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M F Moran

Publications and source records attributed to M F Moran.

At least 37 records · Page 2Linked to original sources
PubMed1995

CaLB: a 43 amino acid calcium-dependent membrane/phospholipid binding domain in p120 Ras GTPase-activating protein.

CaLB was originally observed as a conserved sequence motif in various calcium-responsive signalling proteins and also in p120 Ras GTPase activating protein (p120GAP) (Clark et al. Cell 65: 1043-1051, 1991). Here we show the 43 residue CaLB motif in p120GAP is a functional protein domain that when expressed as a fusion protein in vitro confers Ca(2+)-dependent interactions with cellular membranes and phosphatidylserine and phosphatidylinositol vesicles. p120GAP, but not a mutant lacking the CaLB domain, associates with the particulate fraction of cells in response to elevated intracellular Ca2+ suggesting that p120GAP may be regulated in part by calcium signals. Addition of the p120GAP CaLB domain was able to restore transforming activity and particulate localization to an otherwise transformation-defective and cytosolic mutant v-Sre tyrosine kinase. The CaLB domain appears to be a prevalent protein module that may affect the molecular interactions and subcellular localization of signalling proteins.

3T3 Cells↗
PubMed1995

Efficient cell transformation by the Tpr-Met oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus.

The receptor for hepatocyte growth factor/scatter factor (HGF/SF) was originally identified as an oncogene, Tpr-Met, which consists of the cytoplasmic tyrosine kinase domain of the HGF/SF receptor (Met) fused down-stream of sequences encoded by the tpr gene. As a consequence of this rearrangement the Tpr-Met fusion oncoprotein is localized to the cytoplasm and is a constitutively activated kinase. To identify signalling pathways important for Tpr-Met-mediated cell transformation we have generated tyrosine to phenylalanine mutants of Tpr-Met that are compromised in their ability to transform Fischer rat 3T3 (Fr3T3) cells in culture. We show that a single tyrosine residue in the carboxy terminus of Tpr-Met (residue 489) is essential for efficient transformation of Fr3T3 cells by this oncoprotein. Mutation of tyrosine 489 to phenylalanine does not affect the exogenous kinase activity of the Tpr-Met oncoprotein toward casein, but it impairs the ability of the mutant protein to bind to and activate phosphatidylinositol 3 kinase in vivo and completely abolishes the in vivo association with the Grb2 adaptor protein as well as the association and/or phosphorylation of an unknown protein of 110 kDa. These data are consistent with a single tyrosine residue in the Tpr-Met oncoprotein being essential for the activation of several signalling pathways which lead to the transformation of Fr3T3 fibroblasts.

1-Phosphatidylinositol 4-Kinase↗
PubMed1993

A murine CDC25/ras-GRF-related protein implicated in Ras regulation.

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development.

Amino Acid Sequence↗
PubMed1993

Steroidogenesis-inducing protein stimulates protein-tyrosine kinase activity in rat Leydig cells.

Steroidogenesis-inducing protein (SIP) isolated from human ovarian follicular fluid stimulates steroid production in Leydig cells, human luteal cells, and rat adrenal cells. In addition, SIP is a potent mitogen that stimulates the proliferation of Leydig cells from immature rats to a greater extent than do LH/hCG and other known growth factors. We have shown previously that the actions of SIP on Leydig cells are independent of the adenyl cyclase-cAMP pathway. In the present study we have explored the possibility that SIP, like many growth factors, may exert its effects by activation of tyrosine kinase(s). Stimulation of Leydig cells isolated from immature rats with SIP resulted in an increase in the tyrosine phosphorylation of proteins that were detected with phosphotyrosine-specific antibodies. The phosphorylation of a 90-kilodalton (kDa) protein band, a 65-kDa protein band, and a doublet at 140 kDa was apparent after 5 min. After 30 min, additional SIP-induced phosphotyrosine proteins were detected at 42, 44, 50, 80, 100, and 150 kDa. In addition to phosphorylation at tyrosine residues, all of the proteins isolated from SIP-stimulated cells were phosphorylated at threonine and serine residues. SIP-induced phosphoproteins recovered with phosphotyrosine-specific antibodies were found to have associated protein-tyrosine kinase activity. The major substrate for this kinase activity in vitro was a 140-kDa protein, similar to one of the major phosphotyrosine-containing proteins induced by SIP treatment of intact cells. These observations suggest that SIP influences gonadal cell steroidogenesis and proliferation, presumably by activating cellular protein-tyrosine kinase(s) as part of a phosphorylation-based signalling pathway.

