Review

Leukemia (2004) 18, 189–218. doi:10.1038/sj.leu.2403241

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis

L S Steelman1, S C Pohnert2, J G Shelton1, R A Franklin1,3, F E Bertrand1 and J A McCubrey1,3

  1. 1Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA
  2. 2Department of Biochemistry, Brody School of Medicine at East Carolina University, Greenville, NC, USA
  3. 3Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, NC, USA

Correspondence: JA McCubrey, Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, 600 Moye Blvd, Greenville, NC 27858, USA. Fax: +1 252 744 3104; E-mail: mccubreyj@mail.ecu.edu

Received 29 August 2003; Accepted 24 September 2003.

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Abstract

The roles of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt signal transduction pathways and the BCR-ABL oncoprotein in leukemogenesis and their importance in the regulation of cell cycle progression and apoptosis are discussed in this review. These pathways have evolved regulatory proteins, which serve to limit their proliferative and antiapoptotic effects. Small molecular weight cell membrane-permeable drugs that target these pathways have been developed for leukemia therapy. One such example is imatinib mesylate, which targets the BCR-ABL kinase as well as a few structurally related kinases. This drug has proven to be effective in the treatment of CML patients. However, leukemic cells have evolved mechanisms to become resistant to this drug. A means to combat drug resistance is to target other prominent signaling components involved in the pathway or to inhibit BCR-ABL by other mechanisms. Treatment of imatinib-resistant leukemia cells with drugs that target Ras (farnysyl transferase inhibitors) or with the protein destabilizer geldanamycin has proven to be a means to inhibit the growth of resistant cells. This review will tie together three important signal transduction pathways involved in the regulation of hematopoietic cell growth and indicate how their expression is dysregulated by the BCR-ABL oncoprotein.

Keywords:

JAK, Ras, Raf, PI3K, BCR-ABL

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Hematopoietic cytokines and receptors

Cytokines interact with cell-surface receptors initiating signaling cascades that promote cell growth and division, while inhibiting the pathways of apoptotic cell death.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 The JAK/STAT, Raf/MEK/ERK and PI3K/Akt signaling pathways are activated by a variety of cytokines that function to potentiate or inhibit hematopoiesis. These include IL-3, IL-7, SCF, G-CSF, type I interferons and TGF-beta. Signaling through IL-3/IL-3R interaction has been widely studied as a prototype for how cytokine signaling activates JAK/STAT, Raf/MEK/ERK and PI3K/Akt pathways to modulate normal and abnormal hematopoiesis.

IL-3 is a soluble highly glycosylated 26 kDa protein that belongs to the class of proteins called cytokines. The JAK/STAT, Raf/MEK/ERK and PI3K/Akt signaling pathways elicited by IL-3 promote survival, growth and differentiation of pluripotent hematopoietic cells. A depiction of three pathways is presented diagrammatically in Figure 1. IL-3 was initially characterized by its ability to induce activity of 20-alpha-hydroxy-steroid-dehydrogenase in spleen cells.21 It turns out that IL-3 was being studied by many different investigators, and the diverse bioactivities were known under different names including persisting-cell-stimulating-factor, Thy-1-inducing factor, multilineage hematopoietic growth factor, histamine-producing cell-stimulating factor, multicolony stimulating factor (CSF), CFU-stimulating factor, eosinophil-CSF, megakaryocyte-CSF, erythroid-CSF, burst-promoting activity, neutrophil–granulocyte-CSF and hemopoietin-2, before it was cloned.21 Once it was cloned, it was shown to have all these activities. The human IL-3 peptide consists of 133 amino acids while the mouse peptide is 140 amino acids in length.21,22,23,24 The biological activity of the IL-3 molecule lies in residues 17–118,22 and is not dependent on the glycosylation state of the protein.25 The half-life of the molecule is 40 min with clearance being mainly via re-absorption in renal tubules.26

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Overview of effects of IL-3 on JAK/STAT, Raf/MEK/ERK and PI3K/Akt signaling. The figure depicts some of the signal transduction pathways activated after ligation of the IL-3 receptor. Activation of the IL-3 receptor induces the activity of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt pathways, which results in cell proliferation and prevention of apoptosis. Activation of the JAK/STAT pathway can induce the expression of antiapoptotic genes such as Bcl-XL but it can also induce the expression of CIS and SOCS proteins, which serve as negative feedback regulators. IL-3 can induce the Ras/Raf/MEK/ERK pathway, which results in phosphorylation of transcription factors that can promote cell proliferation. Akt can phosphorylate many targets (Bad, IkappaK, caspase-9, GSK3beta, Foxo3), which have effects on apoptosis and cell cycle progression. Cytokines also induce the tyrosine phosphorylation of the Gab2 protein, which is a docking molecule. Gab2 associates with the Shp-2 phosphatase, Grb-2 and PI3PI3K. Gab2 can regulate PI3K and Ras activities. Cytokines also regulate the expression of phosphates such as Shp2 and SHIP1, which regulate these pathway. Dotted lines indicate potential interactions between the pathways. Solid lines indicate direct paths of signal transduction in the individual pathways.

