Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.     

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.     

Assessment of biological activity of synthetic fragments of transforming growth factor-alpha

Transforming growth factor-alpha (TGF-alpha) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF-alpha that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF-alpha indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF-alpha receptor. Fragments of TGF-alpha encompassing these hydrophilic domains were prepared by using solid-phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF-alpha fragments indicated that none of the fragments significantly inhibited binding of EGF to the receptor, stimulated DNA synthesis of cells, inhibited EGF-induced DNA synthesis of cells, stimulated growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF-alpha is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF-alpha.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

Real-time kinetic studies on the interaction of transforming growth factor alpha with the epidermal growth factor receptor extracellular domain reveal a conformational change model

Transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF), and related factors mediate their biological effects by binding to the extracellular domain of the EGF receptor, which leads to activation of the receptor's cytoplasmic tyrosine kinase activity. Much remains to be determined, however, about the detailed molecular mechanism involved in this ligand-induced receptor activation. The determination of the binding mechanism and the related thermodynamic and kinetic parameters are of prime importance. To do so, we have used a surface plasmon resonance-based biosensor (the BIAcore) that allows the real-time recording of the interaction between TGF-alpha and the extracellular domain of the EGF receptor. By immobilizing different biotinylated derivatives of TGF-alpha on the sensor chip surface, we demonstrated that the N-terminus of TGF-alpha is not directly involved in receptor binding. By optimizing experimental conditions and interpreting the biosensor results by several data analysis methods, we were able to show that the data do not fit a simple binding model. Through global analysis of the data using a numerical integration method, we tested several binding mechanisms for the TGF-alpha/EGF receptor interaction and found that a conformational change model best fits the biosensor data. Our results, combined with other analyses, strongly support a receptor activation mechanism in which ligand binding results in a conformation-driven exposure of a dimerization site on the receptor.     

Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors

TGF-alpha and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a common receptor. Proteolytic cleavage of the transmembrane TGF-alpha precursor's external domain releases several TGF-alpha species. However, membrane-bound TGF-alpha forms remain on the surface of TGF-alpha-expressing cell lines. To evaluate the biological activity of these forms, we modified two cleavage sites in the TGF-alpha precursor coding sequence, making processing into the 50 amino acid TGF-alpha impossible. Overexpression of this cDNA in a receptor-negative cell line, partial purification, and N-terminal sequence analysis indicate the existence of two transmembrane TGF-alpha forms. These solubilized precursors induce tyrosine autophosphorylation of the EGF/TGF-alpha receptor in intact receptor-overexpressing cells, and anchorage-independent growth of NRK fibroblasts. Cell-cell contact between TGF-alpha precursor-overexpressing cells and cells expressing high numbers of receptors also resulted in receptor activation. These findings suggest a role for transmembrane TGF-alpha forms in intercellular interactions in proliferating tissues.     

Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling

Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor beta1 (TGF-beta1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-beta1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-beta1-induced angiogenesis mainly by compromising cell survival. We established that TGF-beta1 stimulated the expression of TGF-alpha mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-beta1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-beta1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-alpha alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-beta1. We therefore propose that TGF-beta1 promotes angiogenesis at least in part via the autocrine secretion of TGF-alpha, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.     

Synthesis of an active hybrid growth factor (GF) in bacteria: transforming GF-alpha/vaccinia GF fusion protein

A hybrid gene encoding for a polypeptide consisting of the first 33 N-terminal amino acid (aa) residues of transforming growth factor-alpha (TGF-alpha) and a C terminus consisting of 20 aa residues of vaccinia growth factor (VGF) was chemically synthesized and expressed as a fusion protein in Escherichia coli. The primary structure of the hybrid gene product maintained the same positioning of the three disulfide bonds found in each parent molecule thus conserving the first two loop regions of TGF-alpha and the third loop region of VGF. After cleavage with CNBr its renatured biological activity was found to be comparable to TGF-alpha and VGF with respect to binding to the epidermal growth factor receptor, stimulation of DNA synthesis and induction of anchorage-independent growth of NRK cells in the presence of TGF-beta. Thus, we suggest that similar domains can be interchanged within the same family of molecules and equivalent functionality maintained.     

