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G Carpenter

Publications and source records attributed to G Carpenter.

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

Identification of cadherin tyrosine residues that are phosphorylated and mediate Shc association.

Previously, we reported association of the adaptor protein Shc through its SH2 domain with the cytoplasmic domain of the adhesion molecule cadherin (Xu et al. [1997] J. Biol. Chem. 272:13463-13466). This association was dependent on tyrosine phosphorylation of cadherin and could be modulated by extracellular Ca(2+) and epidermal growth factor in intact cells. There are six tyrosine residues in the cytoplasmic domain of cadherin. To define the tyrosine residue(s) that mediate Shc recognition, site-directed mutagenesis was employed to alter Tyr851 and/or Tyr883 in cadherin, which both conform to a predicted Shc SH2 domain recognition sequence. Mutation of either Tyr851 or Tyr883, but mostly the latter, decreased Src phosphorylation of cadherin and the binding of Shc to cadherin, as determined by Sepharose bead binding and gel overlay assays. Of the two tyrosine residues, Tyr883 is the major Src phosphorylation and Shc binding site. However, the double mutant (Tyr851, 883 Phe) exhibited less Shc association than the single Tyr883 Phe mutant, suggesting a role for Tyr851 also. In addition, the binding of Shc to the cadherin cytoplasmic domain was competitively inhibited by tyrosine phosphorylated peptides containing either Tyr851 or Tyr883, but not by the corresponding non-phosphorylated peptides. Mutation of Tyr851 and/or Tyr883 did not alter the capacity of the cytoplasmic domain of cadherin to bind beta-catenin in vitro. However, Shc binding to cadherin did negatively influence beta-catenin binding to the same molecule.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The role of individual SH2 domains in mediating association of phospholipase C-gamma1 with the activated EGF receptor.

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Enzyme-Linked Immunosorbent Assay↗
PubMed1999

Focal adhesion kinase promotes phospholipase C-gamma1 activity.

The nonreceptor tyrosine kinase FAK ("focal adhesion kinase") is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Here evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-gamma1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-gamma1 in COS-7 cells increases PLC-gamma1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-gamma1. These observations suggest a role for FAK in recruiting PLC-gamma1 to the plasma membrane at sites of cell-matrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-gamma1 Src homology domains and/or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrin-stimulation of PLC-gamma1.

3T3 Cells↗
PubMed1999

Action of phosphatidylinositol-specific phospholipase Cgamma1 on soluble and micellar substrates. Separating effects on catalysis from modulation of the surface.

The kinetics of PI-PLCgamma1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31P NMR spectroscopy. That cIP is also a substrate (Km = approximately 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLCgamma1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca2+ on PI-PLCgamma1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+ activates the enzyme; however, high Ca2+ inhibits PI-PLCgamma1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLCgamma1 by high Ca2+ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.

Animals↗
PubMed1999

The influence of deletion mutations on phospholipase C-gamma 1 activity.

Phospholipase C-gamma1, a substrate for many growth factor receptor and nonreceptor tyrosine kinases, produces second messenger molecules that are elements of signal transduction pathways related to cell proliferation. The influence of deletion mutations, which do not intrude on the domains required for catalytic function, on the basal activity of this enzyme is reported. Removal of the first 74 amino-terminal residues increases phospholipase C activity, while deletion of the carboxy-terminal 81 residues decreases enzyme activity. Deletion of the SH2-SH2-SH3 central region, which separates the two domains (X, Y) responsible for catalytic function, also increases enzymatic activity. Interestingly, addition of a recombinant SH2-SH2-SH3 fragment of phospholipase C-gamma1 to the holoenzyme inhibits its phospholipase activity at pH 7.0, but not at pH 5.0. However, addition of individual SH2 or SH3 domains does not influence activity of the holoenzyme. All three deletion mutants, in contrast to the holoenzyme, are relatively resistant to V8 proteolysis and activation induced by the epidermal growth factor receptor tyrosine kinase, which require, respectively, specific proteolysis and phosphorylation sites within the SH region. This suggests a conformational change is induced in the SH region by deletion at either the amino- or carboxy-terminus.

Animals↗
PubMed1999

Physiological requirement for both SH2 domains for phospholipase C-gamma1 function and interaction with platelet-derived growth factor receptors.

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.

Animals↗
PubMed1998

SH2 domain-mediated activation of phospholipase Cgamma is not required to initiate Ca2+ release at fertilization of mouse eggs.

