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To appraise the role of TC21 proteins in corpuscle proliferation, we aboriginal bent the transforming action of both the agrarian blazon and the oncogenic TC21 protein in rodent fibroblasts application a focus accumulation assay. These abstracts additionally included transfections with the H-ras oncogene as absolute control. In acceding with antecedent letters (Chan et al., 1994; Graham et al., 1994), it was begin that the TC21G23V aberrant was as transforming as the animal H-ras oncogene (Figure 1a). The TC21 proto-oncogene was additionally transforming, although several orders of consequence beneath than its oncogenic analogue (Figure 1a and b). The adapted phenotype of beef cogent agrarian blazon TC21 was added absolute by barometer their adeptness to abound in an anchorage-independent manner. To this end, several foci of adapted beef were best randomly, expanded, and able in doughy agar plates. Beef acquired from both TC21- and TC21G23V-induced foci were able of developing colonies beneath those conditions, a aftereffect that confirms the transforming action of the agrarian blazon adaptation of TC21 (data not shown). Beef overexpressing agrarian blazon TC21 protein displayed a abstinent advance advantage in allied cultures back compared with the affectionate corpuscle band (Figure 1c). In addition, they showed a apparent morphological change characterized by the actualization of beef with a polygonal appearance that frequently absolute continued cytoplasmic processes (see Figure 5b). This phenotype was added accentuated in beef cogent the TC21G23V protein, as these beef accomplished actual aerial corpuscle densities afterwards all-encompassing confluency (Figure 1c). Moreover, they additionally displayed a added affecting morphological change characterized by the attendance of fusiform beef with rounded, awful refringent corpuscle bodies (see Figure 5c). The announcement of TC21 and TC21G23V was agnate in these cells, advertence that the phenotypic differences empiric are due to the altered activities of these proteins (Figure 1d).



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(a) Transforming abeyant of linearized DNAs encoding agrarian blazon and oncogenic TC21 proteins. (b) Giemsa staining of a adumbrative transfection acceding conducted in the attendance of genomic DNA either abandoned (Genomic) or in aggregate with linearized plasmids absolute the agrarian blazon (1 μg) and oncogenic (0.5 ng) versions of TC21. (c) Advance curves of affectionate (circles), TC21- (squares) and TC21G23V-transformed (triangles) cells. (d) Announcement of TC21 proteins in NIH3T3 cells. Equal amounts of absolute cellular lysates from affectionate beef (None) and corpuscle clones adapted by agrarian blazon and oncogenic TC21 were submitted to anti-TC21 and anti-actin immunoblots. (e) PC12 beef were transfected with vectors (4 μg each) absolute the neo alternative brand (Neo) abandoned or in aggregate with agrarian blazon and oncogenic TC21 and H-Ras proteins. Alternatively, PC12 were transfected with the neo-containing agent additional a beastly a announcement plasmid encoding the abrasion vav oncogene (pJC12) defective neo sequences. Afterwards transfection, beef were split, and adverse denticulate afterwards 20 canicule of culture

Morphology of affectionate (a) and adapted NIH3T3 beef cogent agrarian blazon TC21 (b), TC21G23V (c), agrarian blazon TC21 additional cytosolic Ras GRF (d), the farnesylated catalytic area of rat Ras GRF (d), and the abrasion Vav oncoprotein (e). All pictures were taken at the aforementioned microscope magnification. Scale bar represents 250 μm

The biological action of TC21 and TC21G23V proteins was additionally advised in PC12 cells, a pheochromocytoma corpuscle band that undergoes adverse aloft the addition of elements of the Ras alleyway (Bar-Sagi and Feramisco, 1985). To this end, these beef were transfected with beastly announcement vectors absolute either the TC21, TC21G23V, H-Ras, or H-RasG12V. As a abrogating control, we fabricated alongside transfections with a beastly announcement agent encoding abrasion vav, an oncogene whose arresting transduction alleyway is absolute of Ras action (Bustelo et al., 1994; Crespo et al., 1997). As illustrated in Figure 1e, the oncogenic forms of TC21 and H-ras were able of inducing neurite accumulation in about bisected of the G418 aggressive colonies 20 canicule afterwards transfection. Interestingly, agrarian blazon TC21 additionally induced neurite outgrowth, although at abundant lower levels than the oncogenic forms of H-ras and TC21 (Figure 1e). In contrast, no cogent neurite accumulation was empiric back PC12 beef were transfected with the H-ras proto-oncogene, the pSV2neo, and the abrasion vav oncogene (Figure 1e). Taken together, these after-effects announce that TC21 and Ras proteins abet agnate phenotypic furnishings back bidding in audible beastly corpuscle types.



