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Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.

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Holland-Frei Cancer Medicine. 6th edition.

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Signaling Pathways of Tyrosine Kinase Receptors

, PhD and , MD.

Knowledge of the cascade of biochemical events triggered by ligand stimulation of tyrosine kinase receptors has increased rapidly and has provided further evidence of the importance of their signaling pathways in cancer. For example, nature has created a variety of molecules known as adaptor and scaffolding proteins.206 These proteins play an integral role in intracellular signaling by both recruiting various proteins to specific locations and by assembling networks of proteins particular to a cascade. Adaptor proteins, through protein-protein interactions via specific motifs, provide a link between molecules of a signaling cascade and proteins such as RTKs. One such adaptor, Grb2, is important in the activation of the small G-protein ras (Figure 5-3). These adaptor proteins often contain a variety of motifs that mediate protein-protein interactions. Src homology 2 (SH2) domains are protein motifs that bind to specific phosphorylated tyrosine-containing sequences, dictating particular binding partners. SH3 domains recognize and bind to proline-rich sequences in target proteins.207 Thus, in the case of an adaptor protein such as Grb2, which contains both SH2 and SH3 sequences, an adaptor protein can bring a cytoplasmic protein via its SH3 domain to an activated RTK via an SH2 domain, binding to phosphorylated tyrosine residues of the receptor.

Figure 5-3. Intracellular effectors of receptor tyrosine kinases.

Figure 5-3

Intracellular effectors of receptor tyrosine kinases.

Another form of adapter, a docking protein, provides multiple binding sites on which effector molecules can attach, thereby expanding the magnitude of responses from an activated RTK. One such docking protein, IRS-1, is a substrate of the IR, that has 18 possible tyrosine phosphorylation sites.208 It also contains two other important domains: a pleckstrin homology domain (PH), which binds to specific phosphoinositides (PtdIns), and a phosphotyrosine-binding (PTB) domain, which, like the SH2 domains, binds to phosphorylated tyrosine-containing sequences. These two domains are believed to properly position IRS-1 adjacent to the receptor via the PH domain binding to the plasma membrane and the PTB domain binding to phospho-Tyr of the IR. Proteins that attach to the phosphorylated tyrosine residues that were phosphorylated in response to IR activation include PI3-K, Shp-2, Nck, and Grb-2.209

Another concept that has surfaced in mammalian signaling is the importance of so-called scaffolding proteins in signaling cascades. This idea is not new, and, in fact, it has been known for some time that these proteins exist in the yeast Saccharomyces cerevisiae. Scaffolding proteins allow the formation of multienzyme complexes that are involved in a particular cascade. These are important for two reasons. The first is that the activation of a signaling cascade by a growth factor is an extremely rapid process and is not likely to occur as a result of two proteins randomly floating in the intracellular milieu until they happen to come in contact with each other. Scaffolding proteins ensure the close proximity of the necessary components. The second reason is that several enzymatic components of a particular signaling cascade may be shared, although the substrates of each may differ. Thus, scaffolding proteins ensure the proper routing of signals by preventing unwanted cross talk between pathways.206

The PDGF system has served as the prototype for identification of the components of signaling cascades. Certain molecules become physically associated and/or phosphorylated by the activated PDGF receptor kinase. Those identified to date include phospholipase C (PLC)-γ , phosphatidylinositol-3′-kinase (PI-3-K) regulatory subunit (p85), NCK, the phosphatase SHP-2, Grb2, CRK, RAS p21 GTPase-activating protein (GAP), SRC, and SRC-like tyrosine kinases.210–217 Many of these molecules contain SH2 or SH-3 domains.