The initiation of Ca2+ release at fertilization of mammalian eggs requires inositol trisphosphate (Miyazaki et al., 1992, Science 257, 251-255), indicating that an enzyme of the phospholipase C family is probably activated. Because Ca2+ release at fertilization in echinoderm eggs is initiated by SH2 domain-mediated activation of phospholipase Cgamma (Carroll et al., 1997, J. Cell Biol. 138, 1303-1311), we examined the possible role of PLCgamma in initiating Ca2+ release at fertilization in mouse eggs. Both PLCgamma isoforms, PLCgamma1 and PLCgamma2, are present in mouse eggs and sperm, and stimulation of these enzymes in the egg by way of an exogenously expressed PDGF receptor causes Ca2+ release. Recombinant SH2 domains of PLCgamma1 and PLCgamma2 inhibit PLCgamma1 and PLCgamma2 activation by the PDGF receptor, completely preventing Ca2+ release in response to PDGF when injected at an approximately 20- to 40-fold excess over the concentrations of endogenous proteins. However, even at an approximately 100- to 400-fold excess over endogenous protein levels, PLCgamma1 and PLCgamma2 SH2 domains do not inhibit Ca2+ release at fertilization. These findings indicate that Ca2+ release at fertilization of mouse eggs does not require SH2-domain-mediated activation of PLCgamma. However, activation of PLCgamma in the egg by an alternative pathway, or introduction of activated PLCgamma from the sperm, may be important.

Animals↗
PubMed1998

Analysis of platelet-derived growth factor-induced phospholipase D activation in mouse embryo fibroblasts lacking phospholipase C-gamma1.

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1(-/-) cells could be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells was phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells showed Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells was activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-gamma1, PLD activity was restored. The targeted disruption of Plcg1 did not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase was similar in Plcg1(+/+) and Plcg1(-/-) cells. Because increased plasma membrane ruffles occurred in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs.

Animals↗
PubMed1998

Tyrosine phosphorylation and proteolysis. Pervanadate-induced, metalloprotease-dependent cleavage of the ErbB-4 receptor and amphiregulin.

Enhancement of tyrosine phosphorylation in cells by the application of pervanadate, an extremely potent phosphotyrosine phosphatase inhibitor, provokes the rapid metalloprotease-dependent cleavage of ErbB-4, a transmembrane receptor tyrosine kinase. The pervanadate-induced proteolysis occurs in NIH 3T3 cells expressing transfected human ErbB-4 and in several cell lines that express endogenous ErbB-4. One product of this proteolytic event is a membrane-anchored molecule of approximately 80 kDa, which is heavily tyrosine phosphorylated and which possesses tyrosine kinase catalytic activity toward an exogenous substrate in vitro. This response to pervanadate is not dependent on protein kinase C activation, which has previously been demonstrated to also activate ErbB-4 cleavage. Hence, the pervanadate and 12-O-tetradecanoylphorbol-13-acetate-induced proteolytic cleavage of ErbB-4 seem to proceed by different mechanisms, although both require metalloprotease activity. Moreover, pervanadate activation of ErbB-4 cleavage, but not that of 12-O-tetradecanoylphorbol-13-acetate , is blocked by the oxygen radical scavenger pyrrolidine dithiocarbomate. A second phosphotyrosine phosphatase inhibitor, phenylarsine oxide, also stimulates a similar cleavage of ErbB-4 but, unlike pervanadate, is not sensitive to pyrrolidine dithiocarbomate. Last, pervanadate is shown to stimulate the proteolytic cell surface processing of a second and unrelated transmembrane molecule: the precursor for amphiregulin, an epidermal growth factor-related molecule. Amphiregulin cleavage by pervanadate occurred in the absence of a cytoplasmic domain and tyrosine phosphorylation of this substrate.

Amphiregulin↗
PubMed1998

Epidermal growth factor activation of NF-kappaB is mediated through IkappaBalpha degradation and intracellular free calcium.

The transcription factor NF-kappa-B is normally sequestered in the cytoplasm by its inhibitory subunit IkappaB. Most extracellular signals activate NF-kappa-B through a mechanism involving the phosphorylation and proteasome-dependent degradation of IkappaB. EGF activates NF-kappaB in A-431 carcinoma cells, which overexpress EGF receptors and in mouse embryo fibroblasts, which have a normal complement of receptors. Supershift experiments indicate that the NF-kappa-B complexes induced by EGF are composed of p50/p50 homodimers and p65/p50 heterodimers, but not c-rel. EGF stimulation enhances the degradation of IkappaBalpha, but not IkappaBbeta nor an N-terminal deletion mutant of IkappaBalpha. Treatment of cells with a proteasome inhibitor, such as ALLN or MG132, blocks EGF-mediated NF-kappaB activation, indicating that EGF-induced NF-kappa-B activation requires proteasome-dependent IkappaB degradation. Also, Bapta A/M (a cell-permeable chelator of intracellular calcium) blocks EGF-induced NF-kappa-B activation and IkappaBalpha degradation, suggesting a requirement of intracellular free Ca2+ for this growth factor response. Protein kinase C inhibition, in contrast, did not influence EGF activation of NF-kappaB.