The activation of GTPases during arresting transduction processes requires the barter of GDP by GTP molecules, a footfall catalyzed by enzymes accepted as guanosine nucleotide barter factors. As a above-mentioned footfall to analyze the upstream GEFs complex on TC21 activation, we aboriginal evaluated the built-in nucleotide barter bulk of this protein in vitro. To this end, a GST-TC21 admixture protein antiseptic from baculovirus-infected Sf9 beef was loaded at 32°C with [3H]GDP and again incubated in the attendance of unlabeled GTP (100 μM) for the adumbrated periods of time. Surprisingly, the GST-TC21 protein displayed a actual accelerated absolution of [3H]GTP in the absence of any barter factor, assuming a about complete accident of the radiolabeled nucleotide amid 30 – 60 min (Figure 2a). This accelerated barter of nucleotides was commensurable to that empiric with H-Ras in the attendance of the catalytic area of Ras GRF (Shou et al., 1992) (Figure 3a). In contrast, H-Ras protein showed negligible barter ante beneath the aforementioned altitude (Figure 2a). To added characterize this activity, we conducted [3H]GDP absolution assays in the absence of nucleotides or, alternatively, in the attendance of either GTP or GDP. We additionally advised whether this aftereffect could be due to a aerial alternation of the nucleotide bounden armpit by testing the built-in barter ante of TC21 at altered concentrations of MgCl2. As apparent in Figure 2b, a accelerated absolution of nucleotides was empiric behindhand of the nucleotide and the absorption of MgCl2 present in the reaction. However, no absolution of apprenticed GDP was empiric in the absence of aggressive nucleotide (Figure 2b), demonstrating that the GDP absolution begin on TC21 is due to the accelerated barter of guanosine nucleotides occurring in this GTPase. Titration abstracts adumbrated that the built-in nucleotide barter of TC21 occurred at actual low concentrations of unlabeled nucleotide (1 – 5 μM) (Figure 2c and d) and was temperature-dependent (Figure 2d). Taken together, these after-effects announce that TC21, clashing H-Ras, has a actual accelerated about-face bulk of the associated nucleotides in vitro.

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(a) Agrarian blazon TC21 (circles) and H-Ras (squares) were loaded with [3H]GDP and again incubated at 32°C in the attendance of algid GTP for the adumbrated periods of time. At anniversary time-point, nucleotide barter ante were bent application a clarify apathy assays (Schuebel et al., 1998). (b) Nucleotide and MgCl2 annex for TC21 built-in nucleotide exchange. [3H]GDP-loaded TC21 was incubated with no nucleotides (No Adds), GTP, and GDP (100 μM each) with either 1 mM (closed boxes) or 25 mM (open boxes) MgCl2. Barter ante were bent afterwards a 45 min evolution at 32°C. (c) Built-in nucleotide barter of TC21 in the attendance of accretion concentrations of GTP. [3H]DGP-loaded TC21 was submitted for 45 min to barter assays in the attendance of the adumbrated concentrations of algid GTP. (d) Nucleotide barter of TC21 is temperature-dependent. [3H]GDP-loaded TC21 was submitted to barter assays for 45 min in the attendance of the adumbrated concentrations of algid GTP at 32°C (triangles), 25°C (squares) and 4°C (circles)

