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http://www.unc.edu/courses/2009spring/envr/740/001 slide 14 Begin 02/26/09 SIGNAL TRANSDUCTION In order to prepare for discussion of the first example of transformation of a proto-oncogene to an oncogene, we will review signal transduction. That is because the single study definitively connecting chemically induced mutation of a proto-oncogene to an oncogene leading to generation of a transforming protein involves a signal transduction pathway. In outline, signal transduction is a process in which extra-cellular signals are internalized and transmitted through a cascade of reactions to the nucleus where target genes are then transcribed. In such a cascade, the signal may be amplified, or may constitute part of a confluence of signals required to initiate a transcriptional response or may branch to influence multiple transcriptional events. In our brief review growth factor receptors will be the paradigm. In addition to the fact that the specific protein we are interested in involves a growth factor receptor pathway, a number of other components of growth factor signaling cascades are protooncogenes. The next overhead shows that growth factor receptors are transmembrane proteins that span the membrane a single time, with N-terminal domains projecting into extracellular space and C-terminal domains in the cytoplasm: [OH: growth factor receptors] The N-terminal domain contains a binding site for an extracellular chemical messenger, called a ligand. When the ligand binds, the receptors dimerize. The C-terminal domain changes conformation and in doing acquires a Tyr kinase function and autophosphorylates a Tyr residue in the C-terminal domain. The left panel of the next overhead shows the association can occur via several mechanisms: [OH] (1) the receptor-ligand complex can induce dimerization (2) the receptors can associate through 1 binding domains on a single ligand (3) conformational change resulting in association of Cterminal domains. The center panel defines kinase activity, which is phosphorylation of hydroxyl groups on target residues, which are Tyr, Ser, or Thr. The right hand panel shows that a change in conformation results in phosphorylation of residues in the C-terminal domain. There are two families of kinases: tyrosine kinases, which are predominantly receptors and therefore located in the plasma membrane, as the example in the overhead, and serine/threonine kinases, which are the predominant type of kinase within the cytosol. [OH] The phosphorylation generates recognition sites for target substrates. In the top panel, the substrate may be an “adaptor” protein, containing a recognition domain for a protein to be activated, in the middle panel, phosphorylation directly activates an enzyme, and in the bottom panel the phosphorylated substrate may be another kinase which is then activated. In any event, the activity of the receptor initiates a pathway with one of two results: (1) at some point a small molecule called a second messenger is generated (for example, cyclic AMP) or as pictured on the next overhead, (2) a cascade of kinase activity may be initiated. [OH1; kinase cascade] Activated target proteins of the kinases are called effectors. The phosphorylations within the cytoplasm are accomplished mainly by the Ser and Thr kinases we have just mentioned. The next overhead delineates a specific pathway involving a protein called Ras, named from the Ras protein that is one of the components and, coincidentally is the product of a proto-oncogene that we are interested in. 2 [OH; Ras signaling pathway] The cascade is initiated by activation of a tyrosine kinase receptor, such as EGF (epidermal growth factor). The growth factor activates an adapter molecule called Grb2 containing a recognition domain called SH2, which binds to and activates a second component called the SOS protein (formerly called GNRF [guanine nucleotide release factor], not to be confused with the SOS repair system). The activated SOS contacts and activates Ras, which has lent its name to the pathway, and Ras in turn leads to the activation of the Ser/Thr kinase, Raf, which activates the MEK kinase (MEK, mitogen activated protein kinase/extracellular signal-regulated kinase), a member of the MAPK (from mitogen activated protein kinase) family which in turn activates additional MAP kinases, like ERK (extracellular signal-regulated kiase), which enters the nucleus to phosphorylate and activate transcription factors a number of which are also protooncogenes. The cartoon shows that the signal can branch (example of signal branching) to initiate additional processes. As implied by the acronym MAPK, the signaling pathway is involved in cell proliferation. The next overhead shows in more detail regulation of Ras activity. [OH] The activated receptor binds the adaptor molecule Grb2, which recruits SOS. SOS in turn binds Ras and causes Ras to exchange a bound GDP for GTP. SOS serves as a nucleotide exchange factor, hence the GEF acronym in the right hand panel on the overhead (and also GNRF). Ras belongs to a class of proteins called G proteins so called because they bind guanine nucleotides. Once Ras has bound GTP it interacts with another protein Raf (the next effector in the cascade). Until this point, all the proteins including Raf are membrane bound, but Raf activates MEK kinase, which in turn phosphorylates and activates ERK, which starts the pathway into the cytoplasm. The cartoon shows Raf in the cytoplasm – this is not correct. The right panel shows the regulation of Ras, and also the role of GTP in Ras activity. While bound to GDP, Ras is inactive. Starting from the bottom panel, on exchange of GDP for GTP, Ras becomes activated and activates the next protein in the signaling pathway, Raf. In this process, Ras acts as a 3 GTPase, and becomes inactivated. Ras can also bind to a protein called GAP, which stimulates the GTPase activity of Ras resulting in a dead end for the signaling pathway, so GAP acts as a negative control. So this is the Ras pathway. You need to keep the Ras and Raf reactions in mind, because they will be very important in our discussion of oncogenes. ONCOGENES Having covered the signaling aspect of regulation of cell proliferation with regard to initiation of transcription