http://www.unc.edu/courses/2009spring/envr/740/001 slide 14Begin 02/26/09SIGNAL TRANSDUCTIONIn 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 1binding domains on a single ligand (3) conformational change resulting in association of C-terminal 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 thenext 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 SOSprotein (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 thepathway, 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 3GTPase, 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.ONCOGENESHaving 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