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S J Parsons

Publications and source records attributed to S J Parsons.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mechanism of biological synergy between cellular Src and epidermal growth factor receptor.

Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T1/2 murine fibroblasts that contain kinase-deficient (K-) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR was found to retain its full kinase activity and its ability to activate the adapter protein SHC and extracellular signal-regulated kinase ERK2 in response to EGF, demonstrating that the mitogenic pathway involving phosphorylation of Y845 is independent of ERK2-activation. The application of these findings to the development of novel therapeutics for human cancers that overexpress c-Src and EGFR is discussed.

Amino Acid Substitution↗
PubMed1998

Characterization of human epidermal growth factor receptor and c-Src interactions in human breast tumor cells.

In C3H/10T1/2 murine fibroblasts, overexpression of both c-Src and the human epidermal growth factor (EGF) receptor 1 (HER1) is required for detection of stable complexes between the two molecules and results in hyperactivation of the receptor and synergistic increases in tumor formation in nude mice, as compared with cells that overexpress only one of the pair. Elevated levels or activities of c-Src and HER1 also occur in a subset of later-stage breast cancers, suggesting that interactions between these two molecules could contribute to a more aggressive clinical course. To determine whether stable complexes between c-Src and HER1 occur in human breast cancers under the same conditions as in murine fibroblasts and whether the appearance of such complexes correlates with enhanced signaling through the EGF receptor and increased tumor growth, human breast tumor cell lines and tumor tissues were analyzed for a number of c-Src/HER1-mediated signaling events and tumorigenicity. In a panel of 14 cell lines, 10 overexpressed c-Src, and of these, five contained elevated levels of HER1 and exhibited an EGF-dependent association between HER1 and c-Src. This association was also present in a HER1/c-Src-overexpressing tumor sample from a breast cancer patient. Further analysis of signaling events revealed that phosphorylation of the HER1 substrate, Shc, and its downstream effector, mitogen-activated protein kinase, was increased in EGF-stimulated MDA-MB-468, MDA-MB-231, and BT-549 cells (which overexpress both c-Src and HER1) as compared with MCF7 and ZR-75-1 cells (which only overexpress c-Src). Furthermore, MDA-MB-468 and MDA-MB-231 cells displayed increased tumorigenicity in nude mice. These results support the hypothesis that c-Src/HER1 interactions contribute to tumor progression in certain late-stage breast tumor cells.

Animals↗
PubMed1998

Is a paediatrician needed at all Caesarean sections?

OBJECTIVE: The need for a skilled neonatal resuscitator in the form of a paediatrician or paediatric registrar to attend a vaginal delivery or Caesarean section (CS) is not clearly defined. This study was undertaken in order to ascertain the level of resuscitation a neonate might require dependent on the delivery mode and type of anaesthesia used. METHODOLOGY: We analysed the Tasmanian Obstetric Audit from 1980 to 1989 for the need for intubation and Apgar scores at 1 min of term singleton deliveries by the mode of delivery. RESULTS: The number of singleton term deliveries was 64739. When the data were analysed annually there was a trend for a reduction in the need for intubation in CS deliveries during the first 5 years, although this was not paralleled by an improvement in Apgar scores. Thus the intubation rate data are also presented for the last 5 years of the study. The intubation rate, Apgar score at 1 min of < 4, and Apgar score at 1 min of > or = 4 < 7 for repeat CS under epidural anaesthesia were 0.55% (0.26% for 1985-89) 0.83% and 3.58%, respectively, with relative risks when compared with spontaneous normal vaginal delivery of 1.8 (1.2 for 1985-89), 0.7 and 0.5, respectively. The relative risk of these outcomes was higher than for normal vaginal delivery for all other modes of delivery including repeat CS under general anaesthesia. CONCLUSION: It is concluded that a paediatrician or paediatric registrar is not required to routinely attend repeat CS under epidural anaesthesia, but should be present for repeat CS under general anaesthesia.

Anesthesia, Obstetrical↗
PubMed1998

Effect of butyrolactone I on the producing fungus, Aspergillus terreus.