Since TC21 shows affinity with H-Ras in the arena complex in the alternation with upstream regulators (Boriack-Sjodin et al., 1998), we absitively to investigate whether the nucleotide barter of TC21 could be added by Ras GEFs. To this end, we bent the barter ante of [3H]GDP-loaded TC21 and H-Ras in the attendance or absence of a GST admixture protein absolute the catalytic area of Ras GRF, a Cdc25-related barter agency specific for Ras and R-Ras (Gotoh et al., 1997; Shou et al., 1992). These incubations were performed at 25°C in adjustment to abate the aerial built-in barter ante of TC21 (see Figure 2d). As apparent in Figure 3a (left panel), Ras GRF was able of aesthetic the barter of nucleotides on TC21 alike added calmly than on H-Ras. Ras GRF answer additionally the accelerated assimilation of chargeless [35S]GTP into TC21 molecules ahead loaded with unlabeled GDP molecules (Figure 3b). Titration abstracts showed that the nucleotide barter was detected at molar ratios as low as at 0.6 – 1.2 pmol of Ras GRF/60 pmol of GTPase (Figure 3c), advertence that the action of Ras GRF on TC21 is catalytic in nature. To added authenticate the specificity of these reactions, we conducted in vitro barter reactions with the animal Dbl oncoprotein, a GEF whose catalytic action is directed appear Cdc42 and RhoA GTPases (Cerione and Zheng, 1996). [3H]GDP absolution assays approved that this barter agency was not alive appear TC21 (Figure 3d, larboard panel). As a absolute control, Dbl answer nucleotide barter on Cdc42 (Figure 3d, appropriate panel), acknowledging that this GEF was alive in our assays. Thus, these after-effects announce that the activation of TC21 is apprenticed to associates of the Cdc25 family.

(a) Ras GRF catalyzes nucleotide barter on TC21 and H-Ras. [3H]GDP-loaded TC21 (left panel) and H-ras (right panel) were incubated in the absence (circles) or attendance (squares) of GST-Ras GRF for the adumbrated periods of time. Afterwards anniversary time-point, aliquots were taken in alike and nucleotide barter bent by a clarify apathy technique. (b) Ras GRF facilitates the assimilation of [35S]GTP into GDP-bound TC21. TC21 was preloaded with unlabeled GDP and again incubated for the adumbrated periods of time with 5 μM [35S]GTP-γS in the absence (circles) or attendance (squares) of GST-Ras GRF. (c) The nucleotide barter of Ras GRF assimilate TC21 is concentration-dependent. [3H]GDP-loaded TC21 was incubated either abandoned or in aggregate with the adumbrated amounts of GST-Ras GRF protein. Values represent the allotment of GDP apprenticed to TC21 in the attendance of Ras GRF about to the bulk of GDP apprenticed to TC21 back incubated abandoned for 45 min (value=100%). (d) Lack of barter action of Dbl appear TC21 protein. [3H]GDP-loaded TC21 (left panel) and Cdc42 (right panel) were incubated in the absence (circles) or attendance (squares) of the Dbl oncoprotein for the adumbrated periods of time. At anniversary time-point, the barter action was bent as adumbrated in (a)

To authenticate the physiological acceptation of the TC21/Ras GRF interaction, we aboriginal advised whether Ras GRF could abet the barter of guanosine nucleotides on TC21 in vivo. Since there are not accessible antibodies to TC21 able of blocking its GTPase activity, we absitively to chase alongside the activation of TC21 in vivo by barometer the levels of 32P-labeled GDP apprenticed to TC21 in COS-7 beef afterwards abbreviate incubations with [32P]orthophosphate (Laudanna et al., 1996). These abstracts appear that TC21 had actual aerial levels of nucleotide barter alike in the absence of barter agency (Figure 4a, lane 1). This can be attributed to the aerial levels of built-in barter begin in vitro (see Figure 2). This barter was added added aloft co-expression of TC21 with Ras GRF in COS-7 beef (Figure 4a, lane 2). In acceding with our antecedent in vitro observations, we begin that the built-in ante of nucleotide barter begin for H-Ras were decidedly lower than those empiric for TC21 beneath this in vivo altitude (Figure 4a, lane 3). As expected, co-transfection of Ras GRF led to the activation of nucleotide barter on H-Ras (Figure 4a, lane 4). Western blemish abstracts approved that TC21 and H-Ras were bidding at commensurable levels in those beginning altitude (data not shown).

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(a) Activation of TC21 and Ras by Ras GRF in vivo. COS-7 beef were transfected with agrarian blazon AU5-TC21 (lane 1), agrarian blazon AU5-TC21 additional abounding breadth Ras GRF (lane 2), AU5-H-Ras (lane 3), and AU5-H-Ras additional abounding breadth Ras GRF (lane 4). Afterwards 48 h, beef were fatigued overnight, labeled with 32P-orthophosphate for 20 min, and the assimilation of labeled guanosine nucleotides assimilate TC21 and H-Ras bent by attenuate band chromatography. The advancement of 32P-labeled GDP and the agent (OR) of the chromatography is adumbrated by arrows. (b) Cooperativity of Ras GRF and TC21 in cellular transformation. NIH3T3 beef were transfected with the adumbrated amounts of linearized beastly announcement vectors absolute the catalytic area of rat Ras GRF (without CAAX box) and the TC21 proto-oncogene. (c) Activation of TC21 and Ras in vivo by Ras GRF. Beef were transfected with the catalytic area of rat Ras GRF (without CAAX box, 1 μg), the TC21 proto-oncogene (100 ng), or the H-ras proto-oncogene (500 ng)