PLC-γ is involved in the generation of two important second messengers, inositol triphosphate and diacylglycerol.218 The former causes release of stored intracellular calcium, and the latter activates protein kinase C (PKC). These second messengers appear rapidly in cells following stimulation by growth factors such as PDGF. The relative increase in their synthesis in vivo correlates reasonably well with the ability of a particular receptor kinase to induce tyrosine phosphorylation of PLC-γ.210 In combination with evidence that tyrosine-phosphorylated PLC-γ exhibits increased catalytic activity in vitro, it seems very likely that receptor-induced tyrosine phosphorylation activates this enzyme.219 The actions of a number of tumor promoters are thought to be mediated by PKC, and PKC overexpression or gene alteration has been reported to increase cell proliferation in culture.218,220

PI-3-Kinase and Survival Signaling

The regulation of cell survival and cell death is of extreme importance in both the development of an organism and in the physiologic functions of the adult. During development of a multicellular organism, certain cells are eliminated by apoptosis (programmed cell death), and others permitted to survive. This is essential for organs and systems to form correctly. These processes are undoubtedly complex, with many levels of regulation. As one can imagine, the deregulation of these processes can lead to a variety of malformations resulting in deformities or, in extreme cases, incapability with life. In adulthood, regulation of cell survival is equally important for proper homeostasis. Damaged cells must be removed, and terminally differentiated cells must be sustained. A failure in this to occur may result in either the accumulation of mutations leading to cancer or, alternatively, degenerative diseases.

PI-3-K is a lipid kinase that catalyzes the transfer of the γ -phosphate from ATP to the D3 position of the PtdIns, generating PtdIns3P, 4P2, and PtdIns3, 4, 5P3.221 These lipids can act in a variety of cascades, promoting the activation of several proteins.222 PI-3-K activation has been demonstrated to play an important role in cell survival signaling in a number of cell types.223 There are three classes of PI-3-Ks, which exhibit variability with respect to their method of activation or their preferred lipid substrate.

The prototypical class, 1 PI-3-K, consists of two subunits encoded by two distinct loci: a regulatory and a catalytic subunit.221 The regulatory subunit is a 50 to 85 kDa protein that is tightly associated with the p110 catalytic subunit. The most widely studied regulatory subunit is p85. This subunit has several characteristic protein domains, including two SH2 domains, which can bind to the phosphorylated tyrosines of several RTKs, thereby facilitating its activation, an SH3 domain that binds to proline-rich regions of several proteins and an inter-SH2 region that is essential for its association with the p110 catalytic subunit.224 As is evident, this is a complex molecule, and the exact functions of each domain have not been completely elucidated.

The classic mode of PI-3-K activation involves its binding to the phosphorylated tyrosine residues of RTKs, including PDGFR, EGFR, bFGFR, and TRK A via the two SH2 domains of p85.221 This results in a conformational change that is believed to facilitate activation of the p110 catalytic activity. PI-3-K activates PtdIns by a phosphorylation event. There are several known downstream effectors of PI-3-K. These include RaC, p70s6k, certain isoforms of PKC, and, most relevant to the discussion of cell survival, AKT/PKB.221,223 AKT has been shown to be responsible for PI-3-K-dependent cell survival and is the cellular homolog of the viral oncogene, which was designated v-AKT, based on its isolation from an AKR mouse thymoma.223,225 The three human homologs identified encode 57 kDa serine/threonine kinases that contain an N-terminal PH domain, which binds to the activated PtdIns products of PI-3-K. These lipids are believed to mediate the localization of this cytoplasmic protein to the plasma membrane. In addition, phosphorylation of two residues, a serine and a threonine, is required for full activation. These phosphorylation events are catalyzed by a kinase, PDK1, which also possess a PH domain and is recruited to the membrane, only one of which has been identified. PDK1 (PtdIns3, 4, 5P3-dependent kinase) specifically phosphorylates Thr307 of AKT. An as yet unidentified kinase, PDK2, has been postulated to fully activate AKT.223,225

