Gyöngyi Farkas

Phosphatidylinositol 3-kinase Contributes to Erk1/Erk2 MAP Kinase Activation Associated with Hepatocyte Growth Factor-induced Cell Scattering

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.

Inhibition of DNA binding by the phosphorylation of poly ADP-ribose polymerase protein catalysed by protein kinase C

Purified type II (beta) and type III (alpha) protein kinase C phosphorylates highly purified polyADP-ribose polymerase in vitro whereby 2 mols of phosphate are transferred from ATP to serine and threonine residues present in the 36 and 56 kDa polypeptide domains of the polymerase protein. Calf thymus DNA was a non-competitive inhibitor of the protein kinase C catalyzed phosphorylation of polyADP-ribose polymerase. Coincidental with the phosphorylation of the protein the polymerase activity and DNA binding capacity of polyADP-ribose polymerase were inhibited. These in vitro findings may have possible cell biological significance in cellular signal transduction.

Proteolytic activation of protein kinase C in the extracts of cells treated for a short time with phorbol ester

A 10 min treatment of human neutrophils with phorbol 12‐myristate 13‐acetate (PMA) has been reported to induce accumulation of the proteolytically activated Ca2 +/phospholipid‐independent catalytic fragment of protein kinase C in the cytosol of intact cells [(1986) J. Biol. Chem. 261, 4101‐4105]. We investigated the proteolytic conversion of protein kinase C to the Ca2 +/phospholipid‐independent form in the cytosol and membrane fractions of pig neutrophils. The activity of protein kinase C was measured with its specific oligopeptide substrate Ala‐Ala‐Ala‐Ser‐Phe‐Lys‐Ala‐Lys‐Lys‐amide designed previously. In our experiments the short‐term treatment of neutrophils with PMA did not induce the accumulation of the proteolytically activated form of protein kinase C in the cytosol of intact cells. However, treatment of cells with PMA enhanced the limited proteolysis of protein kinase C during the preparation of cell extracts.

Protein kinase C decreases the hepatocyte growth factor-induced activation of Erk1/Erk2 MAP kinases

HGF and phorbol ester induce the scattering of HepG2 cells. Recently, we have reported that the motility and morphological responses that accompany this process require the activation of Erk1/Erk2 MAP kinases, and phosphatidylinositol 3-kinase contributes to the activation of Erk1/Erk2 in HGF-induced cells. The cell scattering-associated appearance of a high-M(r) (>300 kDa) protein pair has also been observed, and has been proven to be a sensitive marker of the intensity of Erk1/Erk2 activation. Our present study demonstrates that in HGF-induced cells protein kinase C and phosphatidylinositol 3-kinase regulate oppositely the expression of these cell scattering-associated proteins. While in phorbol ester-treated cells the sustained activation of protein kinase C is essential for this expression, in HGF-induced cells the inhibition of protein kinase C with bisindolylmaleimide I stimulates the expression. Protein kinase C reduces the HGF-induced phosphorylation of Erk1/Erk2, and in this way it can limit the intensity of Erk1/Erk2-dependent gene-expression

Possible involvement of protein kinase C-ϵ in phorbol ester-induced growth inhibition of human lymphoblastic cells

Abstract Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional α, β and the novel ϵ isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (0.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5–1.0 ng/ml PMA. The translocation of PKC-ϵ was observed within 30 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and an altered phosphorylation state of PKC-ϵ was seen. In contrast, the conventional α and β isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the α and β isoforms showed a significant down-regulation. The preferential alterations in PKC-ϵ observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca 2+ -independent ϵ isoform in mediating the initial events of the phorbol ester stimulated cellular responses.