It has been ahead apparent that the co-expression of agrarian blazon GTPases with their specific GEFs leads to a synergism in cellular transformation (Bustelo et al., 1994; Schuebel et al., 1998). We advised accordingly whether the co-expression of the catalytic area of Ras GRF and the agrarian blazon TC21 protein could abet in focus accumulation assays. Since the constitutively-active anatomy of Ras GRF (farnesylated at the C-terminus) shows aerial levels of transforming action (Bustelo et al., 1994), we acclimated in these abstracts a beastly announcement agent encoding the cytosolic anatomy of Ras GRF. We accept ahead approved that this protein lacks transforming action unless co-expressed with agrarian blazon Ras (Bustelo et al., 1994). As apparent in Figure 4b, the cytosolic anatomy of the Ras GRF catalytic area finer synergized with TC21, arch to a concentration-dependent access in the cardinal of foci over those acquired back TC21 is transfected alone. This synergism was agnate to that empiric amid Ras GRF and agrarian blazon H-Ras (Figure 4c). As apparent in Figure 5, the TC21/Ras GRF-transformed corpuscle curve (Figure 5d) apparent a morphological transformation identical to those displayed by either TC21G23V – (Figure 5c) or Ras GRF-CAAX-transformed beef (Figure 5e). By contrast, TC21/Ras GRF-transformed beef were absolutely altered in assay from parental, untransformed beef (Figure 5a) and from fibroblasts adapted by the vav oncogene (Figure 5f), a Rac-1-specific GEF (Bustelo, 1996; Crespo et al., 1997). Collectively, these after-effects authenticate that Ras GRF is a bona fide barter agency for TC21.

Activation of Ras leads to its concrete affiliation with Raf proteins, the activation of this serine/threonine kinase, and the consecutive dispatch of MEK (MAPKK) and ERK/MAPK (Marshall, 1996). Like Ras, it has been apparent by others that TC21 induces the activation of the terminal elements of this avenue (Graham et al., 1994). However, antecedent letters accept appropriate that such activation is absolute of Raf action (Graham et al., 1996). In adjustment to analyze the MAPKKK complex in the activation of the MAPK in TC21-transformed cells, we absitively to amend the accessible anatomic alternation amid TC21 and the c-raf-1 proto-oncogene product. This was done by application a TC21/Raf co-immunoprecipitation appraisal in insect cells, a abode acclimated afore to authenticate the alternation amid Ras/Raf proteins (Morrison, 1995). Beneath these conditions, we begin that the co-expression of c-Raf-1 and a GST-TC21 protein in Sf9 beef leads to the able affiliation of these two proteins (Figure 6a, aerial panel). This alternation occurred preferentially back TC21 was in the GTP-bound accompaniment (Figure 6b), advertence that c-Raf-1 binds to TC21 with the aforementioned structural constraints ahead appear for the Ras/Raf interaction.

(a) Affiliation of TC21 with c-Raf-1 in baculovirus-infected Sf9 cells. Corpuscle lysates acquired from Sf9 beef adulterated with the adumbrated combinations of baculovirus were incubated with glutathione-coated chaplet (pull-down) or with anti-c-Raf-1 antibodies (IP). Afterwards bounden and all-encompassing washes, samples were submitted to anti-c-Raf-1 immunoblot analysis. Lower panel, announcement of c-Raf-1 protein in anniversary infection, as bent by anti-Raf immunoblot of c-Raf-1 immunoprecipitates. (b) Bounden of c-Raf-1 to TC21 is nucleotide-dependent. Absolute cellular lysates from c-Raf-1 adulterated Sf9 beef were incubated with either GST or GST-TC21 in the attendance of the adumbrated nucleotides (200 μM). Bounden of c-Raf-1 to these proteins was bent by anti-c-Raf-1 immunoblot. (c) Aerial panel, kinase action of c-Raf-1 in parental, TC21G23V- and H-RasG12V-transformed cells. c-Raf-1 was immunoprecipitated from absolute cellular lysates of NIH3T3 and TC21 and Ras-transformed beef and its action bent as declared in Beginning procedures. Lower panel, levels of autogenous c-Raf-1 in the adumbrated corpuscle lines. Equal amounts of cellular lysates from the adumbrated corpuscle curve were immunoprecipitated with anti-c-Raf-1 antibodies and subjected to anti-c-Raf-1 immunoblot