Animals↗
PubMed1992

Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62.

p62 is a tyrosine phosphoprotein that associates with p21ras GTPase-activating protein (GAP). Purification and cDNA cloning of p62 reveal extensive sequence similarity to a putative hnRNP protein, GRP33. Recombinant human p62 purified from insect Sf9 cells binds to DNA and to mRNA and, like many proteins involved in mRNA processing, recombinant p62 is modified by dimethylation on multiple arginine residues. p62 also binds tightly to p21ras GAP in vitro: this binding depends on phosphorylation of p62 on tyrosine residues and occurs through SH2 regions of GAP. These data suggest that p120-GAP and p62 play a role in some aspect of mRNA processing or utilization and that this role may be regulated by tyrosine phosphorylation, and indirectly, by p21ras.

3T3 Cells↗
PubMed1992

Multiple SH2-mediated interactions in v-src-transformed cells.

The Src homology 2 (SH2) domain is a noncatalytic region which is conserved among a number of signaling and transforming proteins, including cytoplasmic protein-tyrosine kinases and Ras GTPase-activating protein (GAP). Genetic and biochemical data indicate that the SH2 domain of the p60v-src (v-Src) protein-tyrosine kinase is required for full v-src transforming activity and may direct the association of v-Src with specific tyrosine-phosphorylated proteins. To test the ability of the v-Src SH2 domain to mediate protein-protein interactions, v-Src polypeptides were expressed as fusion proteins in Escherichia coli. The bacterial v-Src SH2 domain bound a series of tyrosine-phosphorylated proteins in a lysate of v-src-transformed Rat-2 cells, including prominent species of 130 and 62 kDa (p130 and p62). The p130 and p62 tyrosine-phosphorylated proteins that complexed v-Src SH2 in vitro also associated with v-Src in v-src-transformed Rat-2 cells; this in vivo binding was dependent on the v-Src SH2 domain. In addition to binding soluble p62 and p130, the SH2 domains of v-Src, GAP, and v-Crk directly recognized these phosphotyrosine-containing proteins which had been previously denatured and immobilized on a filter. In addition, the SH2 domains of GAP and v-Crk bound to the GAP-associated protein p190 immobilized on a nitrocellulose membrane. These results show that SH2 domains bind directly to tyrosine-phosphorylated proteins and that the Src SH2 domain can bind phosphorylated targets of the v-Src kinase domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

Amino Acid Sequence↗
PubMed1991

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites.

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.

The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.

Animals↗
PubMed1990

Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors.

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.

ErbB Receptors↗
PubMed1990

Src homology region 2 domains direct protein-protein interactions in signal transduction.

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.

Animals↗
PubMed1989

Radical hysterectomy and tailored postoperative radiation therapy in the management of bulky stage 1B cervical cancer.

Ninety-two patients with Stage IB cervical cancers having a diameter equal to or greater than 4.0 cm were treated with radical surgery. Thirty-two patients received postoperative radiotherapy because of operative findings suggestive of high risk of pelvic recurrence. All 32 irradiated patients were treated with a standard pelvic field. Four patients also received paraaortic radiotherapy, and ten received intravaginal brachytherapy. Postoperative complications were seen in five patients (two nonirradiated, three irradiated). Projected 5-year survival is 79% (71% 5-year survival in irradiated patients, and 83% 5-year survival in nonirradiated patients). Preoperative evaluation of tumor volume was not found to reliably predict histologic high risk factors such as depth of stromal invasion, risk of lymph node metastases, or presence of extracervical/uterine involvement. A primary surgical approach in this group of patients with large-diameter Stage IB cervical cancers allows definition of those patients who might benefit from a combined surgical and radiotherapeutic approach to treatment based on findings at operation.

