PubMed Health⌕ Search

Biomedical subjects

V Kinzel

Publications and source records attributed to V Kinzel.

At least 37 records · Page 2Linked to original sources
PubMed1996

Induced release of cell surface protein kinase yields CK1- and CK2-like enzymes in tandem.

Several types of cell exhibit cell surface protein kinase (ecto-PK) activities with Ser/Thr-specificity. Ecto-PK sharing certain characteristics of protein kinase CK2 can be detached from intact cells by interaction with exogenous substrates (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). However, a detailed molecular analysis of this ecto-PK was hampered by the vanishingly small amounts of labile enzyme protein obtained by substrate-inducible enzyme release. We now describe the stabilization and enrichment of released ecto-PK by precipitation with polyethylene glycol followed by affinity chromatography on heparin-agarose. Ecto-PK is shown to consist of two separate forms released in tandem, ecto-PK I and ecto-PK II. Comparison with cell homogenates as well as cell surface biotinylation experiments excluded contamination with intracellular PK. Purified ecto-PK I and ecto-PK II exhibit respectively selective phosphorylation of CK1- and CK2-specific peptide substrates, a complementary sensitivity to inhibitory agents and a differential use of the cosubstrates ATP and GTP. Ecto-PK I consists of a 40-kDa moiety; the ecto-PK II is an ensemble of three components of 43- and 40-kDa (catalytic subunits) and a noncatalytic 28-kDa subunit. In addition, components of the ecto-PK II react with CK2-specific antibodies. Further, comparative peptide mapping and the results of mass spectrometry in combination with assignment of amino acid sequences confirmed that ecto-PK II is closely related if not identical to the protein kinase CK2. Assays with intact cells that result in the phosphorylation of a variety of endogenous membrane proteins showed that both ecto-PKs participate, and further, certain ecto-PK substrates become preferentially labeled by one or another of the enzymes, whereas others are phosphorylated by both ecto-PK activities.

Amino Acid Sequence↗
PubMed1996

Role of cdc25-C phosphatase in the immediate G2 delay induced by the exogenous factors epidermal growth factor and phorbolester.

Studies on the link between cellular signalling and cell cycle control at the G2 checkpoint have shown that, in HeLa cells, epidermal growth factor (EGF) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly inhibit the G2-M transition by preventing the key component of mitosis-promoting factor (MPF), p34cdc2, from expressing protein kinase activity. The kinase activity of active MPF is not inhibited; rather, the conversion of pre-MPF to MPF, i.e., the activating dephosphorylation of p34cdc2, at tyrosine is rapidly blocked (Barth and Kinzel, 1994, Exp. Cell Res. 212:383-388; 1995, J. Cell. Physiol., 162:44-51). The phosphatase responsible, cdc25-C, is activated by phosphorylation in mitotic cells starting at the G2-M transition in an autocatalytic loop with MPF (Hoffmann et al., 1993, EMBO J. 12:53-63). We now show that, concomitant with the prevention of MPF activation, EGF and TPA induced a reduction of the activity of cdc25-C in synchronized cultures. Furthermore, treatment of mitotic HeLa cells with TPA did not influence the kinase activity of MPF but caused a rapid decrease of the specific enzyme activity of cdc25-C, probably due to dephosphorylation of the enzyme, as indicated by reduced binding of monoclonal MPM-2 antibody specific for phosphoepitopes in M phase. Because of its inability to induce signalling during division, EGF failed to influence the activity of cdc25-C in mitotic cells. The scenario in cells late in G2 that are committed to enter mitosis may be as follows: In those cells where the signalling pathways responding to EGF as well as those responding to TPA are still open, cdc25-C is prevented by dephosphorylation from exceeding the threshold level of activity required to initiate the activation of and the autocatalytic feedback loop with p34cdc2 and to enter mitosis. Therefore, cdc25-C appears to represent part of an interface between cellular signalling and cell cycle control in G2 phase.

CDC2 Protein Kinase↗
PubMed1996

Primary alcohols and phosphatidylcholine metabolism in rat brain synaptosomal membranes via phospholipase D.