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We additionally conducted abstracts in vitro to assay whether the TC21 oncoprotein activates Raf proteins in beastly cells. To abode this point, autogenous c-Raf-1 proteins were antiseptic from quiescent parental, TC21G23V-, and RasG12V-transformed NIH3T3 beef by immunoprecipitation with specific anti-c-Raf-1 antibodies, and their kinase activities evaluated by in vitro kinase reactions application an abeyant anatomy of MEK as substrate. As apparent in Figure 6c (upper panel), c-Raf-1 proteins displayed aerial levels of activation in both TC21G23V- and H-RasG12V-transformed cells. Immunoblot assay accepted that these corpuscle curve bidding commensurable levels of autogenous c-Raf-1 protein (Figure 6c, lower panel).

The coexpression of Ras and Raf proteins leads to transformation of NIH3T3 cells, (Cuadrado et al., 1993). Therefore, we articular that if TC21 is additionally an activator of c-Raf-1 in vivo, their co-expression should aftereffect in a added almighty oncogenic response. To assay this hypothesis, we performed focus accumulation assays in NIH3T3 beef application suboptimal concentrations of the TC21 announcement agent either abandoned or with accretion concentrations of a beastly announcement agent encoding the c-raf-1 proto-oncogene (pXRB38) (Bustelo et al., 1994). As apparent in Figure 7a, the overexpression of c-Raf-1 led to an access in the transforming abeyant of TC21 that was already apparent at low concentrations of the co-transfected c-raf-1-encoding agent (Figure 7a). This aftereffect appears to be abandoned specific for TC21 and Ras proteins, as we could not ascertain such accessory acknowledgment aloft co-transfection of agrarian blazon c-Raf-1 with added agrarian blazon GTPases such as Rac-1, RhoA, and Cdc42 (Figure 7b). Taken together, these after-effects announce that c-Raf-1 participates in the arresting transduction alleyway adapted by TC21.

(a) Cooperativity of TC21 and c-Raf-1 in cellular transformation. NIH3T3 beef were transfected with 100 ng of a linearized plasmid encoding agrarian blazon TC21 either abandoned or in aggregate with the adumbrated amounts of a c-raf-1-expression vector. Foci were bent 15 canicule post-transfection. (b) Cooperativity of c-Raf-1 with GTPases of the Ras superfamily. Values represent the cardinal of foci with anniversary GTPase acquired in the absence (open bars) and attendance (closed bars) of the co-transfected c-raf-1-encoding vector

It has been apparent afresh that Ras activation plays a analytical role in the activation of added associates of the MAPK family, including JNK. This aftereffect appears to be advised by the activation of associates of the Rho subfamily of GTP-binding proteins (Coso et al., 1995; Minden et al., 1995). To actuate whether TC21 could actuate this arresting transduction pathway, we performed in vitro kinase reactions with the autogenous JNK present in affectionate and NIH3T3 beef overexpressing either agrarian blazon or oncogenic TC21 proteins. As absolute control, we acclimated the autogenous JNK immunoprecipitated from beef adapted by the abrasion Vav oncoprotein, a accepted activator of the JNK avalanche (Bustelo, 1996; Crespo et al., 1996). These assays approved that JNK was awful alive in beef cogent the TC21G23V protein (Figure 8, larboard panel). In contrast, this kinase was decidedly beneath alive in beef adapted by the agrarian blazon TC21 protein (n=4 experiments) (Figure 8, larboard panel). The levels of JNK activation induced by TC21G23V were as aerial as those angry by the Vav oncoprotein (Figure 8, appropriate panel). Agnate after-effects were acquired in exponentially-growing beef (data not shown). The protein levels of this kinase were agnate in all the corpuscle curve acclimated (Figure 8, lower panels). Collectively, these after-effects adumbrated that TC21, like Ras, is a all-encompassing activator of MAPK cascades.

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