Adenocarcinoma↗
PubMed1988

Treatment of advanced and recurrent squamous carcinoma of the uterine cervix with constant intraarterial infusion of cisplatin.

Twelve patients with primary or locally recurrent squamous carcinoma of the cervix were treated with constant internal iliac artery infusion of cisplatin (CDDP) via a totally implantable chemotherapy pump. Seven previously untreated patients received standard external and interstitial radiotherapy (RT) in conjunction with CDDP infusion. Five patients with isolated pelvic recurrences received CDDP therapy only. The chemotherapy pump was refilled weekly on an outpatient basis. All nine evaluable patients developed unilateral or bilateral lower extremity pain which responded to dosage reduction. No renal or marrow toxicity was seen. Both of the evaluable patients treated for recurrent tumor died 32 and 60 weeks after initiation of treatment. The seven patients treated primarily with RT + CDDP infusion include one who expired with persistent tumor and one with no evidence of disease (NED) after exenteration for a pelvic recurrence at 48 and 85 weeks respectively. The five remaining patients are NED at 12 to 60 weeks. Constant internal iliac artery infusion of CDDP via an implantable chemotherapy pump can be performed with acceptable toxicity. The preliminary results suggest that further study in previously untreated undergoing concurrent radiotherapy is warranted.

Carcinoma, Squamous Cell↗
PubMed1988

Mutational analysis of a phosphotransfer motif essential for v-fps tyrosine kinase activity.

The catalytic domains of protein-tyrosine kinases such as the P130gag-fps oncoprotein contain the sequence HRDLAARN, followed thirteen residues C-terminal by DFG (P130gag-fps residues 1041-1048 and 1061-1063). These residues define a structural motif conserved among eucaryotic protein kinases (-RD----N, DFG) and shared with several procaryotic 3'aminoglycoside phosphotransferases (H-D----N, D-G). Functional analysis of mutant v-fps proteins employing bacterial and mammalian expression systems indicated that this motif is critical for P130gag-fps kinase activity and oncogenicity. In particular, conservative substitutions of the two invariant aspartates (asp1043, asp1061) with glutamate or asparagine completely eliminated enzymatic activity, suggesting that these residues are essential for catalysis. In contrast, substitution of arg1042 with glutamate decreased but did not eliminate v-fps kinase activity in bacteria. The effects of these and other amino acid substitutions within the phosphotransfer motif and at the nearby autophosphorylation site (tyr1073) of P130gag-fps indicate that these conserved residues are intrinsically essential to the execution or regulation of catalytic activity, and suggest that tight spatial constraints operate within the active centre of the v-fps tyrosine kinase domain.

Amino Acid Sequence↗
PubMed1987

Base excision repair of DNA in gamma-irradiated human cells.

Escherichia coli endonuclease IV was used to incise cellular DNA specifically at apurinic/apyrimidinic (AP) sites prior to alkaline elution to measure the resulting DNA strand breaks. gamma-Irradiated HeLa cells initially contained DNA strand breaks and no AP sites. Upon incubation at 37 degrees C the strand breaks were rapidly repaired and AP sites were generated and subsequently repaired. The transient nature of the AP sites indicates the in vivo operation of a base excision repair pathway whereby damaged bases are removed from DNA by DNA glycosylases to produce AP intermediates that are then substrates for AP endonucleases.

DNA↗
PubMed1986

Effect of cervical spinal cord hemisection and hemidiaphragm paralysis on arterial blood gases, pH, and respiratory rate in the adult rat.

We documented the changes that occur in arterial blood gases, pH, and respiratory rate in rats subjected to a C2 spinal cord hemisection which results in paralysis of the ipsilateral hemidiaphragm. The results indicated that rats compensate for hemidiaphragm paralysis by maintaining arterial pO2 through an increased rate of respiration. This observation is of interest because it implies a form of functional recovery from spinal cord injury which has heretofore not been demonstrated.

Animals↗