Phospholipase D of rat brain synaptosomal membranes was tested with phosphatidylcholine as the substrate for its specificity in the use of primary alcohols as transphosphatidylation co-substrates. The efficiency of the reaction was related to the hydrophobicity and the membrane penetrating capacity of the alcohol molecule. Phosphatidylalcohol formation could be detected up to 1-octanol but not for alcohols with longer hydrocarbon chains (C(9), C(10)). With increasing alcohol concentration the transphosphatidylation activity of the phospholipase D reached an optimum and then declined abruptly. Alcohol concentrations required for maximal transphosphatidylation reaction generally decreased with increasing hydrophobicities of the alcohols. Nevertheless 1-butanol and 4-chloro-1-butanol were the most efficient cosubstrates, sharing identical optimal conditions. Transphosphatidylation works at the cost of phosphatidic acid formation. Phosphatidic acid itself was transformed to diacylglycerol, probably by a contaminating phosphatidic acid phosphohydrolase.

Alcohols↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Epidermal growth factor rapidly impairs activation of p34cdc2 protein kinase in HeLa cells at the G2-M boundary.

Epidermal growth factor (EGF) has been shown rapidly to inhibit the transition from G2 phase to mitosis: beyond this transition point the cells are refractory to EGF (Kinzel et al., 1990, Cancer Res., 50:7932-7936). Using synchronized HeLa cells, EGF has now been shown to induce an overall decrease of the histone H1 kinase activity of p34cdc2 after 20 min of treatment, a time course which correlates with the number of cells in metaphase. The kinase level of actively mitotic cells is not altered by EGF. Neither the amount of p34cdc2 protein present nor that of Cyclin B in influenced by EGF, and the formation of the p34cdc2/Cyclin B complex is also unaffected. The use of antiphosphotyrosine antibodies, however, showed that p34cdc2 from cultures treated with EGF was more intensely stained than that of control cells, indicating that EGF treatment prevents the tyrosine dephosphorylation which is required for expression of the protein kinase activity of the complex. Taken together, the results show that EGF in HeLa cells very rapidly prevents the p34cdc2/Cyclin B complex from expressing kinase activity at the G2-M boundary, which appears to be the cause for the inhibition in G2 phase.

Blotting, Western↗
PubMed1995

Solvent polarity-dependent structural refolding: a CD and NMR study of a 15 residue peptide.

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a beta-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular beta-sheet to a helical secondary structure is known as "switch" behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the "switch." The tetrad has a strong beta-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3(10)-helix suggesting that the tetrad adopts a type III beta-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3(10)-helix at a critical polarity favoring intrachain hydrogen bonds.

Amino Acid Sequence↗
PubMed1994

Major cell surface-located protein substrates of an ecto-protein kinase are homologs of known nuclear proteins.

Cell surface polypeptides serve as substrates for a casein kinase-like ecto-protein kinase activity which is demonstrable under stringent criteria with intact cells using micromolar levels of extracellular [gamma-32P]ATP. Two major 32P-labeled proteins, designated as pp100 and pp120 after their apparent molecular masses on SDS-PAGE under reducing and nonreducing conditions, have repeatedly appeared in the phosphoprotein spectra of different cell types. We have chosen HeLa cells as a source for the biochemical characterization and isolation of pp100 and pp120. Phosphorylation of pp100 and pp120 occurs in their extracellular domains at seryl residues of amino acid side chains. Several criteria deduced from the heparin sensitivity of the ecto-protein kinase and its substrate-induced shedding into the cell supernatant indicated that surface phosphorylation is a function of the ecto-protein kinase. The radioactive phosphorylation of pp100 and pp120 which coincides with their biotinylation on 2D-blots can be reversed by mild trypsination of intact cells. Purification and enrichment of pp100 and pp120 were achieved on the basis of radioactivity detection on and isolation from 1D- and 2D-gels. Amino acid sequence analysis performed on tryptic digests of purified ecto-phosphoproteins in most cases showed significant consensus sequences between pp100 and the nuclear RNA-binding protein nucleolin while pp120 sequences proved to be related to hnRNP U, a nucleoplasmic pre-mRNA-binding protein. Immunochemical analysis using anti-nucleolin and anti-hnRNP U antibodies combined with comparative phosphorylation and characterization of the ecto-proteins with authentic nucleolin and hnRNP U further established the close relationship, suggesting surface membrane versions of the nuclear proteins.

