Atp

promoting the growth and metastatic abilities characteristic of cancer cells.

Constitutive Ras activation can also arise from a recessive loss-of-function mutation in a GTPase-accelerating protein (GAP). The normal GAP function is to accelerate hydrolysis of GTP and the conversion of active GTP-bound Ras to inactive GDP-bound Ras (see Figure 3-29). The loss of GAP leads to sustained Ras activation of downstream signal-transduction proteins. For example, neurofibromatosis, a benign tumor of the sheath cells that surround nerves, is caused by loss of both alleles of NFl, which encodes a GAPtype protein. Individuals with neurofibromatosis have inherited a single mutant NFl allele; subsequent somatic mutation in the other allele leads to formation of neurofibromas. Thus NFl, like RB, is a tumor-suppressor gene, and neurofibro-matosis, like hereditary retinoblastoma, is inherited as an autosomal dominant trait.

Src Protein Kinase Several oncogenes, some initially identified in human tumors, others in transforming retroviruses, encode cytosolic protein kinases that normally transduce signals in a variety of intracellular signaling pathways. Indeed the first oncogene to be discovered, v-src from Rous sarcoma retrovirus, encodes a constitutively active protein-tyrosine kinase. At least eight mammalian proto-oncogenes encode a family of nonreceptor tyrosine kinases related to the v-Src protein. In addition to a catalytic domain, these ki-nases contain SH2 and SH3 protein-protein interaction domains. The kinase activity of cellular Src and related proteins normally is inactivated by phosphorylation of the tyrosine residue at position 527, which is six residues from the C-terminus (Figure 23-17a, b). Hydrolysis of phospho-tyrosine 527 by a specific phosphatase enzyme normally activates c-Src. Tyrosine 527 is often missing or altered in Src oncoproteins that have constitutive kinase activity; that is, they do not require activation by a phosphatase. In Rous sarcoma virus, for instance, the src gene has suffered a deletion that eliminates the C-terminal 18 amino acids of c-Src; as a consequence the v-Src kinase is constitutively active (Figure 23-17b). Phosphorylation of target proteins by aberrant Src oncoproteins contributes to abnormal proliferation of many types of cells.

Abl Protein Kinase Another oncogene encoding a cytosolic nonreceptor protein kinase is generated by a chromosomal translocation that fuses a part of the c-abl gene, which encodes a tyrosine kinase, with part of the bcr gene, whose function is unknown. The normal c-Abl protein promotes branching of filamentous actin and extension of cell processes, so it may function primarily to control the cytoskeleton and cell shape. The chimeric oncoproteins encoded by the bcr-abl oncogene form a tetramer that exhibits unregulated and continuous Abl kinase activity. (This is similar to dimerization and activation of the chimeric Trk oncoprotein shown in Figure 23-15.) Bcr-Abl can phosphorylate and thereby activate many

▲ FIGURE 23-17 Structure of Src tyrosine kinases and activation by an oncogenic mutation. (a) Three-dimensional structure of Hck, one of several Src kinases in mammals. Binding of phosphotyrosine 527 to the SH2 domain induces conformational strains in the SH3 and kinase domains, distorting the kinase active site so it is catalytically inactive. The kinase activity of cellular Src proteins is normally activated by removing the phosphate on tyrosine 527. (b) Domain structure of c-Src and v-Src. Phosphorylation of tyrosine 527 by Csk, another cellular tyrosine kinase, inactivates the Src kinase activity. The transforming v-Src oncoprotein encoded by Rous sarcoma virus is missing the C-terminal 18 amino acids including tyrosine 527 and thus is constitutively active. [Part (a) from F. Sicheri et al., 1997, Nature 385:602. See also T Pawson, 1997, Nature 385:582, and W. Xu et al., 1997, Nature 385:595.]

intracellular signal-transduction proteins; at least some of these proteins are not normal substrates of Abl. For instance, Bcr-Abl can activate JAK2 kinase and STAT5 transcription factor, which normally are activated by binding of growth factors (e.g., erythropoietin) to cell-surface receptors (see Figure 14-12).