AKT promotes survival and prevents apoptosis in various cell types, including cerebellar granule neurons, superior cervical neurons, myeloid cells, and myc-overexpressing fibroblasts. The mechanism of AKT-induced survival has begun to be understood. AKT phosphorylates the pro-apoptotic Bcl-2 family member, BAD, both in vitro and in vivo on serine. When BAD is phosphorylated, it gains affinity for the cytosolic protein 14-3-3 and forms a complex with this protein. Nonphosphorylated BAD can heterodimerize with the anti-apoptotic Bcl-2 family member Bcl-XL. Upon phosphorylation of BAD, its binding to 14-3-3 decreases formation of the BAD-Bcl-XL heterodimer, thus permitting free Bcl-XL to protect the cell from apoptosis (see Figure 5-3).223,225 Although this appears to be a mechanism for PI-3-K-induced cell survival, it is clearly not the only one. For example, the expression of BAD is not ubiquitous, and in certain cell types in which PI-3-K/AKT prevents apoptosis, BAD is not expressed. AKT also phosphorylates the Forkhead-related transcription factor (FKHR), creating a binding site for 14-3-3, which retains FKHR in the cytoplasm and inhibits its transcriptional gene targets including pro-apoptotic proteins such as BIM and Fas ligand. There is also evidence that AKT can indirectly increase the function of the NFkB transcription factor complex, which has pro-survival functions. AKT has been reported to activate IkB kinase (IKK), which induces degradation of the NFkB inhibitor, IkB. Recent studies indicate that AKT may increase p53 degradation through phosphorylation of MDM2.223,225 Thus, AKT has multiple prosurvival functions. There is also evidence that activated AKT and/or PDK1 exert positive effects on cell proliferation and cell growth through phosphorylation of proteins such as p70S6K, involved in protein synthesis, and inhibitory phosphorylation of GSK3, which normally targets cyclin D1 for degradation. Thus, the functions of PI3 kinase are complex and extend beyond prosurvival functions in the coordinated responses of a cell to growth factor signaling (Figure 5-3).223,225

PI-3-Kinase Signaling in Cancer

The striking anti-apoptotic effect of both PI-3-K and its downstream effector AKT, as well as the fact that these two genes were initially found as transforming viral oncogenes, suggested that these two genes might also be involved in human cancer. Indeed, a myristoylated, constitutively active PI-3-K can cause cellular transformation in chicken embryo fibroblasts.226 The genomic locus encoding the p110α catalytic subunit of PI-3-K was found to be amplified in a high percentage of ovarian tumors and ovarian tumor cell lines.227 There is also evidence of AKT involvement in human malignancies. AKT1 was found to be amplified 20-fold in a primary gastric adenocarcinoma.228 Additional studies have shown genomic amplification and overexpression of AKT2 in pancreatic and ovarian carcinoma cell lines, as well as amplification in some of the ovarian and breast carcinomas examined (see Table 5-2).225 Of particular note is the fact that overexpression of AKT2 occurs more frequently in undifferentiated and, thus, more aggressive, tumors.

Further evidence of the involvement of the PI-3-K/AKT pathway in cancer stems from the discovery of the PTEN/MMAC tumor suppressor, a gene inactivated by mutation in a high fraction of glial and endometrial tumors as well as in melanoma, prostate, renal, and small-cell lung carcinomas (see Table 5-2).229,230 PTEN has high sequence homology to dual specificity phosphatases, but its activity on artificial substrates is significantly weaker than other dual specificity phosphatases. However, PTEN was shown to dephosphorylate the 3-position of phosphatidylinositol both in vitro and in vivo.231 Thus, PTEN directly opposes PI-3-K activity by dephosphorylating its activated lipid products. Many PTEN mutations found in human tumors abolish its lipid phosphatase activity, whereas some still retain activity to artificial protein substrates. Therefore, the tumor suppressor activity of PTEN is mediated via its ability to oppose both PI-3-K and AKT, both of which have been shown to be oncogenic.229,230

