Viral and cellular fos proteins are complexed with a 39,000-dalton cellular protein.

The structure of viral and cellular fos gene products and their association with a 39,000-dalton cellular protein (p39) were investigated by using antisera raised against synthetic peptides. The first peptide, termed M, corresponded to amino acids 127 to 152 of the v-fos sequence, a region which is identical in c-fos. The second peptide, termed V, corresponded to the nine C-terminal amino acids of v-fos; this region is not present in c-fos. Rabbit antisera were purified by affinity chromatography against their respective peptides before being used for immunoprecipitation. M peptide antisera precipitated p55v-fos and p55c-fos, whereas V peptide antisera precipitated only p55v-fos. This observation confirms the prediction from nucleotide sequence analysis that these proteins are distinct at their C termini. p39 was precipitated in association with p55v-fos and p55c-fos by M and V peptide antisera. However, V peptide antisera did not precipitate p39 from cells expressing p55c-fos, even though the presence of p39 in such cells was demonstrated with M peptide antisera. Denaturation of cell lysates completely abolished the precipitation of p39, whereas the precipitation of p55v-fos was unaffected. Taken together, the data demonstrate that p39 exists in a complex with p55.     

The structure of the carboxyl terminus of the p21 protein. Structural relationship to the nucleotide-binding/transforming regions of the protein

The carboxyl-terminal region of theras oncogene-encoded p21 protein is critical to the protein's function, since membrane binding through the C-terminus is necessary for its cellular activity. X-ray crystal structures for truncated p21 proteins are available, but none of these include the C-terminal region of the protein (from residues 172–189). Using conformational energy analysis, we determined the preferred three-dimensional structures for this C-terminal octadecapeptide of the H-ras oncogene p21 protein and generated these structures onto the crystal structure of the remainder of the protein. The results indicate that, like other membrane-associated proteins, the membrane-binding C-terminus of p21 assumes a helical hairpin conformation. In several low-energy orientations, the C-terminal structure is in close proximity to other critical locales of p21. These include the central transforming region (around Gln 61) and the amino terminal transforming region (around Gly 12), indicating that extracellular signals can be transduced through the C-terminal helical hairpin to the effector regions of the protein. This finding is consistent with the results of recent genetic experiments.     

The role of the carboxyl-terminal 6 amino acid extension of human TSH beta subunit

The nucleotide sequence of human thyroid stimulating hormone (hTSH) gene can encode a protein of 138 amino acids. However, the mature polypeptide is lacking 6 amino acids of the carboxyl-terminus (C-terminus), suggesting posttranslational cleavage of these residues. To analyze a possible function of these 6 amino acids, we expressed two hTSH beta cDNAs with or without the 6 codons for C-terminal extension, together with alpha subunit cDNA in CHO cells, and determined the amino acid sequence of C-terminus of hTSH beta. hTSH beta propeptides without C-terminal extension were glycosylated, associated with alpha subunit and secreted, as normal propeptides were, and its heterodimer with alpha subunit showed normal TSH bioactivity in FRTL-5 bioassay. These data indicate that the 6 amino acid C-terminal extension is not necessary for the hTSH maturation in the process of the biosynthesis and for its bioactivity.     

An alternative transcript derived from the trio locus encodes a guanosine nucleotide exchange factor with mouse cell-transforming potential

By screening cDNA expression libraries derived from fresh leukemic cells of adult T-cell leukemia for the potential to transform murine fibroblasts, NIH3T3, we have identified a novel transforming gene, designated Tgat. Expression of Tgat in NIH3T3 resulted in the loss of contact inhibition, increase of saturation density, anchorage-independent growth in a semisolid medium, tumorigenicity in nude mice, and increased invasiveness. Sequence comparison revealed that an alternative RNA splicing of the Trio gene was involved in the generation of Tgat. The Tgat cDNA encoded a protein product consisting of the Rho-guanosine nucleotide exchange factor (GEF) domain of a multifunctional protein, TRIO, and a unique C-terminal 15-amino acid sequence, which were derived from the exons 38-46 of the Trio gene and a novel exon located downstream of its last exon (exon 58), respectively. A Tgat mutant cDNA lacking the C-terminal coding region preserved Rho-GEF activity but lost the transforming potential, indicating an indispensable role of the unique sequence. On the other hand, treatment of Tgat-transformed NIH3T3 cells with Y-27632, a pharmacological inhibitor of Rho-associated kinase, abrogated their transforming phenotypes, suggesting the coinvolvement of Rho-GEF activity. Thus, alternative RNA splicing, resulting in the fusion protein with the Rho-GEF domain and the unique 15 amino acids, is the mechanism generating the novel oncogene, Tgat.     