Butyrolactone I [alpha-oxo-beta-(p-hydroxyphenyl)-gamma-(p-hydroxy-m-3, 3-dimethylallyl-benzyl)-gamma-methoxycarbonyl-gamma-butyrolactone] is produced as a secondary metabolite by Aspergillus terreus. Because small butyrolactone-containing molecules act as self-regulating factors in some bacteria, the effects of butyrolactone I on the producing organism were studied; specifically, changes in morphology, sporulation, and secondary metabolism were studied. Threefold or greater increases in hyphal branching (with concomitant decreases in the average hyphal growth unit), submerged sporulation, and secondary metabolism were observed when butyrolactone I was added to cultures of A. terreus. Among the secondary metabolites whose production was increased by this treatment was the therapeutically important compound lovastatin. These findings indicate that butyrolactone I induces morphological and sporulation changes in A. terreus and enhances secondary metabolite production in a manner similar to that previously reported for filamentous bacteria.

4-Butyrolactone↗
PubMed1998

Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP-p120 RasGAP interaction: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation.

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.

Amino Acid Sequence↗
PubMed1998

Gonadotropin-releasing hormone pulses are required to maintain activation of mitogen-activated protein kinase: role in stimulation of gonadotrope gene expression.

The present study examined the effect of alterations in GnRH signal pattern (pulsatile vs. continuous; pulse frequency) on mitogen-activated protein kinase (MAPK) activity and whether MAPK plays a role in regulating gonadotrope gene expression. Pituitary MAPK activity was measured by immunoblot, using a phospho-specific MAPK antibody, corrected to the amount of total MAPK per sample. In vivo studies were conducted in adult castrate testosterone-replaced male rats (to suppress endogenous GnRH). Animals received pulsatile or continuous GnRH (or BSA-saline for controls) via jugular cannulas. Initial studies revealed that pulsatile GnRH stimulated a dose-dependent rise in MAPK activity (30 ng, 2-fold increase; 100 ng, 4-fold; 300 ng, 8-fold) 4 min after the pulse. The effect of pulsatile vs. continuous GnRH was examined by administering 50-ng pulses (60-min interval) or a continuous infusion (25 ng/min) for 1, 2, 4, or 8 h. Pulsatile GnRH stimulated a 2- to 4-fold rise in MAPK activity (P < 0.05 vs. controls) that was maintained over the 8-h duration. In contrast, continuous GnRH only increased MAPK activity (2- to 3-fold; P < 0.05 vs. controls) for 2 h, with MAPK activity returning to baseline at later time points. The effect of GnRH pulse frequency on MAPK activation was determined by giving GnRH pulses (50 ng) at 30-, 60-, or 120-min intervals for 8 h. Maximal increases (3-fold vs. controls; P < 0.05) were seen after 120-min pulses, with faster (30- to 60-min interval) pulses stimulating 2-fold increases in MAPK activity (P < 0.05 vs. controls and 120-min GnRH pulse group). The role of MAPK activation on gonadotrope (alpha, LHbeta, FSHbeta, and GnRH receptor) gene expression was determined in vitro. Preliminary studies demonstrated that the MAPK inhibitor, PD-098059 (50 microM), completely blocked GnRH-induced increases in MAPK activity in adult male pituitary cells. Further studies revealed that PD-098059 blocked gonadotrope messenger RNA (mRNA) responses to pulsatile GnRH (100 pg/ml, 60-min interval, 24-h duration) in a selective manner, with alpha, FSHbeta, and GnRH receptor (but not LHbeta) mRNA responses being suppressed. These results show that a pulsatile GnRH signal is required to maintain MAPK activation for durations of longer than 2 h, and that slower frequency pulses are more effective. Further, MAPK plays a crucial role in alpha, FSHbeta, and GnRH receptor mRNA responses to pulsatile GnRH. Thus, divergent MAPK responses to alterations in GnRH signal pattern may be one mechanism involved in differential regulation of gonadotrope gene expression.

Animals↗
PubMed1997

Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor.

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.

Animals↗
PubMed1997

Role of c-Src tyrosine kinase in EGF-induced mitogenesis.