The chromosomal translocation that forms bcr-abl generates the diagnostic Philadelphia chromosome, discovered in 1960 (see Figure 10-29). The identity of the genes involved was discovered by molecular cloning of the relevant translocation "joint," allowing biochemical study of the Bcr-Abl on-coprotein. If this translocation occurs in a hematopoietic cell in the bone marrow, the activity of the chimeric bcr-abl onco-gene results in the initial phase of human chronic myeloge-nous leukemia (CML), characterized by an expansion in the number of white blood cells. A second mutation in a cell carrying bcr-abl (e.g., in p53) leads to acute leukemia, which often kills the patient. The CML chromosome translocation was only the first of a long series of distinctive, or "signature," chromosome translocations linked to particular forms of leukemia. Each one presents an opportunity for greater understanding of the disease and for new therapies. In the case of CML, that second step to successful therapy has already been taken.

0 After a painstaking search, an inhibitor of Abl kinase named STI-571 (Gleevec) was identified as a possible treatment for CML in the early 1990s. STI-571 is highly lethal to CML cells while sparing normal cells. After clinical trials showing STI-571 is remarkably effective in treating CML despite some side effects, it was approved by the FDA in 2001, the first cancer drug targeted to a signal-transduction protein unique to tumor cells. STI-571 inhibits several other tyrosine kinases that are implicated in different cancers and has been successful in trials for treating these diseases as well. There are 96 tyrosine ki-nases encoded in the human genome, so drugs related to Gleevec may be useful in controlling the activities of all these proteins. I

The Gleevec story illustrates how genetics—the discovery of the Philadelphia chromosome and the critical oncogene it creates—together with biochemistry—discovery of the molecular action of the Abl protein—can lead to a powerful new therapy. In general, each difference between cancer cells and normal cells provides a new opportunity to identify a specific drug that kills just the cancer cells or at least stops their uncontrolled growth.

Inappropriate Production of Nuclear Transcription Factors Can Induce Transformation

By one mechanism or another, the proteins encoded by all proto-oncogenes and oncogenes eventually cause changes in gene expression. This is reflected in the differences in the proportions of different mRNAs in growing cells and quiescent cells, as well as similar differences between tumor cells and their normal counterparts. As discussed in the last section, we can now measure such differences in the expression of thousands of genes with DNA microarrays (see Figure 23-13).

Since the most direct effect on gene expression is exerted by transcription factors, it is not surprising that many onco-genes encode transcription factors. Two examples are jun and fos, which initially were identified in transforming retroviruses and later found to be overexpressed in some human tumors. The c-jun and c-fos proto-oncogenes encode proteins that sometimes associate to form a heterodimeric transcription factor, called AP1, that binds to a sequence found in promoters and enhancers of many genes (see Figure 11-24). Both Fos and Jun also can act independently as transcription factors. They function as oncoproteins by activating transcription of key genes that encode growth-promoting proteins or by inhibiting transcription of growth-repressing genes.

Many nuclear proto-oncogene proteins are induced when normal cells are stimulated to grow, indicating their direct role in growth control. For example, PDGF treatment of quiescent 3T3 cells induces an «50-fold increase in the production of c-Fos and c-Myc, the normal products of the fos and myc proto-oncogenes. Initially there is a transient rise of c-Fos and later a more prolonged rise of c-Myc (Figure 23-18). The levels of both proteins decline within a few hours, a regulatory effect that may, in normal cells, help to avoid cancer. As discussed in Chapter 21, c-Fos and c-Myc stimulate transcription of genes encoding proteins that promote progression through the G1 phase of the cell cycle and the G1 to S transition. In tumors, the oncogenic forms of these or other transcription factors are frequently expressed at high and unregulated levels.

In normal cells, c-Fos and c-Myc mRNAs and the proteins they encode are intrinsically unstable, leading to their rapid loss after the genes are induced. Some of the changes

▲ EXPERIMENTAL FIGURE 23-18 Addition of serum to quiescent 3T3 cells yields a marked increase in the activity of two proto-oncogene products, c-Fos and c-Myc. Serum contains factors like platelet-derived growth factor (PDGF) that stimulate the growth of quiescent cells. One of the earliest effects of growth factors is to induce expression of c-fos and c-myc, whose encoded proteins are transcription factors. [See M. E. Greenberg and E. B. Ziff, 1984, Nature 311:433.]

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