Phorbol-ester-activated protein kinase C-alpha lacking phosphorylation at Ser657 is down-regulated by a mechanism involving dephosphorylation.

Protein kinase C (PKC) is a key enzyme in the intracellular signaling network. Upon activation by 12-O-tetradecanoylphorbol 13-acetate, the alpha-isoform of PKC translocates to the detergent-soluble and the detergent-insoluble fractions. Besides cofactors, the activity and stability of this protein is critically regulated by multisite phosphorylations. At least three distinct sites, Thr497, Thr638 and Ser657, are involved. We have previously shown that the replacement of Ser657 by alanine leads to a premature down-regulation in the detergent soluble compartment of LLC-PK1 cells [Gysin, S. & Imber, R. (1996) Eur. J. Biochem. 240, 747-750]. More detailed analysis revealed that, in contrast to the wild-type molecule, the down-regulation of the mutant protein is in vivo preceded by a rapid dephosphorylation after phorbol-ester-induced translocation to both the detergent-soluble and insoluble compartments. The [Ala657]PKC-alpha mutant protein molecule showed in vitro a strongly increased sensitivity towards protein phosphatase 2A whereas its overall proteolytic sensitivity remained unchanged when compared to wild type. The in vitro studies led to the suggestion that further dephosphorylation of the mutant protein is a prerequisite in order to become proteolytically down-regulated. Therefore phosphorylation of Ser657 controls the duration of activation of this PKC isozyme upon agonist-induced translocation by preventing premature proteolytic down-regulation via protecting the protein from dephosphorylation.

Replacement of Ser657 of protein kinase C-alpha by alanine leads to premature down regulation after phorbol-ester-induced translocation to the membrane.

Protein kinase C (PKC) is activated at the cell membrane by interacting with both the acidic lipid phosphatidylserine and the second messenger diacylglycerol. A direct activation of the kinase is also possible by substituting diacylglycerol with phorbol esters such as the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Transphosphorylation of the activation loop followed by autophosphorylation at sites located on various domains of the protein have been suggested to be required as permissive activation of the alpha and beta isoforms of PKC [Cazaubon, S., Bornancin, F. & Parker, P. (1994) Biochem. J. 301, 443-448; Keranen, L. M., Dutil, E. M. & Newton, A. C. (1995) Curr. Biol. 5, 1393-1403]. Ser657, located near the C-terminus of PKC-alpha, represents a site which is very conserved among the members of the PKC protein family. Circumstantial evidence suggested that this residue represents a possible site of phosphorylation. The conversion of Ser657 to alanine caused a 70% loss of the catalytic activity as well as a drastically increased down regulation upon translocation of this isozyme to the membrane when induced by phorbol ester. The faster electrophoretic mobility of the mutant protein compared to that of the wild-type enzyme suggested that Ser657 represents a phosphorylation site.

Targeted inhibition of tumor-cell growth by recombinant heregulin-toxin fusion proteins.

Fusion of functional domains of proteins by in vitro recombination of gene fragments can be used to generate novel anti-tumor agents. The combination of tumor-cell-recognition functions and toxic functions results in cytotoxic molecules with a high specificity for tumor cells. Human adenocarcinomas are frequently characterized by over-expression of members of the epidermal-growth-factor (EGF) receptor family (ErbB-1, 2, 3 and 4), when compared with normal cells. These tumors are particularly suited to treatment with recombinant toxins. The human heregulins (HRG) and their rat counterparts (neu differentiation factor, NDF) have been identified as ligands for these receptors. Two chimeric heregulin-toxin fusions consisting of the EGF-like receptor recognition domain of the heregulin isoforms HRG alpha and HRG beta I, and the domains II, Ib and III of the Pseudomonas exotoxin A (ETA) were constructed. HRG beta I-ETA is highly cytotoxic for the mammary carcinoma cell lines SK-BR-3 and MDA-MB-453. HRG alpha-ETA was less active than HRG beta I-ETA. The killing activity of the recombinant toxins correlated with the expression levels of ErbB-3 and/or ErbB-4 in the cell lines studied. High expression of ErbB-2 is not sufficient to confer sensitivity towards the HRG-ETA. Treatment of mice with 0.4 mg/kg/day of HRG beta I-ETA caused growth retardation of transplanted human breast tumor cells. Higher levels of HRG beta I-ETA administration resulted in acute hemorrhagic necrosis of the liver.

