G. Steven Martin

The hunting of the Src

The non-receptor tyrosine kinase Src is important for many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first proto-oncogene to be discovered in the vertebrate genome. Src has been important, not only as an object of study in itself, but also as an entry point into the molecular genetics of cancer.The non-receptor tyrosine kinase Src is important for many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first proto-oncogene to be discovered in the vertebrate genome. Src has been important, not only as an object of study in itself, but also as an entry point into the molecular genetics of cancer.

A prudent path forward for genomic engineering and germline gene modification

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed Genome engineering technology offers unparalleled potential for modifying human and nonhuman genomes. In humans, it holds the promise of curing genetic disease, while in other organisms it provides methods to reshape the biosphere for the benefit of the environment and human societies. However, with such enormous opportunities come unknown risks to human health and well-being. In January, a group of interested stakeholders met in Napa, California (1), to discuss the scientific, medical, legal, and ethical implications of these new prospects for genome biology. The goal was to initiate an informed discussion of the uses of genome engineering technology, and to identify those areas where action is essential to prepare for future developments. The meeting identified immediate steps to take toward ensuring that the application of genome engineering technology is performed safely and ethically.

A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCζ signaling and cell transformation

BACKGROUND Rac and Cdc42 are members of the Rho family of small GTPases. They modulate cell growth and polarity, and contribute to oncogenic transformation by Ras. The molecular mechanisms underlying these functions remain elusive, however. RESULTS We have identified a novel effector of Rac and Cdc42, hPar-6, which is the human homolog of a cell-polarity determinant in Caenorhabditis elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKCzeta, and forms a stable ternary complex with Rac1 or Cdc42 and PKCzeta. This association results in stimulation of PKCzeta kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKCzeta-dependent pathway distinct from the pathway mediated by Raf. CONCLUSIONS These findings indicate that Rac/Cdc42 can regulate cell growth through Par-6 and PKCzeta, and suggest that deregulation of cell-polarity signaling can lead to cell transformation.

Structure and Function of Cdc6/Cdc18: Implications for Origin Recognition and Checkpoint Control

Cdc6/Cdc18 is a conserved and essential component of prereplication complexes. The 2.0 A crystal structure of an archaeal Cdc6 ortholog, in conjunction with a mutational analysis of the homologous Cdc18 protein from Schizosaccharomyces pombe, reveals novel aspects of Cdc6/Cdc18 function. Two domains of Cdc6 form an AAA+-type nucleotide binding fold that is observed bound to Mg.ADP. A third domain adopts a winged-helix fold similar to known DNA binding modules. Sequence comparisons show that the winged-helix domain is conserved in Orc1, and mutagenesis data demonstrate that this region of Cdc6/Cdc18 is required for function in vivo. Additional mutational analyses suggest that nucleotide binding and/or hydrolysis by Cdc6/Cdc18 is required not only for progression through S phase, but also for maintenance of checkpoint control during S phase.

Transformation by Rous sarcoma virus: A cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.

Cell signaling and cancer.

During the course of tumor progression, cancer cells acquire a number of characteristic alterations. These include the capacities to proliferate independently of exogenous growth-promoting or growth-inhibitory signals, to invade surrounding tissues and metastasize to distant sites, to elicit an angiogenic response, and to evade mechanisms that limit cell proliferation, such as apoptosis and replicative senescence. These properties reflect alterations in the cellular signaling pathways that in normal cells control cell proliferation, motility, and survival. Many of the proteins currently under investigation as possible targets for cancer therapy are signaling proteins that are components of these pathways. The nature of these signaling pathways and their roles in tumorigenesis were the subject of a recent Beatson International Cancer Conference.

Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function.

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

The road to Src

More than a quarter of a century has elapsed since the identification of the c-src proto-oncogene. During that period, we have learned that cancer arises as the result of mutations in proto-oncogenes and tumor suppressor genes, and we are now seeing the first fruits of these discoveries, in the form of targeted therapies directed against activated tyrosine kinases such as Bcr-Abl, c-Kit and the EGF receptor. But the discovery of the c-src proto-oncogene was in turn based on decades of study on an avian RNA tumor virus, Rous sarcoma virus (RSV). Here I review the work that led up to the identification of the RSV transforming gene and its protein product, and how this information in turn led to the discovery of cellular Src.

Glycoprotein components of avian and murine RNA tumor viruses.

Abstract Two glycoprotein components containing glucosamine, galactose, and fucose were isolated from different strains of Rous sarcoma virus (RSV) and from Rauscher mouse leukemia virus (MLV). The molecular weight of the major glycoprotein of RSV was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) to be between 90,000 and 105,000 daltons, depending on the strain of RSV. The minor glycoprotein had an approximate molecular weight of 37,000. Both glycoproteins have higher molecular weights than the proteins of the group-specific antigen of avian tumor viruses. The glycoproteins represent between 10% and 20% of the radioactive protein of purified virus. Disruption of RSV with SDS yields glycoprotein components with sedimentation coefficients between 4 S and 2 S. The glycoprotein derived from Tween 20 disrupted virus was obtained in a 4-2 S form and in an 8 S form. The 8 S component consists predominantly of a multimeric aggregate of the two viral glycoprotein components, and some group-specific antigen. Because both the 8 S and the 4-2 S glycoprotein derived from Tween 20 disrupted RSV specifically inhibited virus neutralizing antibody and because the glycoproteins of different strains of RSV had different electrophoretic mobilities, it was concluded that the glycoproteins of avian tumor viruses are part of or identical with the viral type- or subgroup-specific antigen.

Protein phosphorylation at tyrosine is induced by the v-erbB gene product in vivo and in vitro

The v-erbB gene product of avian erythroblastosis virus (AEV) has extensive homology with the receptor for epidermal growth factor (EGF). We report here that chicken embryo fibroblasts (CEF) transformed by AEV show enhanced tyrosine phosphorylation of a number of cellular polypeptides, including the 36 kd protein, which is phosphorylated in avian sarcoma virus-transformed fibroblasts, and the 42 kd protein, which is phosphorylated in mitogen-stimulated cells. CEF infected by AEV mutants with deletions in v-erbA showed enhanced tyrosine phosphorylation, whereas CEF infected by mutants with deletions in v-erbB did not. When membranes from AEV-transformed cells were incubated with gamma-32P-ATP, both the v-erbB gene product and the 36 kd cellular protein became phosphorylated at tyrosine. These results indicate that the v-erbB protein induces tyrosine phosphorylation in vivo and in vitro, and suggest that, like the EGF receptor, it possesses tyrosine-specific protein kinase activity.