RAS

Ras proteins are a major point of convergence in RTK signaling and are an important component of the cellular machinery necessary to transduce extracellular signals.232 Ras small GTP-binding proteins are membrane-bound intracellular signaling molecules that mediate a wide variety of cellular functions, including proliferation, differentiation, and survival. This family consists of ten highly conserved proteins, including H-, N-, and K-RAS, R-RAS, RAP1(A and B), TC21 (R-RAS2), and R-RAS3.232,233 Ras proteins are synthesized in the cytosol and become associated with the inner leaflet of the plasma membrane via posttranslational modifications, including a form of fatty acid lipidation, isoprenylation (which encompasses farnesylation and geranylation), on Cys-186. The C-terminal CAAX box (Cys, two aliphatic amino acids, followed by any residue) is an essential motif required for ras function, as it targets the unprocessed protein for this essential modification.234 Two regions of ras, designated switch 1 and switch 2, undergo a conformational change upon GTP hydrolysis. Switch 1 encompasses residues 30 to 38 and constitutes the majority of the effector loop, which mediates binding to its downstream effectors when ras is bound to GTP. Switch 2, encompassing residues 60 to 76, forms the GTP γ phosphate-binding site and may also be important in binding to certain effector proteins. Of considerable interest is the fact that residues 32 to 38 of switch 1 are identical for members of the RAS family excluding ral, indicating that specificity in signaling may lie in other regions of these proteins.232,234

Ras acts as a molecular switch alternating from an inactive GDP-bound state to an active GTP-bound state. The paradigm for ras activation involves the recruitment of a guanine nucleotide exchange factor (GNEF) to the membrane in response to growth factor binding and subsequent activation of an RTK.232 GNEFs promote the release of GDP from the catalytic pocket of ras, and the relative abundance of intracellular GTP as compared to GDP ensures preferential binding of GTP. The best example of a ras GNEF is SOS (son of sevenless), which is brought to the membrane by its stable association with the adaptor protein Grb2.235 Grb2 contains one SH2 domain that binds to a specific motif containing phosphorylated tyrosine residues on several RTKs, including the PDGFR and the EGFR. Grb2 also has two SH3 domains that mediate its binding to SOS via a carboxy terminal proline-rich region in this protein. Alternatively, another adaptor protein, SHC, can bind to the cytoplasmic tail of the receptor through its SH2 domain, resulting in its phosphorylation on tyrosine and subsequently binding Grb2. The exact sequence of binding of adaptors depends on the receptor and cell type.

Once SOS is translocated to the membrane, it can promote the release of GDP from ras, allowing GTP, which is present in excess in the intracellular environment, to bind and ultimately lead to ras activation.236 The crystal structure of h-ras complexed with the catalytic region of SOS has been determined, and the specific structural events involved in the catalytic process resulting in GDP release have been elucidated. Additional ras GNEFs have been cloned and include GRF1 and 2 and ras GRP. The exact specificity of their interactions with different RAS family members and the nature of the stimuli that activate these various exchange factors are presently under investigation.236,237

Although ras is a GTPase, its intrinsic GTPase activity is actually quite low and requires additional proteins, known as GTPase-activating proteins (GAPs), to promote GTP hydrolysis. GAPs can accelerate GTP hydrolysis by several orders of magnitude and are, thus, negative regulators of ras functions.238 The mechanism by which GAP accelerates the GTPase reaction is complex and not completely understood. Currently, several GAPs for ras have been identified, including p120 GAP, NF1-GAP/neurofibromin, and GAP1m, as well as GAPs with preferential activity on related proteins such as R-ras.239 Of particular interest is NF1, as it is found to be frequently inactivated by mutation in patients with the familial tumor syndrome, neurofibromatosis type 1.

Ras Function

RAS appears to have a multitude of functions, which differ depending on factors such as cell type and extracellular environment. It is paradoxical that a single gene product can cause cell cycle entry and DNA synthesis in one cell type, such as fibroblasts, and terminal differentiation in others, such as PC12.234 In other cell types, such as myoblasts, activated ras seems to oppose cell cycle withdrawal and differentiation into myotubes and downregulates expression of muscle-specific mRNA transcripts. Additionally, ras promotes survival in some cell types, such as those of hematopoietic lineages, upon cytokine withdrawal and PC12 cells and primary sympathetic neurons upon removal of NGF or other trophic factors. Although ras mediates such important cellular processes as proliferation, survival, and differentiation, the exact contribution of H-, N-, and K-RAS isoforms is not clear, as targeted knockouts to h- and N-RAS genes resulted in mice that did not exhibit an abnormal phenotype, whereas a K-RAS knockout is an embryonic lethal and exhibits liver and hematopoietic defects.234 Therefore, there may be a certain degree of redundancy between these three ras proteins.

