Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation.

The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457-469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Calpha and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH(2)-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.

Tumor promoter TPA mimics irradiation effects on the cell cycle of HeLa cells.

When asynchronous and synchronous HeLa cells were incubated with small doses (10(-7) M) of tumor promoter 12 O-tetradecanoylphorbol-13-acetate (TPA), a variety of transient alterations in the replication cycle were detected within 24 hours by the use of independent methods. Especially, a delayed passage through the S phase and influences on the G2 phase resemble x-ray irradiation effects on cell cultures. None of these alterations was observed with the hyperplasiogenic but nonpromoting 4-O-methyl-TPA.

Lens culinaris lectin immobilized on sepharose: binding and sugar-specific release of intact tissue culture cells.

Lens culinaris lectin (LCL) covalently linked to 2B Sepharose binds tissue culture cells to the matrix. This is prevented by hapten sugars specific for LCL. Unlike other immobilized lectins, lens culinaris lectin allows the removal of bound cells from the matrix on addition of the specific sugars in a concentration-dependent manner. Binding and release occur under physiological conditions. Released cells continue to grow.

Partial inversion of the initiation-promotion sequence of multistage tumorigenesis in the skin of NMRI mice.

Alterations in NMRI mouse skin induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in "stage I of tumor promotion" are slowly reversible, and this reversibility has a half-time of 10 to 12 weeks. The tumor response observed in the course of an initiation-promotion experiment in vivo is independent of whether stage I of promotion occurs before or after initiation. Since the time interval between treatment with the promoter, and subsequent initiation can be extended up to at least 6 weeks, an enhancement of initiation because of promoter-induced cellular DNA synthesis seems to be unlikely. This result may be inconsistent with the two-stage model of tumor promotion because it indicates that in skin the existence of initiated cells is not required for the induction of cellular alterations that are essential for the stage of skin tumorigenesis called stage I of promotion.

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND: Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS: The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS: The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Radiation with 1 Gy prevents the activation of the mitotic inducers mitosis-promoting factor (MPF) and cdc25-C in HeLa cells.

The mechanism of the transient G2 arrest induced by small doses of ionizing radiation involves the failure to activate the correctly formed pre-mitosis-promoting factor (MPF) complex of cyclin B and p34cdc2 by dephosphorylation at Tyr15 of the latter, as recent studies of other laboratories have indicated. Similar data were obtained with the G2 arrest-inducing agents epidermal growth factor and the phorbol ester 12-0- tetradecanoylphorbol-13-acetate (H. Barth and V. Kinzel, Exp. Cell Res., 212: 383-388, 1994, and H. Barth and V. Kinzel, J. Cell. Physiol., 162: 44-51, 1995). To differentiate the radiation consequences in synchronized HeLa cells from those of 12-0-tetradecanoylphorbol-13-acetate and epidermal growth factor, experiments with a very small dose (1 Gy) have been carried out in cells close to the G2-M border and, for comparison, in mitotic cells. We show that in addition to the failure of p34cdc2 dephosphorylation at Tyr15, radiation with 1 Gy also prevents the activation of the phosphatase cdc25-C, the enzyme catalyzing the MPF activation. In contrast, irradiation of mitotic cells with 1 Gy did not influence that fraction of either MPF or cdc25-C already activated. Moreover, the gain in MPM-2 antigenicity of cdc25-C, usually indicative of an activating phosphorylation, is shown to be prevented by 1 Gy. The data indicate that the initiation of the proposed autocatalytic loop between MPF and cdc25-C becomes interrupted by radiation, but they give no hint at which point.

