Anna Faragó

Protein kinase C in transmembrane signalling

Protein kinase C functions as the transducer of a second messenger, diacylglycerol, and is the major receptor for tumour‐promoting phorbol esters. The enzyme is a family of proteins with closely but distinct structures and individual enzymological properties. Members of the family are differently distributed in particular cell types and limited intracellular locations from lower organisms to mammalian tissues. The enzyme appears to interact with many signalling pathways, and display functions in the processing and modulation of cellular responses to external stimuli. Presumably, each member of the family plays discrete roles in the control of a variety of membrane functions and activation of gene transcription.

Mechanism of Epidermal Growth Factor Regulation of Vav2, a Guanine Nucleotide Exchange Factor for Rac

Vav2 is a member of the Vav family that serves as a guanine nucleotide exchange factor for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the epidermal growth factor (EGF) receptor; however, the mechanism by which Vav2 is activated in EGF-treated cells is unclear. By the means of an in vitro protein kinase assay, we show here that purified and activated EGF receptor phosphorylates Vav2 exclusively on its N-terminal domain. Furthermore, EGF receptor phosphorylates Vav2 on all three possible phosphorylation sites, Tyr-142, Tyr-159, and Tyr-172. In intact cells we also show that Vav2 associates with the activated EGF receptor in an Src homology 2 domain-dependent manner, with Vav2 Src homology 2 domain binding preferentially to autophosphorylation sites Tyr-992 and Tyr-1148 of the EGF receptor. Treatment of cells with EGF results in stimulation of exchange activity of Vav2 as measured on Rac; however, the intensity of the exchange activity does not show any correlation with the level of Vav2 tyrosine phosphorylation. Introducing a point mutation into the Vav2 pleckstrin homology domain or treatment of cells with the phosphatidylinositol 3-kinase inhibitor LY294002 prior to EGF stimulation inhibits Vav2 exchange activity. Although phosphorylation mutants of Vav2 can readily induce actin rearrangement in COS7 cells, pleckstrin homology domain mutant does not stimulate membrane ruffling. These results suggest that EGF regulates Vav2 activity basically through phosphatidylinositol 3-kinase activation, whereas tyrosine phosphorylation of Vav2 may rather be necessary for mediating protein-protein interactions.

Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes.

The Nck adaptor protein links tyrosine kinases or their substrates to proteins containing proline‐rich motifs. Here we show that in activated T cells two tyrosine phosphoproteins of 75 and 120 kDa are co‐immunoprecipitated with polyclonal antibodies against Nck. Analysis of Nck immunoprecipitates with various candidate antibodies revealed that the 75‐kDa tyrosine phosphoprotein is the SH2 domain‐containing leukocyte protein referred to as SLP‐76. In vitro experiments show that the interaction between Nck and SLP‐76 is mediated via the Nck SH2 domain. Using specific phosphopeptides corresponding to the major tyrosine phosphorylation sites of SLP‐76, it was found that Y113 and Y128 phosphopeptides could compete binding of SLP‐76 to the SH2 domain of Nck. In addition, the 120‐kDa tyrosine phosphoprotein was recognized by an antibody raised against Cbl, a proto‐oncogene product that has been previously found to be associated with Nck. These results suggest that the Nck adaptor protein interacts with key signaling molecules and may play an important role in activation of T lymphocytes.

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.

REQUIREMENT OF MULTIPLE SH3 DOMAINS OF NCK FOR LIGAND BINDING

The Nck adaptor protein comprises a single C-terminal SH2 domain and three SH3 domains. The domain structure of Nck suggests that Nck links tyrosine kinase substrates to proteins containing proline-rich motifs. Here we show that Bcr/Abl tyrosine kinase, and three tyrosine phosphorylated proteins (115, 120 and 155 kDa) are co-immunoprecipitated with antibody against Nck from lysates of the human leukaemia cell line K562. By means of affinity purification with the Nck-binding phosphopeptide EPGPY(P)AQPSV, we could also detect the association of endogenous Nck with the proto-oncogene product Cbl. An investigation of the nature of interactions revealed that Bcr/Abl, Cbl, and the 155-kDa tyrosine phosphotyrosine bind exclusively to the SH3 domains of Nck. In addition, none of the single SH3 domains of Nck expressed as glutathione-S-transferase (GST) fusion proteins is able to interact with the proline-rich ligands. However, combined first and second SH3 domains have the capacity to bind Bcr/Abl, Chl and p155. Mutations of conserved tryptophan to Lysine in either of the combined first and second SH3 domains completely abolish ligand binding. These data suggest that cooperation exists among the SH3 domains of Nck for a high-affinity binding of proteins containing proline-rich motifs.

Characterization of interactions of Nck with Sos and dynamin.

One of the adaptor proteins, Nck, comprises a single SH2 domain and three SH3 domains that are important in protein-protein interactions. The in vivo association of Nck with the guanine nucleotide exchange factor Sos has been well documented; however, the precise nature of the interaction is unclear. To determine which SH3 domains are involved in the Nck-Sos interaction, individual SH3 domains of Nck were generated as glutathione S-transferase fusion proteins. We found that exclusively the third (C-terminal) SH3 domain of Nck has the ability to bind to Sos. In addition, in [35S]methionine labelled K562 cells, a 100,000 Mr protein was found to be associated with the third SH3 domain of Nck. This protein was identified as dynamin, a GTP-binding protein that has been implicated in clathrin-coated vesicle formation. Dynamin and Nck co-precipitated when cell lysates were immunoprecipitated with anti-Nck antibody. These data suggest that Nck may contribute to Ras activation and the function of dynamin in membrane trafficking through its third SH3 domain.

Mechanism of arginine biosynthesis in Chlamydomonas reinhardti: II. Purification and properties of N-acetylglutamate 5-phosphotransferase, the allosteric enzyme of the pathway

Abstract 1. 1. The allosteric enzyme of the arginine pathway, N-acetylglutamate 5-phosphotransferase (ATP: N- acetyl - l - glutamate 5-phosphotransferase), which catalyzes the formation of N-acetylglutamate 5-phosphate from N-acetylglutamate and ATP, has been purified 120-fold from cells of the fresh water alga Chlamydomonas reinhardti. 2. 2. The pH optimum of the enzymic reaction is 5.5. The enzyme requires the bivalent ions Mg2+ or Co2+ for activity. The initial velocity data obtained obey the normal Michaelis-Menten kinetics, and do not change in form with variation of pH between 5.5 and 7.5. The apparent Km value of the enzyme at pH 5.5 is 1.5 · 10−2 M for N- acetyl- l -glutamate and 1.6 · 10−3 M for ATP. 3. 3. l -Arginine inhibits the activity of the enzyme, and pH optimum for inhibition is 7.5. The initial velocity data in the presence of l -arginine are compatible with normal Michaelis-Menten kinetics. The kinetic relationship between N- acetyl- l -glutamate and l -arginine is competitive; between ATP and l -arginine it is non-competitive and does not change in form with variation of pH between pH 5.5 and 7.5. The inhibition of enzyme activity by l -arginine is apparently a bimolecular reaction at any pH between pH 5 5 and 7 5. 4. 4. Structural analogs of arginine such as l -canavanine and l -citrulline, in higher concentration as compared with arginine, also inhibit the enzyme activity. 5. 5. Urea at low concentrations reversibly suppresses the inhibitory effect of l -arginine and has no effect on enzyme activity. The kinetic relationship between urea and l -arginine is competitive. 6. 6. l -Arginine protects the enzyme against the inactivating effects of heat and high concentration of urea.

The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca2+-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.

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.