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IL-3 is primarily synthesized by T lymphocytes with a small contribution from mast cells.1,2 IL-3 mRNA stability is regulated by a rapamycin-sensitive pathway.27 This likely involves the target of rapamycin (TOR) and its downstream target p70S6K. Frequent injections of mice with either IL-3 or inoculations of IL-3-producing cells increase the number of myeloid, mast and megakaryocytic cells.28,29

The IL-3 receptor (IL-3R) is a member of the cytokine receptor gene family and is comprised of alpha- and beta-subunits.1 Both subunits contain extracellular domains common to the cytokine receptor family.30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42 The intracellular domains are more variable. The alpha subunit of the IL-3R is divided into three main domains: ligand-binding, transmembrane and intracellular signaling domains. The ligand-binding domain has homology with other cytokine receptors (GM-CSF and IL-5); however, they differ in their binding specificity. The intracellular signaling domain of the alpha subunit lacks intrinsic kinase activity, but is required for activation of STAT5, Raf-1 and p70S6K.30,31,32 Transcriptional regulation of c-Fos and c-Jun is mediated through the interaction of intracellular signaling domain with downstream targets.30

The common beta subunit is a member of the cytokine receptor superfamily, consisting of GM-CSF, IL-3 and IL-5 receptors. The beta subunit also has no intrinsic kinase activity.33,34,35,36 The gene for the beta-subunit gene is located on mouse chromosome 1537 and the alpha-chain gene is located on chromosome 14. The human beta chain is located on chromosome 22q13.1 and the alpha chain is located on chromosome Xp22.3 (Table 1). The cytokine receptor superfamily is characterized by a 200-amino-acid extracellular domain consisting of two fibronectin beta barrels. The highly conserved consensus 'WSXWS box' is characteristic of the membrane proximal region of cytokine receptors.35,38 The transduction of a signal by the cytokine receptor requires conformational changes in the cytoplasmic domain of the beta subunit. This conformational change promotes the binding and activation of downstream targets including JAK, STAT and phosphatidyl-inositol-3-kinase (PI3K) and other shuttling molecules such as Shc, which transduces the signal through Grb2 and SOS to the Ras/Raf/MEK/ERK cascade.1,35 More recently, another docking molecule Gab2 and the associated Shp2 phosphatase have been shown to play important roles in cytokine signaling.38 Cytokines induce the phosphorylation of Gab2, which associates with Shp-2 and Grb-2. Shp-2 has a binding site on the IL-3Rbeta chain.1 The Gab2 docking molecule contains a pleckstrin homology (PH) and two Src homology (Src) domains. The Gab2 docking protein is functionally similar to the insulin-regulated substrate (IRS1) involved in insulin and insulin-like growth factor (IGF) signaling mediated by their receptors. Gab2 serves as a docking site for PI3K and Shp2 and also connects with the Ras/Raf/MEK/ERK pathway via Grb-2. Gab-2 is also a target of BCR-ABL and will be discussed later.


The cytoplasmic portion of the IL-3betac subunit is divided into two regions, which were identified by truncation mutants: the proximal and distal portions.35 The proximal portion of the betac subunit interacts with and activates JAKs, SOCS, CIS, STATs, c-Src, PI3K and Vav.1,35 In the area spanning the distal and proximal regions of the betac chain are the binding sites for the Shc, Shp1 and Shp2 proteins. The distal portion the betac subunit mediates transcription of c-Fos and c-Jun via its ability to promote activation of Raf-1 and p70S6K.30,31 At the very end of the membrane distal domain are regions that inhibit cell growth but promote viability.39

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Roles of hematopoietic cytokine receptors in hematopoietic disease and neoplasia

The A/J strain of mice is nonresponsive to IL-3 and contains a naturally occurring mutation in the IL-3Ralpha-chain gene.40 Knockout mice have been created that lack either the IL-3 or IL-3R genes.41,42 Mice lacking IL-3 or IL-3R or both have a similar phenotype. These mice exhibit a normal population of hematopoietic cells, except for a reduction in eosinophils.41,42 This is likely due to the fact that the IL-3Rbeta chain is also the IL-5Rbeta chain and hence the mice are also deficient in IL-5-mediated responses. The observation that most other hematopoietic cells appear normal suggests that the effects of IL-3 and its receptor can be compensated for by other cytokines.