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus.

We analyzed linker insertion mutations throughout the 3' region of the v-fps gene of Fujinami sarcoma virus to identify tyrosine kinase transforming protein (P130gag-fps) determinants that are important for catalysis and transforming activity and, in particular, to define residues that participate in substrate selection. Mutations that encode kinase-active, transformation-defective v-fps alleles were recovered, defining sites in the transforming protein that may normally facilitate kinase-substrate interaction. Additionally, one region within the catalytic domain of the transforming protein (amino acid residues 1012 to 1020) that tolerates peptide insertions without loss of transforming activity was discovered, although the insertion mutations in this region of v-fps exhibited qualitatively abnormal transforming function. Transformed rat cell lines that express these mutations displayed unusual phenotypes, including giant cells and cells with an extremely fusiform shape. Furthermore, the insertion mutations in this region were temperature sensitive, transformed cells assumed a flat morphology, cellular protein phosphotyrosine was reduced, and the kinase activity of the transforming protein was decreased when cells were incubated at 40.5 degrees C. Point mutations that specify the ancestral chicken c-fps sequence in the insertion-tolerant region were also introduced into v-fps. These back mutations led to a modest decrease in kinase activity, decreased tumorigenic potential in chickens, and an unexpected increase in transforming activity in rat cells. These results indicate that the insertion-tolerant region of P130gag-fps influences the biologic activity and thermostability of the kinase.     

Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities.

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation.     

Differential requirements of gag and gamma-actin domains for transforming potential of Gardner-Rasheed feline sarcoma virus.

The oncogene of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encodes the 70-kilodalton protein containing gag(p15), gamma-actin, and fgr domains. To determine the role of these domains in the biological activity of P70gag-actin-fgr, we have constructed in-frame deletion and insertion mutants of GR-FeSV. We found, first, that the gamma-actin region could be deleted without affecting the transforming ability of these constructs, although an insertion mutant in the middle of the gamma-actin domain (map position 671) was partially defective in transformation and specifically had a reduced level of in vitro autophosphorylation activity. Second, mutations affecting the C-terminal third of the gag region appeared to abolish the ability to transform NIH 3T3 cells and autophosphorylation activity. These results suggest that the gamma-actin domain is not essentially required for the transforming activity of GR-FeSV but that it may take part in maintaining the conformational integrity of P70gag-actin-fgr and that the gag(p15) domain might have a critical role in modulating the function of P70gag-actin-fgr.     

Inhibitory effect of myristylation on transrepression by FBR (Gag-Fos) protein.

The myristylated v-fos product, FBR murine sarcoma virus (Gag-Fos) protein, exhibits a lower level of transrepression of the serum response element (SRE) than does c-fos protein (Fos). Mutation of the N-terminal myristylation site in FBR protein restored SRE transrepression. Replacement of N-terminal viral Gag sequences with the Fos N terminus also restored this activity, providing additional evidence that myristylation inhibits transrepression by FBR protein. However, the myristylated Gag domain did not inhibit SRE transrepression when fused to Fos, indicating that myristylation of a fos protein is not by itself sufficient to prevent SRE transrepression and that C-terminal mutation is necessary to inhibit transrepression by N myristylation. Comparison of transfection results with Fos C-terminal deletion mutants and the Fos/FBR chimeric mutant revealed that the FBR C terminus retained the potential for transrepression despite deletion of the normal Fos C terminus, whereas similar Fos deletion mutants did not. These results indicate that both N- and C-terminal mutations are required to inhibit transrepression by FBR protein and that multiple structural mutations accompanied by posttranslational protein modification alter gene regulation by FBR protein.     