Ras and Cancer

Much of the interest in ras stems from the fact that this gene is involved in a high fraction of human cancers (see Table 5-2). Ras has been shown to be oncogenically activated by mutations in over 15% of all human tumors, and in some cancers, such as pancreatic carcinoma, the frequency is as high as 90%.240 The initial evidence for ras involvement in cancer came from the discovery of transforming retroviruses, Harvey and Kirsten sarcoma viruses, which contained H- and K-ras cellular-derived oncogenes. It was not until later that the first human oncogenes were identified by transfecting genomic DNA from human tumor cell lines into NIH3T3 mouse fibroblasts and isolating the DNA fragments from the transformed foci. These were shown to be the human homologs of the viral ras genes.241

The major hot spots for activating ras mutations are all located in the regions of the protein that are near the bound guanine nucleotide, particularly in proximity to the nucleotide phosphate groups. Naturally occurring mutations in human tumors have been found at residues 12, 13, 59, and 61, with positions 12 and 61 being the most common.234,240 The majority of these mutations decrease the intrinsic rate of GTP hydrolysis by ras and make the molecule significantly less sensitive to GAP-stimulated GTP hydrolysis. Thus, the outcome is a molecule that is predominantly GTP bound and therefore constitutively active. It is now essentially independent of growth factor stimulation and continues to activate downstream pathways in the absence of any stimulation. Oncogenic ras is capable of transforming immortalized rodent fibroblasts or epithelial cells.241 Ras-transformed cells appear refractile and spindle shaped, have disorganized actin filaments, and have a decreased affinity for the substratum. They can proliferate in the absence of adhesion (anchorage independence) or in the presence of low serum concentration. Such cells exhibit a loss of contact inhibition and grow to high saturation density. Of note, ras alone is unable to transform primary mouse or human fibroblasts.241 When oncogenic ras is introduced into such cells by retroviral mediated gene transfer, the cells undergo permanent growth arrest, also termed replicative senescence, characteristic of primary cells passed for multiple generations in culture.242 This senescence response appears to be dependent on the function of certain genes such as p16INK4a and p53, which act as tumor-suppressor genes. The inactivation of these tumor-suppressor genes plays a critical role in cancer development. In fact, inactivation of p53 or p16INK4a allows ras to transform cells, which may help to explain the selective pressure for loss of these tumor suppressor-genes in tumors containing ras oncogenic mutations.242

Additional members of the RAS family of GTP-binding proteins can cause cellular transformation when overexpressed in rodent fibroblasts. These include R-ras, TC21/R-ras2, and R-ras3.243–245 In fact, TC21 has been found to be mutated infrequently in cancers.246 The other transforming members have not been shown to be oncogenically activated in human tumors, although more complete studies need to be performed before this possibility can be excluded.

Signaling Downstream of Ras

Ras mediates its multitude of biologic effects via several downstream effectors. Although many of the signaling pathways that ras perturbs have been extensively studied in mammalian systems, a great deal of the early data has been generated in other organisms, such as Drosophila melanogaster and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe).247 Indeed, all three of these organisms have ras homologs that are activated in response to a diverse array of extracellular signals and, in many cases, signal through similar secondary messengers.