Cyclic AMP-dependent protein kinase stimulates the phosphorylation of phosphatidylinositol to phosphatidylinositol-4-monophosphate in a plasma membrane preparation from pig granulocytes

Plasma membranes prepared from pig granulocytes were incubated in the presence of [gamma-32P]ATP. The dissociated catalytic subunit of cyclic AMP-dependent protein kinase stimulated the incorporation of 32P into both the protein and lipid fractions of the membrane. The SDS gel-electrophoretic analysis of the 32P-labelled proteins showed that the protein kinase phosphorylated preferentially a 24000-Mr protein, though other 32P-labelled proteins were also detected. 32P-labelled membrane lipids were analysed in two different thin layer chromatographic systems. 32P-labelling was found exclusively in polyphosphoinositides. On addition of the protein kinase the 32P-labelling of both polyphosphoinositides was increased but a higher amount of phosphate was incorporated into phosphatidylinositol-4-phosphate than into phosphatidylinositol-4,5-bisphosphate.

Two components of type III protein kinase C with different substrate specificities and a phospholipid-dependent but Ca2+-inhibited protein kinase in rat brain

The activities of rat brain protein kinase C isoenzymic fractions separated by hydroxyapatite chromatography were measured with histone H1 or the oligopeptide Ala‐Ala‐Ala‐Ser‐Phe‐Lys‐Ala‐Lys‐Lys‐amide as substrates. The oligopeptide was a better substrate than histone H1 for nearly all of the protein kinase C fractions. Two subtractions of type III isoenzyme were resolved (IIIa and IIIb); type IIIb was characterized by a very low histone kinase activity compared to its peptide kinase activity. In some brain extracts a phospholipid‐dependent but Ca2+‐inhibited protein kinase was also observed which was eluted from the hydroxyapatite column between type II and III isoenzymes of protein kinase C.

Phosphorylation of poly(ADP-ribose)polymerase protein in human peripheral lymphocytes stimulated with phytohemagglutinin

Intracellular phosphorylation of poly(ADP-ribose)polymerase was assayed in streptolysin-O-permeabilized human lymphocytes. Whereas 32P incorporation from [gamma-32P]ATP into immunoprecipitated enzyme protein was undetectable in resting cells, significant phosphorylation of this enzyme was observed in lymphocytes treated with phytohemagglutinin. The phosphorylation of poly(ADP-ribose)polymerase in permeabilized cells was not stimulated by phorbol ester, while phorbol-induced phosphorylation of other proteins and of a specific oligopeptide substrate of protein kinase C was observed. However, the specific inhibitory pseudosubstrate peptide of protein kinase C blocked the phosphorylation of poly(ADP-ribose)polymerase induced by phytohemagglutinin. Therefore, a potential role of a member of the protein kinase C family in the phytohemagglutinin stimulated intracellular phosphorylation of poly(ADP-ribose)polymerase is conceivable.

Isoenzyme patterns of protein kinase C and a phospholipid-dependent but Ca2+-inhibited enzyme fraction in the crude extracts of different tissues.

We compared the protein kinase C isoenzyme patterns of crude extracts of rabbit brain, cerebellum, spleen, thymus and human and pig granulocytes. The isoenzymes were fractionated by hydroxyapatite chromatography and the protein kinase C activity was determined with a synthetic oligopeptide substrate. In the extracts of several tissues we also observed an enzyme fraction which was activated by phosphatidylserine+diacylglycerol but inhibited by Ca2+.

The 38 kDa Ca2+/membrane-binding protein of pig granulocytes needs a high Ca2+ concentration to be phosphorylated by protein kinase C

The 38 kDa Ca2+/membrane-binding protein reported to be the dominant substrate of protein kinase C in the extracts of pig neutrophil granulocytes was purified partially and its phosphorylation was investigated. In pig granulocytes type II protein kinase C was the major isoform, while type III isoenzyme was present only as a minor activity. Phosphorylation of the 38 kDa protein was performed with rat brain protein kinase C. Each of the three isoenzymes purified from rat brain was able to phosphorylate this protein, though on the conditions used in our experiments it was phosphorylated most intensively by type II protein kinase C. A phospholipid-dependent, but Ca2(+)-independent, form of protein kinase C was demonstrated with the aid of a synthetic oligopeptide substrate. Phosphorylation of the 38 kDa protein by the Ca2(+)-independent enzyme proceeded exclusively in the presence of Ca2+. The Ca2+ concentration necessary for the phosphorylation of the 38 kDa by either form of protein kinase C was by orders of magnitude higher than that required for the activation of protein kinase C.