Overexpression of the IL-3R chains was first observed in murine hematopoietic cells, which grew in either the absence of exogenous IL-3 or in relatively low concentrations of IL-3.17 Mutations that result in constitutive activation or overexpression of the IL-3/GM-CSF/IL-5 receptors have been detected in human and murine leukemias.43,44,45,46,47 Some naturally occurring cytokine receptor mutants act as dominant negative (DN) mutations, which may serve to dampen the response to cytokines.47 Experimentally we have demonstrated that overexpression of the IL-3Ralpha or IL-3Rbeta chains will lower the concentrations of cytokines necessary to promote proliferation of cytokine-dependent cells.5,17

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JAK/STAT pathway

The JAK/STAT pathway consists of three families of genes: the JAK, or Janus family of tyrosine kinases, the STAT (signal transducers and activators of transcription) family and the CIS/SOCS family, which serves to downregulate the activity of the JAK/STAT pathway.48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 The JAK/STAT pathway involves signaling from the cytokine receptor to the nucleus. JAKs are stimulated by activation of a cytokine receptor. Stimulation of JAKs results in STAT transcription factor activity.

JAKs are a family of large tyrosine kinases, having molecular weights in the range of 120–140 kDa (1130–1142 aa). Four JAKs (JAK1, JAK2, JAK3 and Tyk2) have been identified in mammals.49 JAK proteins consist of seven different conserved domains (JH1–JH7). The JH1 constitutes a kinase domain, while JH2 is a pseudokinase domain. Many possible roles have been proposed for the different domains of the JAK proteins:54, 55,56, 57, 58, 59, 60, 61 (1) JH2 inhibits JH1; (2) JH2 promotes STAT binding; (3) JH2 is required for kinase activity of JH1; and (4) JH6 and JH7 are necessary for association of JAKs with cytokine receptors. A diagram illustrating the key features of the JAK, STAT and SOCS proteins is presented in Figure 2.

Figure 2.
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Structure of the JAK, STAT and SOCS proteins. The JAK family of proteins is centrally involved in cytokine-mediated signal transduction (see Kisseleva et al49 and Krebs and Hilton50 and references therein). The JAK family consists of JAK1, JAK2, JAK3 and TYK2. The JAK family of kinases contains seven JAK homology (JH) regions. Shown in this diagram is the JAK3 protein with the amino-acid residues indicated in the JH regions. The JH1 region is the kinase domain that contains a regulatory tyrosine residue. The STAT family of proteins consists of at least seven STAT proteins, which all have a STAT dimerization domain, a coiled-coil domain, a DNA-binding domain, an SH2 domain for interaction with tyrosine-phosphorylated proteins and finally a transactivation domain. The SOCS (suppressor of cytokine signaling proteins) consists of at least six SOCS proteins and a CIS (cytokine-inducible SH2-containing) protein. These proteins contain an amino-terminal domain which inhibits JAK activity, an SH2 domain which binds tyrosine-phosphorylated proteins and a SOCS box which promotes protein degradation via interaction with the proteosome.

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Loss of JAK1 produces prenatal lethality due to neurological disorders,53 while JAK2-/- results in embryonic lethality due to defects in erythropoiesis.54,55 JAK3 expression is limited to hematopoietic cells, and JAK3 knockout mice have developmental defects in lymphoid cells.64,65,66

Aggregation of cytokine receptors following activation allows formation of receptor homodimers and heterodimers.67 Receptor aggregation allows transphosphorylation of receptors and activation of the associated JAKs and STATs. The best evidence of transphosphorylation is JAK1 mutant cell lines, which cannot activate Tyk2 after stimulation with interferon alpha/beta.52 Another example of this transphosphorylation is that IL-2 cannot activate JAK1 in the absence of JAK3.53,68 Together these data indicate that receptor aggregation and transphosphorylation are important in activation of the JAKs.

The STAT gene family consists of seven proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6) ranging in molecular weights from 75 to 95 kDa (748–851 aa). The structure of the STAT family proteins consists of an amino-terminal oligomerization domain, a DNA-binding domain in the central part of the protein, an Src homology 2 (SH2) domain and a transactivation domain near the carboxyl terminus (Figure 2). The transactivation domain is the most divergent in size and sequence and is responsible for activation of transcription. The oligomerization domain contains a tyrosine that is rapidly phosphorylated by JAKs, allowing the phosphotyrosine product to interact with the SH2 domains of other STAT proteins. Formation of STAT dimers promotes movement to the nucleus, DNA binding and activation of transcription, as well as increased protein stability. Threonine (T) phosphorylation has been proposed to play a further role in the regulation of STAT activity.69,70 This may be mediated by ERK,70 indicating a point of interaction between the Raf/MEK/ERK and JAK/STAT pathways.

