S.G. Rhee

Phospholipase C-γ is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro

Abstract Phospholipase C-γ (PLC-γ) was rapidly phosphorylated on tyrosines and serines following PDGF and EGF treatment of quiescent 3T3 mouse fibroblasts and A431 human epidermoid cells, respectively. PDGF treatment increased PLC-γ phosphorylation within 30 sec. This lasted for up to 1 hr, and occurred at high stoichiometry. Continuous receptor occupancy was required to maintain this phosphorylation. Three major sites of tyrosine phosphorylation were detected in PLC-γ, two of which were phosphorylated in EGF-treated A431 cells. Under certain conditions PDGF receptor coimmunoprecipitated with PLC-γ, suggesting that PDGF receptor can phosphorylate PLC-γ directly. Indeed, purified PDGF or EGF receptor phosphorylated purified PLC-γ on tyrosines identical to those phosphorylated in vivo. Tyrosine phosphorylation of PLC-γ was not induced by bombesin, TPA, or insulin. Stimulation of PLC-γ tyrosine phosphorylation and the reported ability of PDGF and EGF to induce phosphatidylinositol turnover in different cells were strongly correlated. We propose that tyrosine phosphorylation of PLC-γ by PDGF and EGF receptors leads to its activation, and a consequent increase in phosphatidylinositol turnover.

EGF induces tyrosine phosphorylation of phospholipase C-II: A potential mechanism for EGF receptor signaling

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.

Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels

Highly fluorescent thiosemicarbazide and hydrazide prepared by reaction of fluorescein isothiocyanate with hydrazine or adipic acid dihydrazide have been used to monitor the presence of carbonyl groups in oxidatively modified proteins. After oxidation, proteins react with these reagents under anaerobic conditions in the dark to yield fluorescent protein conjugates (presumably thiosemicarbazones or hydrazones) which can be visualized as fluorescent bands following electrophoresis (0-4 degrees C) on lithium dodecyl sulfate-polyacrylamide gels. These reagents do not react with unoxidized proteins. The conjugates formed dissociate readily at room temperature but are fairly stable at pH 6-9, 0 degrees C. Current data suggest that these reagents will be useful in the detection and quantitation of oxidatively modified proteins in biological systems.

Two forms of phosphatidylinositol-specific phospholipase C from bovine brain.

Two immunologically distinct forms of phosphoinositide-specific phospholipase C were purified to near homogeneity from bovine brain. Their molecular weights determined by SDS-PAGE are 150,000 (enzyme I) and 145,000 (enzyme II), respectively. Under a nondenaturing condition, purified enzyme I exists mainly in dimeric form and as tetramer to a small extent, while enzyme II is predominantly in monomer, to a small extent as dimer and to a very small extent as trimer. Multiple forms of phosphoinositide-specific phospholipase C in brain tissue described in the literature might be, therefore attributed to the oligomerization of the two independent forms.

Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase β family in NIH 3T3 cells

Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.

Developmental changes in the activities of phospholipase C, 3-kinase, and 5-phosphatase in rat brain

The specific activities of phospholipase C, 3-kinase, and 5-phosphatase were measured in brain homogenates from rats at different developmental stages. The activities of 3-kinase and 5-phosphatase increased by 14-fold and 2-fold, respectively, during development from fetus to adult, while PLC activity remained constant. These results suggest that the metabolism of inositol phosphates varies widely during development. In young brain stimulated by an agonist, it is predictable that Ins(1,4,5)P3 lasts longer and its average concentration is higher than in adult brain. The opposite is true for both the lifetime and concentration of Ins(1,3,4,5)P4. These developmental changes will invariably affect the property of Ca2+ oscillation and the effective time during which cells respond to the Ca2+-mobilizing agonists.