No tumor-specific expression levels of protein kinase C isoenzymes and of c-fos in human breast cancer cell cultures.

Epithelial cells derived from 46 human breast tissue samples of patients suffering from breast cancer have been cultivated. Twenty-five of these cell cultures stemmed from normal and 21 from tumor tissues. Moderate to large variations of protein levels of three protein kinase C (PKC) isoenzymes (alpha, delta and epsilon) were found among the various cell cultures. The cell cultures also exhibited very heterogeneous basal as well as inducible levels of c-fos mRNA. However, none of these variations could be correlated with the character of the original tissue nor with any clinical parameter of the respective patient. Our results suggest that altered levels of PKC isoenzymes or of the protooncogene c-fos per se cannot serve as an indication for a transformed behavior of the epithelial cell fraction of human breast tissue.

Unphosphorylated alpha-PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro.

Expression of the alpha-isoform of protein kinase C (alpha-PKC) in E. coli yielded the unphosphorylated 74 kD precursor molecule. This precursor form exhibited phospholipid- and calcium-dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity. In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of alpha-PKC is not required for binding of substrates, cofactors, or activators. These results, therefore, suggest that posttranslational phosphorylation of alpha-PKC is not needed for the formation of a functional enzyme-substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.

Growth-promoting effect of a protein-free hemodialysate used in situations of hypoxia and for tissue repair as measured via stimulation of S6-kinase.

Solcoseryl is the low-molecular weight fraction of calf blood as manufactured by counterflow dialysis. This hemodialysate (HD) is in clinical use in situations involving hypoxia and for the normalization of tissue repair. The influence of the HD on ZR-75 cells was tested. These cells preferably express receptors for Epidermal Growth Factor (EGF)/Transforming Growth Factor alpha (TGF-alpha) or Somatomedin C (SMC = insulin like growth factor I resp. ILA-I) and react upon stimulation by enhancement of their S6-kinase activity, the latter being a prerequisite for growth. The functional presence of one or several of these peptide growth factors should therefore reflect in a stimulation of S6-kinase activity.

Failure of wild-type or a mutant form of protein kinase C-alpha to transform fibroblasts.

A mutant form of the alpha-isoform of protein kinase C (PKC) was recently isolated from an ultraviolet radiation-induced murine fibrosarcoma cell line and reported to transform mouse BALB/c 3T3 fibroblasts on transfection. Four point mutations in the regulatory domain were assumed to be responsible for its oncogenicity and unusual preference for membrane localization. Here, we report that overexpression of the reported mutant PKC alpha complementary DNA in three fibroblast cell lines, including BALB/c 3T3, does not enable these cells to grow in soft agar or nude mice. In addition, this mutant PKC alpha form seems to be indistinguishable from the wild-type PKC alpha with respect to its dependence on cofactors, phorbol ester binding, subcellular distribution and its effects on growth and morphology. These results fail to confirm the previous study and indicate that overexpression of either the wild-type or the reported mutant form of PKC alpha does not transform rodent fibroblasts.

Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets.

Protein kinase C (PKC), a family of related but distinct enzymes whose cellular functions are poorly understood, acts in synergy with Ca2+ mobilization for the activation of platelets. Using specific antibodies for the different isoforms, immunoblot analysis revealed the presence in human platelets of three different PKC subtypes which specifically react with alpha, beta and zeta-PKC antibodies. Whereas the subcellular distribution of the alpha PKC remained unaffected, incubation of platelets with 1 microM PMA for 2 min resulted in a significant subcellular distribution from cytosol to membrane of beta-PKC (25%) and zeta (15%). The beta-PKC isoform is more sensitive than alpha and zeta-PKC to PMA, since 100 nM PMA resulted in a translocation of 85%, 64% and 66% respectively of a maximum translocation observed with 1 microM PMA.

Tumor-promoting phorbol ester and activated Ha-ras synergistically reduce the interleukin 3 requirement in a mast cell line.