Phorbol ester-induced G2 delay in HeLa cells analyzed by time lapse photography.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown previously to mimic X-irradiation in altering cell cycle parameters in Hela cells [Kinzel, V., Richards, J., and Stöhr, M., Science (Wash. DC), 210: 429-431, 1980]. These changes include a delay in G2 phase from which cells recover in the presence of TPA, which suggests an involvement of cellular mediators. In order to obtain information on the onset and the duration of the G2 delay, as well as the onset and rate of recovery, a time-lapse study has been carried out. The analysis of cells in prophase shows that at 10(-6) M and 10(-7) M concentrations, TPA and 12-O-retinoylphorbol-13-acetate (RPA) cause a G2 delay which lasts on the order of 3.5 to 4 h. Below 10(-7) M of RPA and below 10(-8) M of TPA a clear-cut inhibition of HeLa cells in G2 is no longer detectable by this method. These results for a given phorbol ester are dose dependent within a certain range but unlike the case of X-rays are not proportional to dose. Within the dose range studied the recovery rate follows the opposite order. At 10(-6) M TPA and RPA an indication of a parasynchronous burst is observed. At smaller concentrations or with less biological activity of phorbol ester, the cell multiplication rate approaches that of the control or remains even smaller. Possible reasons are discussed. The determination of the transition points seems to indicate that the cellular events inhibited in G2 occur shortly before visible prophase.

Proposed role of phosphatidic acid in the extracellular control of the transition from G2 phase to mitosis exerted by epidermal growth factor in A431 cells.

Epidermal growth factor (EGF) has been shown to cause an inhibition of A431 cells in G2 phase within approximately 10 min, i.e., shortly before mitosis (Kinzel et al., Cancer Res., 50: 7932-7936, 1990). This system has been used to study the proposed role phospholipid metabolites, particularly phosphatidic acid (PA), may play (Kaszkin et al., Cancer Res., 51: 4328-4335, 1991) in the extracellular control of cells at the physiological restriction site in G2 phase. A431 cells responded to EGF with a dose-dependent formation of phosphatidic acid (PA) which correlated with the dose-dependent G2 delay as well as with their time courses. The G2 delay induced by EGF as well as PA mobilization were effected in conditioned medium or in fresh medium containing bovine serum albimun instead of serum, i.e., under the conditions necessary for precursor studies to be carried out. The major pathway of PA formation was probably via phospholipase C-mediated breakdown of phosphatidylinositol and diacylglycerol kinase: (a) the dose response of PA formation correlated with that of total inositol phosphate accumulation; (b) little diacylglycerol was found and then only at a high EGF concentration; (c) prelabeling with [1-14C]arachidonic acid resulting in a large specific labeling of phosphatidylinositol led to an EGF-induced, dose-dependent formation of radioactive arachidonyl-PA (correlated with that of total PA and inositol phosphate), but in the presence of a primary alcohol not to the formation of radioactive phosphatidylalcohol; (d) prelabeling with [1-14C]oleic acid led to the EGF-induced formation of labeled PA, which in the presence of a primary alcohol was only slightly reduced to the advantage of very low levels of labeled phosphatidyl alcohol, thus demonstrating that an EGF-effected activation of phospholipase D did occur but contributed little to the general PA level. An alternative mobilization of PA was attempted with the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA), which was shown to activate phospholipase D in A431 cells and to elicit PA from a phospholipid pool which was not significantly labeled with radioactive arachidonic acid. The TPA-induced degree of PA formation and of the G2 delay correlated. Both phenomena were considerably larger with fresh medium containing 0.5% bovine serum albumin instead of serum than in conditioned medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Early effects of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate on the cell cycle traverse of asynchronous HeLa cells.

Within 24 hr after incubation of synchronous HeLa cell cultures with small nontoxic doses of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) or 10(-8) M), a variety of transient alterations in the cell cycle traverse was detected by different techniques. The measurement of thymidine incorporation rates into DNA, of labeling and mitotic indices, and of flow cytometry revealed (a) an inhibition of cells in G1 shortly prior to their entering S phase, (b) a reduced rate of DNA synthesis and a delayed passage of cells through S phase which were in this phase on addition of TPA, (c) a delayed passage through G2 of a portion of cells which were somewhere in the first half of the S phase on addition of TPA, and (d) an instant but short-lasting blockage in G2 immediately before mitosis. The effects of TPA are reminiscent of published results on X-irradiated cell cultures. None of these effects was noticed with the hyperplasiogenic but nonpromoting phorbol ester 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10(-6) M). The data were confirmed by experiments with synchronized HeLa cells as described in an accompanying paper.