Animals↗
PubMed1998

Epidermal growth factor signaling and mitogenesis in Plcg1 null mouse embryonic fibroblasts.

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-gamma1, a ubiquitous tyrosine kinase substrate. Plcg1(-/-) embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1(+/+) embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1(-/-) cells respond equivalently to PLcg1(+/+) cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-gamma1 for many responses to growth factors.

Animals↗
PubMed1997

Constitutive proteolysis of the ErbB-4 receptor tyrosine kinase by a unique, sequential mechanism.

The heregulin receptor tyrosine kinase ErbB-4 is constitutively cleaved, in the presence or absence of ligand, by an exofacial proteolytic activity producing a membrane-anchored cytoplasmic domain fragment of 80 kD. Based on selective sensitivity to inhibitors, the proteolytic activity is identified as that of a metalloprotease. The 80-kD product is tyrosine phosphorylated and retains tyrosine kinase activity. Importantly, the levels of this fragment are controlled by proteasome function. When proteasome activity is inhibited for 6 h, the kinase-active 80-kD ErbB-4 fragment accumulates to a level equivalent to 60% of the initial amount of native ErbB-4 (approximately 10(6) receptors per cell). Hence, proteasome activity is essential to prevent the accumulation of a significant level of ligand-independent, active ErbB-4 tyrosine kinase generated by metalloprotease activity. Proteasome activity, however, does not act on the native ErbB-4 receptor before the metalloprotease-mediated cleavage, as no ErbB-4 fragments accumulate when metalloprotease activity is blocked. Although no ubiquitination of the native ErbB-4 is detected, the 80-kD fragment is polyubiquitinated. The data, therefore, describe a unique pathway for the processing of growth factor receptors, which involves the sequential function of an exofacial metalloprotease and the cytoplasmic proteasome.

3T3 Cells↗
PubMed1997

Interaction of the adaptor protein Shc and the adhesion molecule cadherin.

In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Using several experimental approaches we demonstrate that Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras.

3T3 Cells↗
PubMed1997

Heregulin degradation in the absence of rapid receptor-mediated internalization.

Heregulin receptors are unable to mediate the rapid internalization of bound ligand as demonstrated in cells transfected with chimeric or wild-type ErbB-2, -3, or -4 receptors (Baulida et al., 1996, J. Biol. Chem. 271, 5251-5257; Pinkas-Kramanski et al., 1996, EMBO J. 15, 2452-2467). This observation is now extended to include mammary carcinoma cell lines (SK-BR-3 and MDA-543) which express endogenous ErbB-2 and ErbB-3 receptors. Also, the fate of receptor-bound heregulin is examined. While receptor-bound heregulin is not rapidly internalized, the ligand is subject to a slow process of inactivation and degradation, which requires heregulin incubation at 37 degrees C with cells that express heregulin receptors. The degradation of heregulin is blocked to a significant extent by chloroquine, an inhibitor of endosome fusion with lysosomes, indicating that heregulin is slowly internalized and degraded. However, this process is not sufficiently rapid to produce ligand-dependent down-regulation of heregulin receptors.

3T3 Cells↗
PubMed1997

Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development.

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E9.0. These results clearly demonstrate that PLC-gamma1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

Animals↗
PubMed1997

Mapping of the molecular determinants involved in the interaction between eps15 and AP-2.

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans approximately 80 amino acids. At least three molecular determinants, located at residues 650-660, 680-690, and 720-730, are involved in the binding. AP-2 binds to eps15 through its alpha subunit (alpha-adaptin); in particular, the COOH-terminal region of alpha-adaptin, the so-called alpha-ear, contains the eps15 binding region.

3T3 Cells↗
PubMed1997

Phospholipase C-gamma1: regulation of enzyme function and role in growth factor-dependent signal transduction.

Phospholipase C(gamma)1 (PLC-gamma1), a tyrosine kinase substrate, is a multi-domain molecule that modulates the intracellular levels of the second messenger molecules: Ca2+ and diacylglycerol. Although a wide variety of growth factor receptor tyrosine kinases phosphorylate and activate PLC-gamma1, the biological role and necessity of this signal transduction element in mitogenesis has remained unclear. Recent results, however, point to a more essential role than was suggested by initial studies. Also, biochemical studies have indicated a putative means for the intramolecular repression of PLC-gamma1 activity and provide a means for interpreting activation signals through a derepression mechanism.

Animals↗