Several proteins have been shown to directly bind to ras either in vivo or in vitro in a GTP-dependent manner by such methods as yeast 2-hybrid or co-immunoprecipitation. These include A-, B-, and C-raf, ral GDS, RGL II, the PI-3-K, MEKK1, AF6, and PKCζ.232 Whether they are all true physiologic effectors of ras remains uncertain. It has been demonstrated that PI-3-K can be activated independently of RTKs by ras, providing a direct connection between ras and PI-3-K prosurvival signaling.248

Ras > Raf > MAP Kinase Cascade

The most well-studied effector of ras is the serine/threonine kinase raf. Raf has been shown to bind to ras and, in many cases, has been demonstrated to be indispensable for some ras functions, such as cellular transformation.249,250 In fact, activated raf, or v-raf, a truncated form of raf, was initially isolated as a retroviral oncogene. There are three known mammalian raf isoforms, designated A-, B-, and C-raf (also known as raf-1). C-raf is ubiquitous in its tissue expression, whereas A-raf and B-raf expression are more restricted. A-raf is expressed mainly in steroid-responsive tissues, particularly in urogenital tissues, whereas B-raf is restricted to neural-derived tissues. There is a high degree of conservation between these isoforms, particularly in the regions defined as CR1, CR2, and CR3. CR1 and CR2 are located at the N-terminus of the protein, which has a negative regulatory role in raf activation.251 The CR3 region is in the C-terminus of the protein and contains the kinase domain. RAS-mediated activation of RAF requires binding to two regions of this cytoplasmic kinase, both of which are located at the amino terminus (within CR1). These include residues 55 to 131, known as the RAS-binding domain, and a cysteine-rich domain that additionally binds the membrane phospholipid, phosphatidylserine (PS).252

Although RAS appears to mediate the activation of RAF, it is not entirely sufficient, as other factors are necessary for maximal activation of its kinase activity both in vivo and in vitro. It is believed that interaction of ras switch 1 with the ras-binding domain of raf allows translocation of raf to the membrane (see Figure 5-3), where additional steps leading to its full activation can occur.232 These include binding of the cysteine-rich domain to PS, which serves to promote interaction of ras switch 2 with this second region of interaction. Several phosphorylation events on both serine-threonine and tyrosine residues are believed to have a role in the full activation of raf as well. The complexity of these phosphorylations is enormous, with both inhibitory and stimulatory sites being described. In addition, there are major differences in certain phosphorylation sites between B-RAF and C-RAF, indicating that regulation of these two isoforms may differ significantly.253

Once activated, raf can phosphorylate MEK (mitogen/extracellular-signal-regulated kinase kinase), also known as Map Kinase Kinase (MKK), a dual-specificity kinase, on Ser218 and Ser222, leading to its activation.254 Partial activation can be seen by phosphorylation on only one serine (see Figure 5-3). There are two isoforms of MEK, designated MEK1 and MEK2, both of which are expressed ubiquitously with an approximate sequence identity of 80%. MEK, once activated, can, in turn, activate MAP Kinase or extracellular signal-regulated kinase (ERK).254 Activation occurs via tandem phosphorylations on both threonine and tyrosine (Thr183-Glu-Tyr185), with the phosphorylation on tyrosine occurring first. There are two ERK isoforms (1 and 2), ubiquitously expressed and with very similar sequence (90%). These proteins, 44 and 42 kDa, respectively, translocate to the nucleus, where they can activate a variety of proteins through phosphorylation on serine or threonine.

ERK can phosphorylate several of the members of the ETS family of transcription factors, explaining its apparent ability to activate transcription of certain genes.254 The ETS transcription factors are helix-turn-helix proteins. A member of this family, p62TCF/Elk-1, in complex with the serum response factor (SRF), transactivates the serum response element (SRE), which can be found in several promoters, including that of c-fos. Phosphorylation of ElK-1 by ERK dramatically increases c-fos transcription.255 ERK can also activate a variety of protein kinases via phosphorylation. For example, p90 RSK is a serine-threonine kinase that has a role in protein translation and has been shown to be a substrate for the ERKs.254

In addition to positive regulation of the MAP kinase pathway by phosphorylation, there are negative regulatory mechanisms that serve to attenuate activation of this cascade. A principal mode of this negative regulation is through a variety of phosphatases, a majority of which are dual specific, meaning that they can dephosphorylate both serine-threonine and tyrosine residues.254 This is consistent with the knowledge that ERK must be phosphorylated on both threonine and tyrosine to achieve maximal activation. There are several known MAP Kinase phosphatases that differ in terms of substrate specificity.