Responses of synchronized HeLa cells to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate as evaluated by flow cytometry.

A flow cytometric study was carried out on the effects of tumor-promoting 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-8) M) on HeLa cells synchronized by amethopterin for DNA synthesis. Cells treated with TPA at the time of release from the amethopterin block showed a delayed passage through S phase and partially through G2 in their immediate life span as measured 24 hr after release. This late G2 delay was not observed when TPA was added to cells during late S or G2 phase. In this case, however, a direct inhibition of cells in G2 became evident as observed about 15 hr after release from block. None of these effects was caused by the nonpromoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10(-6) M). These data support observations obtained with asynchronous cultures. The TPA effects resemble those reported after X-irradiation of cell cultures.

Radiomimetic activity of phorbol esters exerted in HeLa cells in comparison with their tumor-promoting capacity.

The biological activities exerted in mouse skin by three closely related phorbol esters were compared with the effects of these compounds on HeLa cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate has been shown previously to influence various cell cycle parameters of these cells, thereby mimicking X-irradiation [Kinzel, V., Richards, J., and Stöhr, M. Science (Wash. D. C.), 210: 429-431, 1980]. Qualitatively similar effects were exerted by the mitogenic and irritant but almost nonpromoting "incomplete" phorbol esters 12-O-tetradeca-2-cis-4-trans-6,8-tetraenoylphorbol-13-acetate and 12-O-retinoylphorbol-13-acetate. Cell cycle parameters were analyzed by measuring thymidine incorporation rates, labeling indices, DNA histograms gained through flow cytometry, and mitotic activity. In every case, 12-O-tetradecanoylphorbol-13-acetate was more effective than 12-O-tetradeca-2-cis-4-trans-6,8-tetraenoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate. The analysis of the influence of phorbol esters in G2 phase showed that, in order to reach the effectiveness of 10(-8) M 12-O-tetradecanoylphorbol-13-acetate, approximately 10 times the concentration of either 12-O-tetradeca-2-cis-4-trans-6,8-tetraenoylphorbol-13-acetate or 12-O-retinoylphorbol-13-acetate has to be applied. Therefore, the susceptibility of replicating HeLa cells to these phorbol derivatives reflects the promoting rather than the mitogenic and irritant capacity of these compounds.

Epidermal growth factor inhibits transiently the progression from G2-phase to mitosis: a receptor-mediated phenomenon in various cells.

An immediate effect of epidermal growth factor (EGF) on the cell cycle is described. EGF, when given to replicating cells such as HeLa, A431, and D HER 14, very rapidly inhibits the transition from G2 phase to mitosis (M) in a transient fashion. The influence of EGF (10(-10) to 10(-7) M concentrations) on the G2-M transition of individual cells has been analyzed by time-lapse photography in cell lines carrying intact, mutated, or no EGF receptor. The G2-M transition of cells devoid of EGF receptor or carrying an EGF receptor devoid of most of the cytoplasmic domain was not influenced by EGF. In cells carrying intact EGF receptor, EGF caused a transient and dose-dependent delay in G2 phase which could last for greater than 2 h. Cells were inhibited in G2 within less than 10-20 min prior to prophase. A parasynchronous recovery from G2 inhibition was observed at large EGF concentrations; the G2-M transition rate exceeded that of the controls. The system described may represent a model for the mechanistic analysis of a ligand-induced transient restriction of the cell cycle, particularly at the G2-M border.