Amino Acid Sequence↗
PubMed1994

Inhibitors of the conformational switch involved in CD4 binding by the env glycoprotein gp120 from human immunodeficiency virus type 1 (HIV1).

A 15-residue fragment within the major continuous domain of gp120 from HIV1 that can bind independently to the CD4 receptor conserves the property of behaving as a polarity-triggered conformational switch despite displaying over 50% variability between strains. As this switch behavior (the ability to flip abruptly from beta-sheet to alpha-helix as the medium polarity is lowered past a critical point) is closely linked to CD4-binding ability, it presents a potential strain-independent target for intervention. A number of compounds have been tested for their ability to function as switch inhibitors. All those that displayed switch inhibitory activity also have been shown to act to prevent CD4 binding and/or viral infectivity. In addition, all compounds testing positive as switch inhibitors have certain chemical characteristics in common. The groundwork has thus been established for the design of strain-independent blockers of CD4 binding based on the strategy of switch inhibition.

Amino Acid Sequence↗
PubMed1994

Phorbol ester TPA rapidly prevents activation of p34cdc2 histone H1 kinase and concomitantly the transition from G2 phase to mitosis in synchronized HeLa cells.

HeLa cells in G2 phase are temporarily inhibited and prevented from entering mitosis by treatment with the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate), whereas cells in mitosis are refractory to TPA and divide. In this study the possibility was tested that TPA may interfere with the regulatory cycle of MPF (mitosis promoting factor), the rate-limiting protein kinase for cell division. MPF, consisting of the catalytic subunit p34cdc2 and the regulatory subunit Cyclin B, is known to be activated at the transition from G2 phase to mitosis through dephosphorylation at Tyr15 and to become inactivated after metaphase by proteolysis. Treatment of HeLa cells (synchronized around the G2-M transition) with TPA (10(-7) M) has now been shown to induce an overall decrease of the histone H1 kinase activity associated with anti-p34cdc2 immunoprecipitates after about 20 to 30 min. In metaphase cells, the histone H1 kinase activity of p34cdc2 was shown to remain unaffected by TPA treatment. In cultures enriched in G2 cells neither the amount of p34cdc2 protein nor that of Cyclin B was influenced by TPA. Moreover, the p34cdc2/Cyclin B complex formation was also unaffected. However, p34cdc2 from cultures treated with TPA was more intensely stained by anti-phosphotyrosine antibodies than that of control cells, indicating that TPA treatment probably prevented the tyrosine dephosphorylation required for expression of the histone H1 kinase activity of the complex. The results indicate that TPA treatment of HeLa cultures rapidly stops the G2-M transition because it very rapidly prevents the p34cdc2/Cyclin B complex in G2 cells from developing histone H1 kinase activity.

CDC2 Protein Kinase↗
PubMed1994

Evidence for CKI and CKII at the cell surface.

Ser/Thr-protein kinases at the cell surface (ecto-PK) use physiological concentrations of extracellular ATP for phosphorylation of endogenous cell surface proteins, as well as of soluble protein substrates in the extracellular environment (Kübler et al., 1982, 1989). One abundant ecto-PK component is believed to be a protein kinase CKII since it phosphorylates phosvitin and casein, is sensitive to heparin at low concentrations, and can use both ATP and GTP as cosubstrate. This ecto-PK activity can be detached from the surface of intact cells through interaction with exogenous substrates, a process termed "shedding" (Kübler et al., 1983). This study reports a method for the purification and identification of shedded ecto-PK. Affinity chromatography of the concentrated ecto-PK through a heparin-matrix resolved two phosvitin/casein kinase activities upon elution with a NaCl gradient, termed as peak I and peak II. Relative to the total protein load of the cells employed for ecto-PK shedding, the specific activities increased by a factor of about 10(4) times. The use of peptide substrates specific for CKI and CKII, of ATP and GTP, as well as of antibodies specific for CKII subunit, clearly identified one of the enzymes as a CKI-like entity and the other one as CKII-like. Although the spatial arrangement on the cell surface of the two related ecto-PKs is unknown, their tandem appearance together in the cell supernatant might suggest the possibility of a functional unit.