Cyclic and noncyclic inositol phosphates are formed at different ratios by phospholipase C isozymes

The cyclic inositol phosphate content in the product of PLC-beta, gamma, and delta mediated cleavage of three phosphoinositides, PtdIns, PtdIns(4)p, and PtdIns(4,5)P2, was measured under several different experimental conditions. The ratio of cyclic to noncyclic product generally decreased in the order PLC-beta greater than PLC-delta greater than PLC-gamma. For all three enzymes the ratio decreased in the order PtdIns greater than PtdIns(4)P greater than PtdIns(4,5)P2. For all combinations of the three enzymes and three substrates cyclic product content was always higher at pH 5.5 than at pH 7.0. The effect of Ca2+ on the ratio of cyclic to noncyclic was also measured. The ratio remained constant between 0.5 microM and 2 mM for PtdIns. For PtdIns(4)P and PtdIns(4,5)P2, the ratios were unchanged between 0.5 and 500 microM, but increased abruptly at millimolar Ca2+ concentrations.

Direct evidence for separate binding sites for L-Glu and amino acid feedback inhibitors on unadenylylated glutamine synthetase from E. coli.

Abstract Glutamine synthetase from E. coli is modulated by adenylylation of a tyrosine residue on each subunit of the dodecamer, as well as by feedback inhibition. With the stopped-flow fluorometric method, the binding constants for L-Glu, L-Ala, D-Val, and Gly to E1.0—Mg, E ∗∗ , in the absence or presence of ATP or ADP, and NH3 were evaluated at pH 7.0, 15°. Strong synergistic effects between the amino acids and the nucleotide were observed. The fluorescence amplitude observed due to either simultaneous or sequential addition of 2 different amino acids to E or E·ATP indicate that L-Glu can bind to the enzyme simultaneously with L-Ala, Gly and D-Val; L-Ala can coexist with D-Val, Gly or D-Ala. NMR method also shows that L-Glu and L-Ala can bind simultaneously. Therefore, within our experimental conditions, the unadenylylated enzyme possesses allosteric site(s) for the amino acid inhibitors.

New enzymic assays for glutamine synthetase adenylyltransferase and its regulatory protein PIIA

Abstract Glutamine synthetase from some gram-negative bacteria is regulated mainly by a covalent modification cascade. The activity of the enzyme is dependent on its state of adenylylation, which is catalyzed by adenylyltransferase (ATase), whose activity is modulated by a regulatory protein, P II , which exists in two interconvertible forms, P IIA and P IID . In this report, two simple enzymic assays for ATase and P IIA are described. These methods are based on the knowledge that unadenylylated and adenylylated glutamine synthetase exhibit different pH optima in the γ-glutamyl transferase reaction and that l -alanine, a noncompetitive inhibitor, inhibits them to different extents. These assays have been used in the purification of ATase and P IIA and in mechanistic studies on adenylylation. The scope and limitations of these assays are discussed.

New methods for the colorimetric assay of PIID regulatory protein, uridylyltransferase, and uridylyl-removing enzyme in glutamine synthetase cascade

Abstract Adenylylation and deadenylylation of glutamine synthetase (GS) are catalyzed by the same adenylyltransferase (ATase). The ability of ATase to catalyze adenylylation is markedly stimulated by the unmodified form of a regulatory protein, PIIA, whereas its capacity to catalyze deadenylylation is stimulated by the uridylylated form (PIID) of the regulatory protein. Interconversion between PIIA and PIID is catalyzed by uridylyltransferase (UTase) and uridylylremoving enzyme (UR). New colorimetric methods were developed for the assays of PIID, UTase, and UR activities. The PIID activity is monitored by its unique ability to stimulate the ATase catalyzed formation of unadenylylated subunits from adenylylated GS. The inerease of unadenylylated subunits is determined by measuring the γ-glutamyltransferase activity of GS under conditions where the activity of an unadenylylated subunit is about 15 times greater than that of an adenylylated subunit (i.e., at pH 8.0 in the presence of Mn2+). Assays for UTase and UR enzyme are derived by coupling the PIID assay to the UTase and UR reactions. For the UTase reaction, the formation of PIID from PIIA is measured, whereas the decrease in PIID is followed for the UR assay. These assays have been applied to follow the activities of these proteins during their purification procedures, to the mechanistic studies on the deadenylylation reaction, and to determine the activities of these proteins in mutants produced during the genetic study of glutamine synthetase cascade. The problems evolved from these assays are discussed.