A proposed common mechanism by which phorbol esters and epidermal growth factor delay the progression from G2 phase to mitosis of HeLa cells through phospholipid metabolites.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; also called phorbol myristate acetate) and epidermal growth factor both rapidly inhibit the transition of HeLa cells from G2 phase to mitosis in a reversible fashion with characteristics of a receptor-mediated response. It was proposed that an intracellular mediator was responsible for this inhibition. In searching for a common mediator elicited by the action of both ligands, the time course of generation of phospholipid metabolites was compared with the cell cycle response of the G2 cohort monitored by time lapse analysis. The time course and the degree of mobilization of diacylglycerols (DG) effected by TPA and by epidermal growth factor and that of phosphatidic acid correlated to a great extent with the onset and the duration of the G2 inhibition caused by both agonists. Further evidence for the proposed role of DGs and/or phosphatidic acid was obtained by the observation that membrane-penetrating 1,2-DG inhibited HeLa cells in G2 phase directly. The concentration of DG required correlated with that elicited by TPA or epidermal growth factor metabolically. This cellular response to 1,2-DG was also seen in cells after down-regulation of protein kinase C using TPA. The data indicate that cellular DG and/or phosphatidic acid may contribute to a restriction of cells in G2 phase possibly by changing membrane properties.

The human papillomavirus type 16 E5 protein modulates phospholipase C-gamma-1 activity and phosphatidyl inositol turnover in mouse fibroblasts.

The human papillomavirus type 16 E5 (HPV16-E5) protein is a membrane protein that has been associated with malignant growth. The protein affects growth factor-mediated signal transduction in a ligand-dependent manner. We show now that E5 expression in A31 fibroblasts results in an increased level of diacylglycerol (DAG) and inositol phosphates. Immunoprecipitation of phospholipase C-gamma-1 (PLC-gamma-1) with specific antibodies and immunoblotting with anti-phosphotyrosine antibodies reveal a large increase in tyrosine phosphorylation of the enzyme in E5-expressing cells compared to control vector-transfected cells. This activation of tyrosine phosphorylation is growth factor independent. In addition, an enhanced formation of phosphatidic acid (PA) was observed in E5 cells. This increase did not result from activation of phospholipase D (PLD), although the enzyme was activatable by treatment with phorbol ester Thus, a phosphohydrolase-mediated DAG synthesis from PLD-produced PA can be excluded. The observed effects were not further enhanced by EGF showing that the presence of the growth factor is not necessary for maintaining permanent activation of PLC-gamma-1 in E5-expressing cells. The DAG- and inositol phosphate-mediated signal cascade within the cells is thus effectively uncoupled from external control via EGF and its receptor in the presence of E5 protein.

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.

Phosphorylation by cell surface protein kinase of bovine and human fibrinogen and fibrin.

Human and bovine fibrinogen as well as fibrin are shown to be phosphorylated by Co631 (monolayer, hamster) and RPL12 (suspension, chicken) cells by their surface protein kinase of the casein kinase II type. The phosphate label is introduced into the alpha-peptide. The kinase system phosphorylates serine residues and utilizes GTP equally well as ATP. The participation of intact cell surfaces indicates the possibility of phosphorylation of extracellular fibrinogen independently of the site of its biosynthesis.

Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts.

A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP-dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis.

Mobilization of diacylglycerol in intact HeLa cells by exogenous phospholipase C from Cl. perfringens is accompanied by release of fatty acids including arachidonic acid.