of the proteins involved, we can describe how oncogenes fit into the picture of chemical carcinogenesis using the activity of mutant Ras as an example. Oncogenes were initially discovered in transforming retroviral genomes and identified as mutated cellular genes or pieces of cellular genes through sequencing. It was established that the transforming capability of the viruses resided within the genetic information that had been acquired from host cells, hence the name oncogenes was applied. In a quick aside - the transduction of cellular information by retroviruses is a rare event that can occur during the viral cycle. The genome of a retrovirus is RNA and in order to integrate the viral genome into the DNA of the host, which is a prerequisite for viral replication, the RNA genome must first be copied into DNA. Retroviral RNA is first processed as mRNA, to synthesize the proteins necessary to achieve integration into host DNA. During the cutting and splicing that accompanies the processing of the viral RNA, it is possible for the viral message to become fused with cellular mRNA. The evidence for acquisition or transduction of cellular information during processing rather than at the point of integration of the DNA form of the viral genome into the host genome lies in the fact that genes of transforming viruses contain only exonic portions of cellular mRNA - intronic sequences have not been identified in any of the transduced cellular information characterized to date. (One of the proteins coded for by the viral RNA is a reverse transcriptase, which performs the synthesis of DNA from the RNA template.) To distinguish the viral information from the wild-type cellular genes that are not transforming, the cellular genes are designated with the prefix c-, and the viral genes with the prefix v-. 4 As molecular biology techniques developed to determine genetic changes caused by chemical carcinogens, it was found that the same transforming ras gene was active in both virally and chemically transformed cells. Since further elucidation of mechanisms of carcinogenesis involves understanding the function of the oncogene products, the investigation into mechanisms of chemical and viral carcinogenesis is completely parallel beyond this point. The objective of both tracks becomes to determine the nature of the activity of oncogene products. The most often applied strategy to understand the function of oncogenes starts with mechanistic knowledge of the function of the non-transforming cellular counterparts- the proto-oncogenes. This research defines a specialized area in the field of genomics. At the moment, the Ras signal transduction cascade is one of the best characterized systems relating an oncogene to protooncogene function, and the only system for which activation can be directly tied to a chemically induced mutation. In previous overheads we described the normal function of cellular Ras; i.e., the wild-type nontransforming cellular protooncogene product. It turns out that a high proportion of lung tumors induced by benzo[a]pyrene (a carcinogenic PAH) in the A/J mouse, which is an animal model for lung cancer, have activated K-Ras oncogenes (the K-Ras designation comes from KirstenRas, because of homology of the transforming A/J Ras sequence to a transforming Ras protein isolated from a line of human cells derived from a cancer patient named Kirsten). The DNA from mouse lung tumor cells has been extracted the mutational spectrum induced by BaP in tumors expressing the oncogenic form of K-Ras has been characterized. The mutations along with the corresponding amino acid substitutions and the distribution of the mutations, which constitute the mutational spectrum are shown in columns 1 – 3 of the table on the next overhead. The transforming mutations were point mutations in codon 12, which codes for Gly, in c-Ras: [OH120; 3 Tables of mutations in codon 12] Column 1 gives the DNA mutation; col. 2, gives the relative frequency of each mutation in the tumor DNA that has been sequenced and defines the mutational spectrum; col. 3 gives the amino 5 acid substitution and col. 4, the relative transforming potency. From the Table, you see that this has been done for two mutations, Gly Val and GlyAsp. The experiment to determine transforming potency is to transfect the mutant Ras genes into an established cell line, which was the NIH 3T3 mouse line. The cells which express the mutant Ras proteins are then scored for the number of transformed colonies or foci that appear. The last column suggests that the mutations can vary widely in relative transforming power. Similar studies have been performed with the carcinogenic PAH 7,12-dimethylbenzanthracene (DMBA) in trout embryos. In trout, codon 12 of ras is GGA, rather than GGT, but GGA is one of the redundant codons for Gly, so the WT amino acid sequence is the same. As the slide shows, two mutations in codon 12 were characterized and found to occur with equal frequency. Mutations in codon 12 Mutation frequency aa substitution rel. transforming potency GGA AGA (G•C A•T transition) 50% Gly Arg 1.0 GGA GTA (G•C T•A transversion) 50% Gly Val 0.6 They were a G•C → A•T transition at position 1, resulting in Gly → Arg mutation and a G•C → T•A transversion in position 2 resulting in a Gly → Val mutation. The relative potency of the two different amino acid substitutions in the trout embryos was been characterized. Again, it turns out that the mutations vary in relative transforming potency, although in this case the difference isn’t particularly striking. In this table, the substitution Gly Arg was taken as the standard and assigned the value 1.00. If we go back to the results from the top table, and compare all of the transforming potencies (possible because Gly → Val was tested in both systems), then Gly Arg becomes the most potent transforming mutation, with a value of 1.67. A point mutation in codon 61 of the trout embryos was also shown to generate a transforming protein. In codon 61, only a single mutation has been observed. The transforming potency has not been assessed for this mutation relative to the mutations in codon 12. 6 Mutations in codon 61 Mutation CAG CTG (A•T T•A transversion) frequency aa substitution 100% Gln [Short lecture because of projector problem] 7 Val rel. potency ?