The BCR gene encodes a novel serine/threonine kinase activity within a single exon.

Sequences encoded by the first exon of BCR that bind to the ABL SH2 domain are essential for the activation of the ABL tyrosine kinase and transforming potential of the chimeric BCR-ABL oncogene. The normal cellular BCR gene encodes a 160,000 dalton phosphoprotein associated with a serine/threonine kinase activity, but it shows only weak dispersed homologies to protein kinases. p160c-BCR was purified to apparent homogeneity as an oligomer of greater than 600,000 daltons that contains autophosphorylation activity and transphosphorylation activity for several protein substrates. A region containing paired cysteine residues within the 426 amino acids encoded by the first exon of BCR is essential for its novel phosphotransferase activity, which overlaps with the strong SH2-binding regions. The recent demonstration of a GTPase-activating function within the C-terminal portion of BCR suggests that the protein kinase and SH2-binding domains may work in concert with other regions of the molecule in intracellular signalling processes.     

Structural requirements for Arabidopsis beta1,2-xylosyltransferase activity and targeting to the Golgi

Characterization of a beta1,2-xylosyltransferase from Arabidopsis thaliana (AtXylT) was carried out by expression in Sf9 insect cells using a baculovirus vector system. Serial deletions at both the N- and C-terminal ends proved that integrity of a large domain located between amino acid 31 and the C-terminal lumenal region is required for AtXylT activity expression. The influence of N-glycosylation on AtXylT activity has been evaluated using either tunicamycin or mutagenesis of potential N-glycosylation sites. AtXylT is glycosylated on two of its three potential N-glycosylation sites (Asn51, Asn301, Asn478) and the occupancy of at least one of these two sites (Asn51 and Asn301) is necessary for AtXylT stability and activity. Contribution of the N-terminal part of AtXylT in targeting and intracellular distribution of this protein was studied by expression of variably truncated, GFP-tagged AtXylT forms in tobacco cells using confocal and electron microscopy. These studies have shown that the transmembrane domain of AtXylT and its short flanking amino acid sequences are sufficient to specifically localize a reporter protein to the medial Golgi cisternae in tobacco cells. This study is the first detailed characterization of a plant glycosyltransferase at the molecular level.     

Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros.

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was delected; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity.     

Truncation of the human EGF receptor leads to differential transforming potentials in primary avian fibroblasts and erythroblasts.

The transforming capacity of the normal and mutant human EGF receptor (EGFR) was investigated in primary chicken cells. In fibroblasts, both N- and C-terminal truncations resulted in a weak, additive oncogenic activity. However, not even double truncations caused a v-erbB-like phenotype. Upon EGF-binding, on the other hand, both normal and C-terminally truncated EGFRs resembled v-erbB in their fibroblast transforming potential. In erythroblasts, N-terminal truncation was sufficient to induce constitutive self-renewal, which was enhanced by deletion of 32 C-terminal amino acids but abolished by a larger truncation of 202 amino acids. In contrast to the normal EGFR, the receptor lacking 32 C-terminal amino acids resembled v-erbB in conferring erythropoietin independence for spontaneous differentiation to the transformed erythroblasts. Our results indicate that the C-terminal domain of the EGFR is non-essential in fibroblast transformation, but seems to be crucial for both self renewal induction and specificity of receptor function in erythroblasts.     

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.     

Distinctive effects of the carboxyl-terminal sequence of the insulin-like growth factor I receptor on its signaling functions.