Differential effects of epidermal growth factor, transforming growth factor-alpha, and vaccinia virus growth factor in the positive regulation of IFN-gamma production

We have recently shown that epidermal growth factor (EGF) is capable of positive regulation of IFN-gamma production, thus establishing a functional relationship between nonhemopoietic growth factors and the immune system. In order to study this relationship further, EGF and the EGF-related growth factors transforming growth factor-alpha (TGF-alpha) and vaccinia virus growth factor (VGF), which stimulate cellular proliferation via binding to the EGF receptor, were studied for their functional and physicochemical effects on IFN-gamma production. In contrast to the positive signal of purified murine EGF and recombinant human EGF (both at 1 nM), neither synthetic TGF alpha nor recombinant VGF were capable of restoring competence for IFN-gamma production by Th cell-depleted spleen cell cultures. TGF-alpha and VGF, in molar excess, also failed to block the helper signal of EGF for IFN-gamma production. Thus TGF-alpha and VGF failed to functionally compete for the EGF receptor in the murine spleen cell system. Both TGF-alpha and VGF stimulated murine 3T3 cell proliferation at concentrations similar to those of EGF, and thus their failure to provide help for IFN-gamma production was not due to a general lack of biologic activity. Binding studies with 125I-EGF suggest that the EGF receptor on murine lymphocytes is not constitutively expressed, but inducible by the T cell mitogen staphylococcal enterotoxin A. TGF-alpha did not compete with 125I-EGF for the induced receptor. The data suggest that lymphocytes express a novel inducible EGF receptor that differs from that expressed on cells such as 3T3 fibroblasts.     

The transmembrane domain of TACE regulates protein ectodomain shedding

Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.     

Epidermal growth factor and transforming growth factor-alpha: differential intracellular routing and processing of ligand-receptor complexes.

Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. We have compared the intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha in two different cell systems. We found that TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha in contrast to the mostly degraded EGF. In addition, TGF-alpha did not induce a complete down-regulation of cell surface receptors, as observed with EGF, which is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation, in the case of TGF-alpha. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

Epidermal growth factor-like proteins in breast fluid and human milk

Epidermal growth factor (EGF), and the transforming growth factor-alpha (TGF-alpha) family of proteins, which also bind to the EGF receptor, have been associated with human breast cancer. The total EGF-like proteins were determined by a radioreceptor assay, and TGF-alpha by radioimmunoassay, in human milk and breast fluid samples. The breast fluids were collected by nipple aspiration from healthy premenopausal women. Both the 24 milks and 18 breast fluids assayed contained EGF-like proteins, at concentrations ranging from 32-600 ng/ml (median, 140 ng/ml), and 62-654 ng/ml (median, 205 ng/ml) respectively. Immunoreactive TGF-alpha proteins were detected at higher levels in 21 breast fluids (range, 0-50.0; median 5.1 ng/ml) than in 24 milk samples (range, 0-8.4; median, 0.8 ng/ml).     

Transforming growth factor-alpha binds to the epidermal growth factor receptor in gastric mucosa.

The ability of transforming growth factor-alpha (TGF-alpha) to interact with the gastric mucosal epidermal growth factor (EGF) receptor was investigated using a mucosal membrane preparation. TGF-alpha inhibited specific binding of [125I]EGF to its receptor, but the IC50 for TGF-alpha was at least 100 fold greater than that observed for unlabeled EGF. Cross-linking studies revealed no attachment of [125I]TGF-alpha to EGF-receptor size components, and the unlabeled TGF-alpha was only weakly effective in inhibiting cross-linking of [125I]EGF to the 170 kDa receptor. However, when the cytosolic fraction was reconstituted with the membrane preparation, an enhancement in binding of [125I]TGF-alpha to the EGF receptor occurred in a manner dependent on the concentration of cytosolic protein. Hence the binding characteristics of TGF-alpha to the EGF receptor in gastric mucosa are different from those for EGF.     

