Mutational activation of

We conclude that mutational K-ras activation may be an important early event in the pathogenesis of adenocarcinoma of the lung but that amplification of ras genes or mutational activation of H-ras or N-ras does not play a major part in non-small-cell lung cancer. Abstract To define the role of cellular oncogenes in human cancers, we studied the prevalence of mutational activation of ras oncogenes in untreated non-small-cell lung cancer.

Publication types Research Support, Non-U. Journal List Mol Cell Biol v. Mol Cell Biol. Author information Copyright and License information Disclaimer.

Copyright notice. This article has been cited by other articles in PMC. Abstract A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Images in this article Image on p. Image on p. The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus. Nucleic Acids Res. Potential metal-binding domains in nucleic acid binding proteins.

The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. Negative regulation of c-myc transcription involves myc family proteins. Oncogene Res. Replacement of lys in the ATP binding domain of Pgag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus.

EMBO J. A comprehensive set of sequence analysis programs for the VAX. The steroid and thyroid hormone receptor superfamily. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Am J Pathol. Detection of a raf-related and two other transforming DNA sequences in human tumors maintained in nude mice.

Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X mutant, with a specific nucleotide deletion, at very high efficiency. A new technique for the assay of infectivity of human adenovirus 5 DNA.

J Biol Chem. Myristyl amino-terminal acylation of murine retrovirus proteins: an unusual post-translational proteins modification. Actively transcribed genes in the raf oncogene group, located on the X chromosome in mouse and human.

Plasmin cleaves after accessible lysines or, occasionally, arginines. Four major fragments of TyrRS were generated by limited plasmin digestion, and the identity of each fragment was determined by mass spectrometry and N-terminal sequencing Figure 2A left. This analysis established that the four fragments arose from cleavages at three sites, located at K, K and K K is a minor cleavage site located on a long internal loop of the CP1 insertion that splits the Rossmann nucleotide binding fold Figure 2B.

Therefore, because the major cleavage at K and K is within the linker loop, plasmin can liberate mini-TyrRS from the C-domain, and thereby activate the cytokine activities imbedded in TyrRS. B The cleavage sites generated by plasmin magenta and elastase cyan are marked on the structure.

Limited elastase cleavage also generated four major fragments, arising from three cleavages located at V, A and S Figure 2A left and B. Although the sequence specificity of elastase is different than that of plasmin, the three elastase cleavage sites are essentially juxtaposed with those of plasmin. This observation-of the same spatial locations for three distinct cleavage sites with two different proteases-gave strong confidence that the protease probes were primarily revealing structural information and not information that was idiosyncratic to the protease.

Those additional fragments corresponded to YA-specific cleavage sites at K, K and K by plasmin, and L and A by elastase, respectively. With the broken tether that holds the C-domain close to the ELR motif, we imagined that the YA mutation would generate a more extended overall conformation.

We thought that the conformational difference between YA and wild-type TyrRS might be sufficient to be detected by small angle X-ray scattering. These values support the hypothesis that YA is more open, and therefore more extended than, the wild-type enzyme. The positions and magnitudes of the peaks are independent of the D max value used in the analysis. Therefore, these observations further support the idea of a more open conformation for the mutant protein at the level of tertiary structure.

The complex process of angiogenesis involves endothelial cell activation, proliferation, and migration. Previous work established that mini-TyrRS induced chemotaxis of endothelial cells in vitro , and also simulated angiogenesis in vivo [ 17 ].

To test our hypothesis that the YA substitution activates mini-TyrRS-like activity in full-length TyrRS, we first tested our mutant protein for stimulation of endothelial cell proliferation and migration in vitro , and then went on to test its activity in vivo in two animal models. First, we investigated the stimulation of proliferation of bovine aortic endothelial cells BAECs. Endothelial cell proliferation and migration assays.

A YA mutation activated full-length TyrRS for stimulating proliferation of bovine aortic endothelial cells. Cell proliferation was measured in five randomly chosen HPFs by the percentage of cells with positive bromodeoxyuridine BrdU incorporation. B YA mutation activated full-length TyrRS for stimulating migration of human umbilical vein endothelial cells. Migration of untreated monolayers was normalized to 1.

Similar results were observed in an endothelial cell migration assay. In these experiments, wounds were created in monolayers of human umbilical vein endothelial cells HUVECs. Our proteins were added in the media to test for their activities in wound closure by stimulating cell migration. Next, we tested our proteins in vivo in the mouse matrigel Figure 5A and chick chorioallantoic membrane CAM Figure 5B assays for angiogenesis. In the mouse matrigel model, a collagen plug containing PBS or an added protein is injected subcutaneously.

When t here is an added pro-angiogenic factor, blood vessels perfuse into the plug. Upon injection of a fluorescent endothelial-cell binding lectin, the blood vessel density in each plug can easily be quantified by spectrophotometric analysis. Mouse matrigel and chick chorioallantoic membrane angiogenesis assays. A YA mutation activated full-length TyrRS for angiogenesis in the mouse matrigel angiogenesis assay. The blood vessel content in the matrigel plug with PBS control was normalized to 1.

B YA mutation activated the full-length TyrRS for angiogenesis in chick chorioallantoic membrane angiogenesis assay. As expected and as also reported earlier, full-length TyrRS was inactive in this assay. To confirm the results seen in the matrigel assay, we further tested the proteins in the CAM assay. Thus, these data independently support results from the mouse matrigel assay.

We collected and analyzed media at the 6-hour time point of this assay, where we saw that only YA TyrRS, and not wild-type TyrRS, stimulated wound closure. Using western blot analysis with polyclonal anti-TyrRS antibodies, we found that the majority of the wild-type and YA TyrRS proteins were full-length. These results provide no support for the idea that the activity of YA TyrRS is from serendipitous cleavage of the mutant versus the wild-type version of TyrRS.

