CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a SH3-binding protein. The nucleotide sequence of 4.1-kb C3G cDNA contains 3.2-kb open reading frame encoding 121-kDa antibodies against have shown detect 130-140 kDa. carboxyl terminus peptide homologous GNRPs for Ras, expression this...

Whether calcium is the only major intracellular activator of calpain has not yet been established. Here we demonstrate that polyphosphoinositides may play critical roles in activation process mu-calpain. Experiments with purified enzyme, substrate (fodrin), and phospholipids show but other lipids significantly promote action physiological range 10(-7) to 10(-6) M. The effect polyphosphoinositide exerted through both a reduction concentration required for autolysis an increase Vmax...

We developed a monoclonal antibody specific to phosphatidic acid (PA). Using this antibody, novel method quantify trace amounts of PA was achieved. With the method, can be measured in range 20-500 pmol. applied changes levels Balb/c 3T3 cells stimulated by platelet-derived growth factor. contents were very low quiescent and dramatically increased with time up 15 min. On other hand, biphasic diacylglycerol (DG) increase found. The early phase showed transient small peak DG at 30 s followed...

Western blot analysis of Balb/c 3T3 cell lysates by an antibody specific to phosphatidylinositol 4,5-bisphosphate (PIP2) showed that several proteins exist in a PIP2-bound form. Among them, two proteins, 100 and 115 kDa molecular mass, were detected as PIP2 abundant proteins. These identified alpha-actinin vinculin their antibodies. In cells, the cytoskeleton contains PIP2, while cytosol does not. The levels bound decrease response platelet-derived growth factor (PDGF). Similarly, is...

The effect of prostaglandin E, (PGE,), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, Ca2+ mobilization was investigated.The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced release in neutrophils significantly inhibited by PGE,, CAMP.Among them, PGE2 found to be the most potent inhibitor.However, when were stimulated CaZ+ ionophore A23187, such inhibitory these agents less marked.PGE, also suppressed enhanced...

A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced. PLC IV is distinct from the other three types I, II, III) with respect to primary structure tissue distribution its mRNAs. contains two homologous regions included commonly in III most similar II (identity: 50.2%). IV, common a sequence N-terminal regulatory domains nonreceptor tyrosine kinases src-family oncogenes. Using an Escherichia coli expression system, we succeeded...

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in variety of functions, although its activation mechanism vivo is not yet fully understood. To evaluate the signaling pathways PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) (PDGFR), we used series PDGFR "add-back" mutants. Activation mutant (Y40/51) that binds activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation...

The protein ASH (for abundant Src homology), composed of one homology region (SH) 2 and two SH3 domains, was cloned by screening human rat cDNA libraries with an oligonucleotide probe directed to a consensus sequence the SH2 domains. rat-derived peptide comprised 217 amino acids molecular mass 25-28 kDa found be ubiquitous in tissues. A clone also code for part same protein, suggesting that is common rat. acid strikingly similar Sem-5, product nematode cell-signaling gene, most probably...

j a p n Two types of phosphatidylinositol (PI) 3-kinase (PI3K) have been purified 6250-fold (PI3KI) and 1250-fold (PI3KII) from the cytosol fraction bovine thymus.Purified PI3KI PI3KII were found to apparent molecular masses 110 190 kDa, respectively, by gel filtration.On other hand, on sodium dodecyl sulfate-polyacrylamide electrophoresis, while mass was again estimated as showed two bands with 85 suggesting heterodimer form.Peptide mapping analysis demonstrated that 110-kDa protein in same...

Epidermal growth factor (EGF) receptor (EGFR) can induce cell and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with capacity activated to transformation, we truncated human just after residue 1011, removing all three major sites (DEL1011). Further, point mutation was introduced at another site, Tyr-992-->Phe (DEL1011+F992). The wild-type mutant receptors were stably expressed NIH 3T3 variant line that expresses an extremely low...

Ash/Grb-2 is an adaptor protein composed only of Src homology (SH) 2 and SH3 domains that considered to be essential for Ras activation. To clarify the downstream Ash signaling, we investigated Ash-bound proteins. Ash-glutathione S-transferase (GST) fusion proteins were used affinity-purify bound Ash. We found 180-, 150-, 100-, 70-kDa GST-Ash, among which 100 kDa was dynamin by amino acid sequencing Western blot with anti-dynamin antibody. Next, in vitro vivo associations between examined...

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be enhanced in cells transformed by some types oncogenes and tumor viruses, but it is still unknown whether the breakdown PIP2 plays a role oncogene-induced cell proliferation. For examination this problem, monoclonal antibody specifically directed was injected into cells. This bound endogenous so inhibited its intracellular breakdown. Injection ras-transformed cultured presence serum caused reversible...

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract rat brain. The purification procedures consisted chromatography both enzymes on Affi-Gel blue cellulose phosphate, followed by three sequential high performance liquid steps, which were for the two enzymes. resultant preparations each contained homogeneous enzyme with Mr 85,000 as determined sodium dodecyl sulfate-polyacrylamide gel...

The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding signaling proteins containing Src homology 2 domains. We determined sites Grb2/Ash, an domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF mutants in which one autophosphorylation was retained. In intact cells, amount Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines...

The inositol phospholipid metabolism is one of the main pathways signal transduction in cells. We measured activities its key enzymes v-Ha-ras-transformed 208F rat fibroblasts. In ras-transformed clones, incorporation [32P]Pi into intermediates was stimulated. phosphatidylinositol and phosphatidylinositol-4-phosphate kinases transformed clones were about 35-50% more than untransformed cells, indicating increased metabolism. However, activity diacylglycerol kinase their membrane fraction...

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play an important role in the signal transduction of cell growth. It is also suggested that it involved cytoskeletal reorganization. We have found alpha-actinin copurifies with PI from bovine thymus. The antibody against 85 kDa subunit (p85) co-immunoprecipitates lysates NIH/3T3 cells. In addition, anti-alpha-actinin coprecipitates activity. This coprecipitation was observed even after depolymerization actin fibres, suggesting...

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including two proteins, Shc and Grb2, are thought to lie upstream from the ras protooncogene in a signaling cascade. The phosphorylation molecular of these were evaluated PC-12 pheochromocytoma cells treated nerve growth (NGF), epidermal (EGF), insulin. Both NGF EGF stimulated their subsequent association receptors. In contrast, insulin had no effect on...

A tyrosine kinase inhibitor, genistein, caused the subcellular translocation of phosphoinositide-specific phospholipase C (PLC) activity from membrane fractions to cytosolic in rat 3Y1 fibroblasts and their transformants by Rous sarcoma virus, SR-3Y1. The ratio PLC activities associated with those homogenate was greater SR-3Y1 (32.6%) than (20.8%) whereas membrane-associated were strikingly reduced same levels both cells treatment genistein. Moreover, it found immunoblotting analyses that...

After adding insulin to cells overexpressing the receptor, activity of phosphatidylinositol (PI) 3-kinase in anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for receptor tyrosine kinase one mediators signal transduction. However, it is unclear whether or not activated directly phosphorylates PI at residue(s) stimulates specific 3-kinase. We reported previously that 85-kDa subunit purified phosphorylated by vitro. To examine...

Spontaneously hypertensive rats (SHR), which develop hypertension approximately 10 weeks after birth, are considered to provide a good animal model for human essential hypertension. We report here that the abnormal activation of phospholipase C delta 1 (PLC-delta 1) may be one main causes Levels second messengers inositol 1,4,5-trisphosphate and diacylglycerol found higher in aortas 12-week-old SHR than age-matched normotensive Wistar-Kyoto (WKY), although levels 7-week-old SHR, have normal...