Infection of the bone marrow-derived mast cell line PB-3c with a retrovirus carrying oncogenic c-Ha-ras or v-Ha-ras reduced the interleukin 3 (IL-3) growth requirement and induced a state of tumorigenicity. In contrast, normal c-Ha-ras had no effect on the IL-3 requirement of this cell line nor did the cells become tumorigenic. A factor reduction similar to that caused by activated Ha-ras was transiently obtained with 12-O-tetradecanoylphorbol-13-acetate in the PB-3c cells expressing normal c-Ha-ras. The analogous stimulation of protein kinase C (PKC) in PB-3c cells producing oncogenic Ha-ras led to an additional reduction of the IL-3 requirement during the first 24 h. In the absence of IL-3, the prolonged exposure of the cells to 12-O-tetradecanoylphorbol-13-acetate for 72 h resulted in a stimulation of growth when activated but not when normal Ha-ras was expressed. PB-3c cell lines expressing activated Ha-ras neither revealed differences in the amounts nor in the subcellular distribution of PKC activity but displayed elevated levels of immunoreactive beta-PKC compared to the parental PB-3c cells. Upon 12-O-tetradecanoylphorbol-13-acetate treatment, a protracted down-regulation of the immunodetectable alpha-PKC as well as constitutively high levels of c-fos mRNA were observed when oncogenic Ha-ras was expressed. These data suggest the involvement of specific PKC subtypes and of c-fos in the reduction of the IL-3 requirement caused by activated Ha-ras in this particular hematopoietic cell line.

DNA-binding properties and primary structure of HB protein from Bacillus globigii.

The binding of Bacillus globigii HB protein to synthetic deoxyoligonucleotides of different length and sequence has been studied by polyacrylamide gel electrophoresis. Without detectable sequence specificity the protein binds to single-stranded and double-stranded DNA. Under the conditions employed, binding of HB protein to deoxyoligonucleotides with six or less nucleotides per strand cannot be detected while eight or more nucleotide units per strand of single-stranded DNA or base pairs of double-stranded DNA are sufficient for binding. The complete amino acid sequence of HB protein has been determined by manual Edman degradation of tryptic peptides. Like most DNA-binding proteins of its class, HB protein does not contain cysteine, tyrosine or tryptophan residues. The primary structure of HB protein shows 84% homology with the sequence of the related DNA-binding protein II from Bacillus stearothermophilus.

Regulation of expression of the cloned repE gene from the F plasmid of Escherichia coli.

The repE gene of the Escherichia coli F plasmid has been fused to an N-terminal fragment of the Salmonella typhimurium araB gene in the plasmid expression vector pING1. A fusion protein is expressed at high levels upon addition of arabinose to E. coli hosts containing the recombinant plasmid and has been purified to homogeneity. Antibodies prepared against the fusion protein react with both araB' and repE-coded proteins and can be used to detect their synthesis by immunoblotting methods. In vitro as well as in vivo expression of the repE gene from chimeric plasmids indicate a very tight control of the expression of this replication initiator protein.

Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).

M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates.

M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.

Purification and characterisation of a small DNA-binding protein, HB, from Bacillus globigii.

We have purified a small, heat-stable DNA-binding protein from Bacillus globigii: HB protein. The protein binds cooperatively to double-stranded DNA and the DNA-protein complexes are destabilised by the presence of moderate (0.1 M) levels of monovalent cations or by low levels (10 mM) of Mg2+. We have also purified two small DNA-binding proteins from Escherichia coli, NS1 and NS2 [Suryanarayana, T. and Subramanian, A.R. (1978) Biochim. Biophys. Acta, 520, 342-357], also known as HU protein [Rouvière-Yaniv, J. and Gros, F. (1975) Proc. Natl Acad. Sci. USA, 72, 3428-3432] that have been extensively studied by others. We have prepared antibodies against all three proteins and have shown that all three are, by immunological criteria, related.

Purification and properties of the restriction endonuclease BglII from Bacillus globigii.

The restriction endonuclease BglII from Bacillus globigii has been purified to homogeneity. The enzyme is a dimer of two subunits of Mr = 27000. The reaction mechanism does not involve the accumulation of a DNA intermediate nicked in one strand and the enzyme is not affected by superhelical twists in the substrate DNA, indicating that DNA binding does not involve either winding or unwinding of the double helix. Antibodies were prepared against BglII. These antibodies did not cross react with any other restriction endonucleases tested, including other enzymes from B. globigii or from closely related strains. It is thus unlikely that type II restriction enzymes represent a closely related group of proteins.

A simple, general procedure for purifying restriction endonucleases.

A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.

Dermabrasion for adenoma sebaceum.

Two patients with tuberous sclerosis but without mental retardation were dermabraded for adenoma sebaceum. The treatment was effective and cosmetically satisfying.