Evidence for a novel Cdc42GAP domain at the carboxyl terminus of BNIP-2

We recently identified BNIP-2, a previously cloned Bcl-2- and E1B-associated protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite the lack of homology to previously described catalytic domains of GTPase-activating proteins (GAPs). BNIP-2 contains many arginine residues at the carboxyl terminus, which includes the region of homology to the noncatalytic domain of Cdc42GAP, termed BNIP-2 and Cdc42GAP homology (BCH) domain. Using BNIP-2 glutathione S-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into alpha-helical bundles similar to the topology of the catalytic GAP domain of Cdc42GAP. Alignment of exposed arginine residues in this domain helped to identify Arg-235 and Arg-238 as good candidates for catalysis. Arg-238 matched well to the arginine "finger" required for enhanced GTP hydrolysis in homodimerized Cdc42. Site-directed mutagenesis confirmed that an R235K or R238K mutation severely impaired the BNIP-2 GAP activity without affecting its binding to Cdc42. From deletion studies, a region adjacent to the arginine patch ((288)EYV(290) on BNIP-2) and the Switch I and Rho family-specific "Insert" region on Cdc42 are involved in the binding. The results indicate that the BCH domain of BNIP-2 represents a novel GAP domain that employs an arginine patch motif similar to that of the Cdc42-homodimer.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Expression of transforming growth factor alpha in plutonium-239-induced lung neoplasms in dogs: investigations of autocrine mechanisms of growth

We have previously shown that 47% of radiation-induced lung neoplasms in dogs exhibit increased expression of epidermal growth factor receptor (EGFR). In this study, we investigated the expression of transforming growth factor alpha (TGF-alpha), a ligand for EGFR, to determine if an autocrine mechanism for growth stimulation was present in these tumors. As determined by immunohistochemistry, 59% (26/44) of the lung neoplasms examined had increased expression of TGF-alpha. Expression of TGF-alpha was not related to the etiology of the tumor, e.g., spontaneous or plutonium-induced; however, it was related to the phenotype of the tumor. Statistical analysis of the correlation of EGFR and TGF-alpha expression within the same tumor did not show a positive association; however, specific phenotypes did have statistically significant expression of EGFR or TGF-alpha, suggesting that overexpression of either the ligand or its receptor conferred a growth advantage to the neoplasm. Twenty-seven percent (32/117) of radiation-induced proliferative epithelial foci expressed TGF-alpha, and a portion of those foci (8/32) expressed both EGFR and TGF-alpha. This supports the hypothesis that these foci represent preneoplastic lesions, and suggests that those foci exhibiting increased expression of the growth factor or its receptor are at greater risk for progressing to neoplasia.     

Understanding the catalytic mechanism of GTPase-activating proteins: demonstration of the importance of switch domain stabilization in the stimulation of GTP hydrolysis

Cdc42, a member of the Rho family of GTP-binding proteins, has been implicated in a variety of biological activities, including the organization of the actin cytoskeleton, changes in cell morphology and motility, intracellular trafficking, cell cycle progression, and cellular transformation. The cycling of Cdc42 between its on (GTP-bound) and off (GDP-bound) states is essential for its stimulation of cell growth and transformation, with an important aspect of this cycle being the regulation of the GTP hydrolytic activity of Cdc42 by its GTPase-activating protein (Cdc42GAP). On the basis of the structural determinations of the Cdc42-Cdc42GAP complex, as well as the Ras-RasGAP complex, it has been proposed that an arginine residue provided by the GAP (called the "arginine finger") stabilizes charges developing on the guanine nucleotide during the transition state for GTP hydrolysis and is an important contributor to GAP-stimulated catalysis. However, the 85 kDa regulatory subunit (p85) of the phosphoinositide 3-kinase (PI-3K) is homologous with the Cdc42GAP and contains the essential arginine residue, but is ineffective as a GAP. This argues that the introduction of the arginine finger is insufficient for GAP activity and that the GAP must fulfill an additional function, one possibility being the engagement and stabilization of the conformationally sensitive switch regions of Cdc42. In the study presented here, we have tested this idea by examining three residues within the Cdc42GAP, which are missing in the GAP homology domain of the 85 kDa regulatory subunit (p85) of the PI 3-kinase and are involved in specific interactions with switch domain residues of Cdc42. We show that the mutation of all three residues, as well as individual mutations of each of these residues, yields GAPs that are defective in stimulating GTP hydrolysis. We further demonstrate that the switch I residue tyrosine 32 plays an important role in GAP interactions and in the regulation of both intrinsic and GAP-stimulated GTP hydrolysis. Taken together, these findings indicate that stabilizing the switch domains of GTP-binding proteins is an important part of GAP-stimulated catalysis, and that the inability of p85 to participate in these interactions may at least in part explain its ineffectiveness as a GAP.