insulin. Similar results were obtained with other colon tumor-promoting bile acids such as cholate, chenodeoxycholate and taurocholate. In contrast to these bile acids, 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis fully without FGF in the presence of insulin. DOC did not affect TPA-induced DNA synthesis. Prolonged treatment of the cells with phorbol-12,13-dibutyrate caused the down-regulation of the phorbol ester receptor and rendered the cells unresponsive to TPA. In these cells, FGF still induced DNA synthesis in the presence of insulin, but the maximal level was reduced to about one third of that in the control cells. DOC did not enhance this DNA synthesis any more. DOC did not alter the binding of FGF to the cells. These results indicate that colon tumor-promoting bile acids enhance the mitogenic action of FGF and thereby stimulate DNA synthesis, although the phorbol ester substitutes for the mitogenic action of FGF.     

A possible role of protein kinase C in signal-induced lysosomal enzyme release

In platelets, activation of protein kinase C and mobilization of Ca2+ were selectively induced by the addition of 1-oleoyl-2-acetyl-glycerol and a low concentration of A23187, respectively (Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704). Using this procedure evidence was obtained suggesting that the protein phosphorylation and Ca2+ mobilization were both essential and synergistically effective to cause release of lysosomal acid hydrolases such as N-acetylglucosaminidase. A similar observation was made for the lysosomal enzyme release from rat neutrophils.