This observation suggested that the cytokine activation of TyrRS by the YA mutation was not a result of proteolysis. Arrows point to the mini form of TyrRS. Each protein was incubated with a protease, and the time course of each digestion was monitored Figure 6B. Plasmin digested away both full-length enzymes in 2 hours. The C-domain was more resistant to plasmin digestion, so that even at 18 hours some C-domain remained.

This band, which was previously identified as comprised of residues Figure 2A , remained in the digestion mixture for more than 6 hours. As with the plasmin digestion, a fragment slightly smaller than mini-TyrRS remained for at least 6 hours. This fragment was identified as comprising residues Figure 2A. Thus, with both plasmin and elastase digestions, no evidence for an intrinsically labile and protease-susceptible fragility of YA TyrRS could be obtained.

Because of the localized nature of the conformational change, as supported by the protease digestion study, and the subtlety of the overall conformational change suggested by small angle X-ray scattering analysis, we imagined that it was unlikely that the YA mutant would affect the region around the critical heptapeptide of the C-domain, and therefore the chemotaxis activity of the C-domain would not be activated by the YA mutation.

To further explore this question, we tested the C-domain activity in a cell migration assay, where only the C-domain, and not native TyrRS and mini-TyrRS, stimulate migration of monocytes. Thus, by this functional assay, the critical heptapeptide unit in the C-domain appears to still be masked in YA TyrRS.

Monocyte chemotaxis assay. Each protein 0. Chambers was incubated for 3 hours and then migrating cells were counted in five HPFs in each case.

Given that dominant disease-causing mutations occur in genes for at least two human tRNA synthetases [ 3 , 4 ], our work shows how in principle such mutations can occur. That is, for a tRNA synthetase that is also a procytokine, any mutation that serves to bypass the normal cytokine activation pathway such as alternative splicing or proteolysis , like a mutation analogous to the YA allele of the gene encoding TyrRS, could in principle activate the imbedded cytokine activity and confer a constitutive gain-of-function.

Although dominant disease-causing mutations in genes for human tRNA synthetases are known, the alternative functions associated with disease are not well understood. Detailed studies of the aminoacylation activities of various CMT disease-causing mutant GlyRSs, and of a genetically constructed mouse model, have established that CMT disease can occur without defects in aminoacylation activity and, instead, associate the disease to changes in the dimer interface [ 10 , 11 , 30 ].

These changes in the dimer interface, in a way that is not known, may affect an alternative function of GlyRS that is associated with neurogenesis. At least one of these mutant proteins EK appears to be fully active in aminoacylation. Because the mutations are dominant, they are reminiscent of the work presented here, which demonstrates a mutational gain-of-cytokine function. Thus, the dominant mutations connected to CMT disease may be unrelated to the cytokine activation and signaling pathways investigated here.

Still, it remains of interest to test whether the CMT-causing mutations can be linked in any way to the cytokine activities known for human TyrRS and, conversely, whether the YA mutation would give rise to a CMT disease-like condition. In addition to determining whether a gain-of-function mutation in a gene for a human tRNA synthetase can be created, another goal of our work was to understand the mechanism of activation of synthetase procytokines.

Our data show how steric shielding-deshielding of a critical motif imbedded within TyrRS can regulate its cytokine activity.

TrpRS, a close homolog of TyrRS, is activated by alternative splicing or natural proteolysis that removes a vertebrate-specific N-terminal extension of the polypeptide chain [ 20 ]. Thus, this extension is thought to regulate the cytokine activity.

When the extension is removed, the resulting fragment, known as T2-TrpRS, acts through the VE-cadherin receptor on the surface of endothelial cells and triggers the Akt signaling pathway [ 31 ]. The fragment has been used in animal models for macular degeneration and cancer, and in gene therapy applications [ 19 , 32 , 33 ].

Unlike TyrRS, where the ELR motif imbedded in the catalytic domain has been shown to be critical for the ILlike cytokine activity, the determinants in TrpRS needed for cytokine activity have not been identified. However, by analogy with the results presented here, we speculate that activation of the TrpRS procytokine may come from steric deshielding of a critical motif imbedded in the core protein.

In this instance, the shield would be provided by the N-terminal extension. Our work also showed that only subtle changes in structure are needed for cytokine activation. The protease probes Figure 2 and small angle X-ray scattering Figure 3 proved to be sufficiently sensitive to detect the alteration caused by the YA mutation. At the same time, the change in conformation was small enough that TyrRS was not destabilized and subjected to proteolysis Figure 6.

This conformational change has maintained most of the catalytic activity see above. When the essential aminoacylation function and protein stability are both retained, mutations that activate a cytokine function can be vertically transmitted and accumulated in the population, where the mutant alleles may be associated with a non-lethal pathological condition.

That the YA TyrRS example shows how cytokine activation can occur, without disruption of stability or the essential function in protein synthesis, makes plausible the idea that more examples of disease-associated mutations in genes for tRNA synthetases will be identified in the population.

This work addresses the question of the mechanism of how an expanded function of a tRNA synthetase is activated. A proposed model is supported by a series of biological assays and physical characterizations. The results also raise new possibilities for understanding why mutations in genes for tRNA synthetases are causally linked to human diseases. In this connection, the work is original in demonstrating the idea that gain-of-function can be achieved by mutation in these enzymes.

All proteins were expressed with a C-terminal His-tag to facilitate purification. Recombinant proteins were expressed in E. Cells were grown to an OD of 0.