ERK activation can lead to increased DNA synthesis and cell proliferation. In fact, cyclin D1 expression is induced by activated forms of ras, raf, and MEK.256 Dominant negative mutants of members of this cascade can also block this induction in response to growth factor stimulation. Of particular interest is the fact that cyclin D1 can be rearranged or amplified in human tumors and tumor cell lines (see Table 5-2), thus implicating a role for this G1 cyclin in human cancer.257 Mutationally activated forms of raf or MEK1/2 can transform rodent fibroblasts and form tumors in nude mice, although they are not as potent as oncogenic ras.232 This is not unexpected, as it is now understood that ras transformation occurs through multiple pathways.

Raf and Cancer

Recent studies, taking advantage of high-throughput genomic sequencing, have uncovered a major role for B-raf in human cancer. Knowledge of the importance of RTK signal transduction in cancer has recently led to screening of tumor cell lines for mutations in these pathways. Davies and colleagues identified B-raf mutations in around 66% of human melanoma cell lines and primary tumors (see Table 5-2).258 Of note, the nucleotide changes observed were not consistent with mutations typically induced by ultraviolet light (UV). Lower frequencies of analogous mutations were observed in colon carcinoma and small-cell lung cancer (SCLC)(see Table 5-1).258 These mutations were further shown to oncogenically activate B-raf, as determined by NIH3T3 transfection analysis, although the transforming efficiency was significantly lower than observed with ras. These new findings further increase the number of human tumors that exhibit mutational activation of growth factor signal transduction pathways.258

Other MAP Kinases

In addition to the ERKs, there are other MAP Kinases belonging to distinct MAPK cascades but with different upstream activators and downstream effectors. The c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 MAP Kinase have been demonstrated to modulate cellular responses to a wide variety of extracellular stimuli including mitogens, inflammatory cytokines, and UV irradiation.259

In contrast to its ability to activate the MAPK/ERK cascade, h-ras only minimally perturbs JNK/SAPK. However, overexpression of the constitutively activated mutants of the small G-proteins, rac and Cdc42, leads to the robust stimulation of JNK/SAPK activity.260 The pathways leading to JNK activation mirror those seen for ERK. Thus, a variety of MKKs have been discovered that can phosphorylate the various JNK isoforms.254,259

As with the ERKs, the end result of JNK activation is the phosphorylation of certain transcription factors within their activation domains, increasing the transcriptional activity of promoters containing response elements for these factors. JNK can phosphorylate ATF2, ATFa, c-Jun, and Jund, as well as ElK-1 and SAP1.259 Phosphorylation of c-Jun by JNK increases its half-life, preventing ubiquitin-mediated degradation of this short-lived protein.261 Evidence from experiments performed using knockout cells of an upstream activator of JNK (MKK4) have demonstrated that JNK plays a role in AP-1 transcription-dependent events in response to stress.262

MAP Kinase cascades also activate transcription factors such as c-fos and c-Jun. Of note, these genes were initially discovered as retroviral oncogenes in mice and chickens, respectively. The FBJ and FBR murine viruses contain the fos sequence under the transcriptional control of the viral LTR promoter and exhibit changes in regulatory phosphorylation sites that make them more active than the protooncogene.263 ASV17 is a chicken retrovirus containing a Jun oncogene fused to the viral Gag sequence and has lost regulatory phosphorylation sites.264 Overexpression of c-fos can cause transformation of cells as well.265 Fos and Jun together comprise the AP-1 transcription factor. This dimer, in response to UV irradiation, environmental stresses, and PKC activation, binds to AP-1 target sequences such as 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements.266

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2003, BC Decker Inc.
Bookshelf ID: NBK12569

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