c-Src, the prototype of the cytoplasmic, membrane-associated,non-receptor tyrosine kinases, is a co-transducer of mitogenic signals emanating from a number of tyrosine kinase polypeptide growth factor receptors. Examples of such receptors include those that bind the platelet-derived growth factor (PDGF), colony stimulating factor-1 (CSF-1), and epidermal growth factor (EGF). Investigations into the mechanisms by which c-Src contributes to receptor signaling suggest that interactions between the two proteins are bidirectional, i.e., that c-Src can bind, phosphorylate, and activate the receptor, and vice versa. The consequences of these interactions appear to be enhanced phosphorylation of specific substrates. Delineating which cellular proteins are substrates of which tyrosine kinase and determining the consequences of tyrosine phosphorylation on the function of specific substrates are the goals of current investigations. Utilizing the murine C3H10T fibroblast model, in which a panel of wild type and mutant c-Src/EGF receptor overexpressors has been studied for temporal and spatial second messenger responses to EGF, distinctions between substrates of c-Src and the EGF receptor and the effects of tyrosine phosphorylation on substrate function are beginning to emerge. In the 10T model, preferred substrates of c-Src are almost exclusively comprised of those molecules that associate with the actin cytoskeleton or with focal adhesions, such as cortactin, p190RhoGAP, and p130CAS, while preferred substrates of the EGF receptor include the receptor itself, SHC, phospholipase C-gamma and p62DOK. While the major mitogenic signaling pathway is thought to proceed directly from the receptor (through SHC/GRB2/SOS/Ras/Raf/MEK/MAPkinase/Elk1), more evidence is accumulating to suggest that proteins involved in regulating the actin cytoskeleton (such as c-Src substrates) also participate in mitogenesis, either as unique transducers of growth signals and/or as monitors of anti-apoptotic conditions (substratum attachment). How c-Src may contribute to the EGF mitogenic response through tyrosine phosphorylation of or association with its specific substrates is discussed. Cellular Src (c-Src), prototype for a family of intracellular membrane-associated tyrosine kinases, is required for mitogenesis initiated by multiple growth factor receptors, including the receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), colony stimulating factor-1 (CSF-1), and the basic fibroblast growth factor (bFGF). C-Src is also overexpressed and/or activated in many of the same human carcinomas that overexpress members of the EGF receptor (EGFR) family, suggesting that the two types of tyrosine kinases can cooperate during the genesis of human tumors. This review focuses on the role of c-Src in EGF-dependent mitogenesis and tumorigenesis, i.e., on the interactions between c-Src and the receptor and on identification of c-Src substrates, their functions, and the effects of tyrosine phosphorylations on their functions. A synopsis of other mitogenic and signaling systems is also included for comparative purposes.

Animals↗
PubMed1997

Phosphotyrosine phosphatase activity associated with c-Src in large multimeric complexes isolated from adrenal medullary chromaffin cells.

Chromaffin cells, which secrete catecholamines in response to acetylcholine, express high levels of the Src-family tyrosine kinases. These kinases contain protein-protein interaction domains which bind signal transduction proteins that participate in a variety of cellular processes. To determine if signalling proteins bind c-Src in chromaffin cells, we examined c-Src immunocomplexes for co-precipitating proteins. We discovered a phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) activity which associates with specific subcellular pools of c-Src in vivo and which preferentially binds the SH2 (Src homology 2) domain of c-Src in vitro. Known PTPases were not identified by blotting of c-Src immunocomplexes with a panel of anti-PTPase antibodies, suggesting that the PTPase may be a novel family member. The c-Src-PTPase complex is enriched in the plasma membrane fraction and exists in several large complexes, as revealed by gel-filtration analysis. This PTPase activity is altered rapidly following stimulation by secretagogues, decreasing within 30 s and returning to basal levels by 60 s of stimulation. Both the subcellular localization and rapid activity changes suggest that the c-Src-associated PTPase may function in early signalling events emanating from the nicotinic acetylcholine receptor. In support of this is the co-precipitation of a PTPase activity with the nicotinic acetylcholine receptor and co-chromatography of this receptor with one or the c-Src-PTPase complexes.

Adrenal Medulla↗
PubMed1997

A novel negative imaging technique for accurate localization of stainable proteins on complex two-dimensional autoradiograms.

This paper describes a fast, simple, and accurate method for localization of protein antigens in complex two-dimensional (2-D) autoradiograms where the precise position and identity of the protein is required. The method involves the creation of negative images on an autoradiogram through arrangement of image-intensifying screens. By placing a chromogenically stained 2-D gel or blot between the intensifying screen and the film, photons emitted from the intensifying screen are obstructed by the stained spots, thus creating a negative image on the film. The technique can be used in autoradiograms of proteins labeled with either 32P or (125)I radioisotopes. The technique permits analysis of radiolabeled, gold-stained and immunoreacted proteins on a single film and offers versatility by combining analysis of total protein patterns with specific identification of radiolabeled and/or immunoreacted protein spots. The technique is especially useful when selecting a subset of specifically radiolabeled proteins from the total protein pattern in 2-D gels or membranes for microsequencing.