We have shown previously that the extracellular sequences of the human insulin receptor (IR) and the insulin-like growth factor I receptor (IGFR) have an inhibitory effect on protein tyrosine kinase (PTK) activity and on the biological functions of their respective Gag-receptor fusion proteins. To study the role of IGFR carboxyl sequence in modulation of the Gag-IGFR PTK and biological activities, five mutants, CM1, CM2, CM3, CM4, and CM5, containing carboxyl deletions of 17, 27, 47, 67, and 88 amino acids (aa), respectively, were constructed from the parental virus UIGFR encoding the Gag-IGFR. Deletion of up to 27 aa had little effect on the cell-transforming and PTK activities of UIGFR. Deletions of 47 aa in CM3 abolished PTK and transforming activities. Surprisingly, a further deletion of 20 aa in CM4 beyond that in CM3 reactivated the kinase and transforming activities. CM5, containing a deletion of 20 aa beyond that in CM4, had only marginal transforming and PTK activities. We conclude that deletion of the carboxyl region of the Gag-IGFR inactivates, instead of activating as in the case with Gag-IR, its transforming activity and the amino acid sequence 1250 to 1310 is essential for PTK and transforming activities. Analysis of the ability of the full-length IGFR and its mutant receptors described above to associate with phosphatidylinositol 3 kinase indicated that the association required PTK activity and tyrosine phosphorylation of the receptors and correlated well with their transforming activities. The carboxyl 88 aa are not essential for the association.     

Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein.

RNA helicase II/Gu (RH II/Gu) is a nucleolar protein that unwinds dsRNA in a 5' to 3' direction, and introduces a secondary structure into a ssRNA. The helicase domain is at the N-terminal three-quarters of the molecule and the foldase domain is at the C-terminal quarter. The RNA folding activity of RH II/Gu is not a mere artifact of its binding to RNA. This study narrows down the RNA foldase domain to amino acids 749-801 at the C-terminus of the protein. Dissection of this region by deletion and site-directed mutagenesis shows that the four FRGQR repeats, as well as the C-terminal end bind RNA independently. These juxtaposed subdomains are both important for the RNA foldase activity of RH II/Gu. Mutation of either repeat 2 or repeat 4, or simultaneous mutation of Lys792, Arg793 and Lys797 at the C-terminal end of RH II/Gu to alanines inhibits RNA foldase activity. The last 17 amino acids of RH II/Gu can be replaced by an RNA binding motif from nucleolar protein p120 without a deleterious effect on its foldase activity. A model is proposed to explain how RH II/Gu binds and folds an RNA substrate.     

Partial identification of carbohydrate-binding sites of a Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

The Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades (MOA) contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three identical subdomains (alpha, beta, and gamma) and a C-terminal domain of unknown function. Here, we investigate the structure-function relationship of MOA to define the number and location of its carbohydrate-binding sites. Based on the sequence alignment of MOA to the ricin B-chain lactose-binding sites, we systematically constructed mutants by site-directed mutagenesis. We have used precipitation and hemagglutination assay for the primary analyses, and surface plasmon resonance for the kinetic analysis. Among amino acid residues at the putative carbohydrate-binding sites, Gln(46) in the alpha subdomain and Trp(138) in the gamma subdomain have been identified to be important amino acid residues directly or indirectly involved in carbohydrate recognition. By surface plasmon resonance, Q46A and W138A were 2.4- and 4.3-fold less active than that of the wild-type MOA (K(a) = 2 x 10(7)), respectively. A double-site mutant (Q46A/W138A) had activity similar to W138A. The C-terminal deletion mutant MOADeltaC showed hemagglutination and precipitation activity, although its binding constant was 12.5-fold less active (K(a) = 1.6 x 10(6)) than that of the wild-type MOA. A C-terminal deletion mutant with mutations at both Gln(46) and Trp(138) (MOADeltaC-Q46A/W138A) was 12,500-fold less active (K(a) = 1.6 x 10(3)) than that of the wild-type MOA. On the basis of this observation, we conclude that both alpha and gamma subdomains are most probably involved in carbohydrate binding, but the beta subdomain appears to be inactive.     

Role of the C-terminus in the biological activity of human interleukin 5

Alterations of the C-terminal amino acid sequence of recombinant human interleukin 5 (rhIL-5) caused significant changes in its biological activity. Removal of eight amino acids from the C-terminus of rhIL-5 by CNBr treatment led to a complete loss of biological activity as determined by the BCL1 cell IgM-inducing assay. Oxidation of Met residue located at C-terminus also resulted in a loss of activity. These results suggest that the C-terminal amino acids of rhIL-5 are crucial for its biological actions.