Adenosine Triphosphate↗
PubMed1993

Primary structure elements responsible for the conformational switch in the envelope glycoprotein gp120 from human immunodeficiency virus type 1: LPCR is a motif governing folding.

The ability to undergo a particular conformational switch on moving from a polar to a less polar environment has been shown to be conserved at the CD4-binding domain of the envelope glycoprotein gp120 from human immunodeficiency virus type 1 despite considerable variability in primary structure and is essential for the process of binding to the T-cell receptor CD4. The elements necessary to the expression of this behavior have been examined in synthetic peptides using circular dichroism and have been found to include a tetrad, LPCR, plus a tryptophan at a position 8 residues C-terminal to it. In the absence of the tryptophan the conformational change from beta-sheet to alpha-helix as medium polarity decreases does not occur abruptly but, rather, in a linear fashion. In the absence of the LPCR tetrad, no transition to alpha-helix occurs even at 100% trifluoroethanol. These two domains interact to control not only the beta-->alpha transition but also both its cooperativity and the critical point on the polar-->apolar gradient at which it occurs. Sequence similarity searches of the protein data banks suggest that an LPCR tetrad, governing the folding behavior of subsequent residues, may occur as a conserved motif in proteins in general. Synthetic peptides with the sequence of non-gp120 proteins that contain the tetrad do in fact display a similar pattern of folding response to decreasing polarity, with a sharp, cooperative transition from beta-sheet to alpha-helix. The LPCR tetrad appears to be a motif that controls secondary structure in a manner supplementary to that predicted by folding algorithms.

Amino Acid Sequence↗
PubMed1993

Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24).

The crystal structure of the porcine heart catalytic subunit of cAMP-dependent protein kinase in a ternary complex with the MgATP analogue MnAMP-PNP and a pseudosubstrate inhibitor peptide, PKI(5-24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine-rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three-dimensional space. The structure supports an in-line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.

Adenosine Triphosphate↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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)

Drug Synergism↗
PubMed1992

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)

Arachidonic Acid↗
PubMed1992

Epidermal-growth-factor-induced production of phosphatidylalcohols by HeLa cells and A431 cells through activation of phospholipase D.

In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.

Diglycerides↗
PubMed1992

Atrial natriuretic peptide is phosphorylated by intact cells through cAMP-dependent ecto-protein kinase.

Recently we demonstrated the presence of cell-surface-located cAMP-dependent protein kinase (ecto-PK A) activity in a number of different cell types [Kübler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J. and Kinzel, V. (1989) J. Biol. Chem. 264, 14549-14555]. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto-PK A of the human atrial natriuretic peptide ANP99-126, a hormone released by cardiac cells. This 28-amino-acid peptide carries the phosphorylation consensus sequence Arg-Arg-Ser-Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on cAMP; cAMP could not be replaced by cGMP. The phosphorylation was inhibited by the PK A-specific inhibitory peptide and increased linearily for up to 15 min and with a Km value of 3-5 microM for ANP. At higher ATP concentrations (greater than 100 microM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)-1 min-1. The rise of intracellular cAMP in HEL30 (an epidermal cell line) after application of the beta-adrenergic receptor agonist isoproterenol led to an approximately three-fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular cAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto-PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate cAMP-dependent phosphorylation of the peptide hormone analogue ANP99-126 by intact cells through ecto-PK A, an intriguing mechanism for post-translational processing of ANP.

Adenosine Triphosphate↗