Animals↗
PubMed1997

Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells.

Both the Ca2+/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca2+ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca2+ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC50 1-5 microM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC50 approximately 10 microM) than that induced by nicotine (IC50 approximately 30 microM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca2+ ionophore and the participation of both PKC and MEK/MAPK in optimal, secretion induced by nicotine.

Adrenal Medulla↗
PubMed1996

Activation of Fyn tyrosine kinase upon secretagogue stimulation of bovine chromaffin cells.

Adrenal medullary chromaffin cells derive from the neural crest during embryogenesis and differentiate into dedicated secretory cells that release catecholamines in response to acetylcholine in vivo or nicotinic agonists in vitro. Previous studies have indicated that tyrosine kinases participate in early secretagogue-induced events in these cells and are required for exocytosis. Abundant levels of the cytoplasmic tyrosine kinases, c-Src and c-Yes, have been detected in chromaffin cells, thereby implicating them as kinases relevant to these events. However, c-Src has been found to undergo a decrease in activity following secretagogue-stimulation, and c-Yes appears to exist in a constituitively low activity state, suggesting that other tyrosine kinases are involved. Furthermore, other members of the Src family of tyrosine kinases have been implicated as playing roles in secretion in a variety of cell types. Therefore, we sought to determine if other Src family members were present in chromaffin cells, and if so, to examine them for subcellular localization and changes in activity following treatment with nicotinic agonists. To this end, antibodies for Fyn, Lck, Lyn, and Fgr were assembled and used in immunoprecipitation, in vitro autokinase, and Western immunoblotting assays. Of these four kinases, only Fyn was found to be expressed at detectable levels. Differential centrifugation studies revealed that Fyn resides predominantly (> 95%) in the crude plasma membrane fraction and undergoes nicotinic-and carbachol-induced activation. This activation is reduced by the nicotinic antagonist, mecamylamine, is not elicited by muscarine, and is dependent upon the presence of extracellular Ca2+. These results suggest that Fyn is involved in signalling through the nicotinic receptor and may be one of the relevant kinases responsible for at least some of the tyrosine phosphorylations detected after stimulation.

Acetylcholine↗
PubMed1996

Phospholipase A2 and protein kinase C contribute to myofilament sensitization to 5-HT in the rabbit mesenteric artery.

1. Calcium (Ca2+, 0.1-100 microM) stimulated concentration-dependent contractions in small strips from the rabbit mesenteric artery in which the smooth muscle cells had been permeabilized with Staphylococcus aureus alpha-toxin. 2. 5-Hydroxytryptamine (5-HT) and phenylephrine, each in the presence of 10 microM guanosine 5'-triphosphate (GTP), concentration-dependently stimulated additional contractions in strips sub-maximally contracted by the presence of a buffered concentration of calcium (0.3 microM). All the additional contraction was abolished with the selective inhibitor of protein kinase C, Ro 31-8220 (10 microM). 3. Quinacrine (10-50 microM), an inhibitor of phospholipase A2, selectively inhibited the sensitization to 5-HT, but did not alter the sensitization to either phenylephrine or GTP. 4. Myofilament sensitization to calcium was mimicked by exogenous arachidonic acid (300 microM, in the presence of indomethacin, miconazole and BW755c) and the stable analogue of arachidonic acid, 5,8,11,14-eicosatetrayonic acid (ETYA, 100 microM), and in both cases did not require the additional presence of GTP. Ro 31-8220, but not quinacrine, reduced the sensitization to arachidonic acid by around 30%. 5. These results indicate that G protein-linked myofilament sensitization to calcium in the mesenteric artery that follows the activation of 5-HT receptors, but not alpha 1-receptors, involves phospholipase A2. The sensitization stimulated by each of these different receptors, and a component of the response to arachidonic acid, also appears to involve the activation of protein kinase C.

Animals↗
PubMed1996

Tyrosine kinases are required for catecholamine secretion and mitogen-activated protein kinase activation in bovine adrenal chromaffin cells.

Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by > 90% in cells stimulated by nicotine, 55 mM KCl, or the Ca2+ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie downstream of Ca2+ influx and upstream of MEK activation. This Ca(2+)-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca(2+)-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.

Adrenal Glands↗
PubMed1995

Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein.

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.

Animals↗