The second messenger diacylglycerol (DAG), chiefly derived from phosphatidylcholine (PC) or from phosphatidylinositol (PI), through the activation of specific phospholipases C (PLC), plays a key role in cellular stimulation. The activation of a particular PLC was simulated in intact HeLa cells by treatment with exogenous PC-PLC (Cl. perfringens) or with PI-PLC (B. cereus). Both enzymes rapidly mobilized DAG. However, only PC-PLC led, in Hela cells, to morphological changes (which were reversible on enzyme removal within the time frame of the experiments) and to an increase of intracellular calcium concentration with a lag of > 10 min. In cells prelabeled with [1-14C]arachidonic acid only PC-PLC but not PI-PLC induced the release of labeled fatty acid with a lag of > 10 min. Upon prelabeling of cells with [1-14C]oleic acid, PC-PLC led to a release of radioactive oleic acid. The release of arachidonic acid (AA) required a threshold dose of PC-PLC and a minimum time of treatment beyond which the AA release continued for a certain period, even in the absence of the exogenous enzyme. Under the conditions used, neither PLA2 nor DAG lipase activity were detectable in the PC-PLC preparation. Therefore, AA release was due to activation of a cellular enzyme, probably cellular PLA2 activity. The PC-PLC-induced AA release could be inhibited to a certain extent by EGTA and by quinacrine but not by the glucocorticoid fluocinolone acetonide. Only PC-PLC (but not PI-PLC) caused, in addition, an increase of the level of monoglycerol, which paralleled the appearance of AA. An increase of labeled monoglycerol was detectable in HeLa cells prelabeled with radioactive oleic acid or with 1-[1-14C]palmitoyl-lyso-PC but not in cells prelabeled with radioactive AA, thus indicating that the fatty acid originated from sn-2 position of the glycerol moiety. The 1-monoacylglycerol was probably generated from lysophospholipids by the bacterial PC-PLC. This enzyme preparation has been shown to catalyze such breakdown of lysophosphatidylcholine in vitro. PC-PLC-induced AA release occurred also after down-regulation of protein kinase C by an overnight pretreatment with phorbol ester TPA (TPA-pretreated cells, but not control cells, on treatment with PC-PLC, metabolized AA to prostaglandins).(ABSTRACT TRUNCATED AT 400 WORDS)

Promoter of the gene encoding the bovine catalytic subunit of cAMP-dependent protein kinase isoform C beta 2.

Genomic sequences flanking the 5' end of the cDNA encoding isoform C beta 2 of the catalytic subunit of bovine cAMP-dependent protein kinase were cloned, sequenced and analyzed for promoter activity and transcription initiation sites. A region of 913 bp upstream the translation initiator ATG was amplified from genomic DNA by vectorette polymerase chain reaction. In primer extension reactions and RNase protection assays, residues C (at position -91), T (-71) and G (-70) were found to serve as transcription initiation sites of the gene. Amplification products and sub-fragments thereof were ligated upstream of the reporter gene chloramphenicol acetyltransferase to test for promoter activity. Constructs were transiently transfected into a Chinese hamster ovary cell line which was shown to express endogenous C beta 2 mRNA. The genomic sequence upstream the C beta 2 cDNA does have promoter activity. The region from position -51 to -292 proved sufficient to drive efficient transcription of the reporter gene. The promoter is AT rich (68%), does not contain a TATA box within 50 bp upstream of the first initiation site and possesses putative binding sites for several transcription factors such as PEA-3 and a glucocorticoid receptor.

Investigation of the structural components governing the polarity-dependent refolding of a CD4-binding peptide from gp120.

The conformational switch at the principle CD4-binding domain of gp120 from HIV1 exhibits a highly cooperative folding transition from beta-sheet to helix triggered within a very narrow range of solvent polarity. The physical basis of this folding behaviour is of interest because it is unusual and because it is closely connected with biological function, i.e. binding to the CD4 receptor. Previous work revealed two primary structural elements, an N-terminal LPCR tetrad and a tryptophan residue eight residues C-terminal to this, that were essential for the helical and for the beta-sheet conformation, respectively. Attempts to construct synthetic "switch" domains using the characteristics so far identified produce peptides undergoing the transition at much higher polarity and involving fewer residues than the natural domain, in essence a lower stability of the beta-fold to apolar conditions. Introduction of a tryptophan residue reduced at the C(2)-C(3) linkage demonstrates clearly that the aromatic system of the tryptophan residue is central to beta-sheet stabilization. Residues with side-chains that might participate in electrostatic or aromatic interactions with the pi-electron system of Trp were sequentially altered to alanine. The results indicate that the "switch" properties of the CD4-binding domain arise from a poised tension between multiple interactions with the Trp aromatic ring stabilizing the beta-structure and the tendency of the LPCR tetrad to act as a template for a helical fold. Under polar conditions the former dominate. Lowering the polarity alters this both by weakening the aromatic interactions and by simultaneously increasing the helical propensities of the isoleucine and valine side-chains. Tryptophan seems uniquely suited to act as a polarity-sensitive conformational sensor.