The roles of the STAT and Jak proteins in hematopoietic growth have been investigated by the creation of knockout strains of mice.71,72,73 STAT3-/- mice have severe developmental problems resulting in fetal death.73 Cytokine signaling abnormalities are associated with other STAT knockout models, but all mice are viable.

Constitutive STAT activity is associated with viral infections, but only STAT3 is known to have oncogenic properties.74, 75,76, 77, 78 v-Abl and BCR-ABL induce constitutive STAT4 activity.74,75 STAT transcription factors can induce antiapoptotic gene expression including Bcl-XL.

The JAK/STAT pathway is negatively regulated by the suppressors of cytokine signaling (SOCS) and cytokine-induced SH2-containing (CIS) family of proteins. The more accepted name for this family is SOCS. This protein family consists of SOCS1–SOCS5 and CIS1.79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 This family of genes has a conserved SH2 domain and SOCS box79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 (Figure 2). The SOCS box, the carboxy-terminal 40 amino acids, is implicated in stability and degradation of the protein by targeting it to the proteosome.82,83,84 CIS inhibits STAT5 activation by competing for its receptor-binding site.85,86 SOCS1 directly binds and inhibits the kinase domain (JH1) of JAK2.86,87,88,89 Gene ablation studies have indicated that the SOCS proteins have important roles in regulating the effects of interferon-italic gamma, growth hormone and erythropoietin. SOCS1 and SOCS3 knockout mice are lethal, whereas SOCS2 knockout mice are 30% larger than their wild-type counterparts.89,90,91,92

There are other mechanisms to downregulate JAK/STAT signaling. Protein phosphatases, including CD45 and PTPalt epsilonC, are also implicated in the negative regulation of JAK–STAT signaling.93,94

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Roles of JAK/STAT pathway in neoplasia

A chromosomal translocation forming the TEL-JAK2 fusion protein that results in constitutive kinase activity has been observed in a limited number of patients with ALL and CML.95,96 This chimeric protein has been shown to abrogate the cytokine dependence of certain hematopoietic cell lines.95,96,97,98 On the other hand, the partner TEL gene (translocated ETS in leukemia) is often rearranged in human leukemia.99 TEL rearrangement partners include Abl, PDGF-R and JAK, all of which encode tyrosine kinases. The fusion products encode constitutively active tyrosine kinases involved in human leukemia. Activation of JAK in TEL-JAK is due to the oligomerization domain provided by the TEL transcription factor, which results in the constitutive, ligand-independent activation of the JAK kinase domain.95,96,97,98 A diagram illustrating the breakpoints and resulting TEL-JAK fusion protein is presented in Figure 3.95,96,97,98,99 Activated JAK proteins have been made from the TEL-JAK rearranged chromosomal translocation.

Figure 3.
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Structure of TEL-JAK proteins. (a) Breakpoints in chimeric TEL-JAK proteins and (b) chimeric TEL-JAK proteins that have been observed in different types of leukemias. Note that the chimeric TEL-JAK proteins contain the TEL-encoded oligomerization domain and the JAK-encoded kinase. The TEL oligomerization domain makes the JAK kinase domain constitutively active. This diagram was derived in part from the work published by Drs Bernard and Gilliland and others.95,96,97,98,99

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STAT overexpression is frequently observed in human cancers.74, 75,76, 77, 78, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 The STAT3 protein can function as an oncogene,78 and other STAT proteins may function in oncogenic transformation.105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 The STAT molecules provide novel therapeutic targets for oncogenic transformation.114,115 Activating mutants of STAT5a have been made, which will abrogate the cytokine dependence of hematopoietic cells.116,117,118

Some STAT mutants were isolated based on their ability to abrogate the cytokine dependence of hematopoietic cell lines.95, 96, 97, 98, 99, 116, 118 A diagram of these retroviral constructs is presented in Figure 4. Use of these constructs has allowed determination of the ability of STAT5 to interact with other oncoproteins to transform primary hematopoietic cells. Interestingly, one activated STAT5 retrovirus contains the necessary activating mutation in the SH2 domain (N642H) and the other activated STAT5a construct requires two mutations in different domains (the coiled-coil) and the transactivation effector domains to be able to abrogate the cytokine dependence of hematopoietic cell lines.

Figure 4.
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TEL-JAK and mutated STAT5 retroviruses. The cDNAs encoding the TEL-JAK oncoprotein and activated STAT5a mutants have been inserted into retroviruses encoding neor.95, 96, 97, 98, 99, 116, 118 These retroviruses allow the relatively easy transfer of the genes into hematopoietic cells. Thus the effects of activated TEL-JAK and STAT5a proteins on other signal transduction pathways as well as the prevention of apoptosis can be determined. The amino-acid substitutions that result in STAT5 activation are indicated above the different STAT5 mutants. These constructs were made in the laboratories of Drs Bernard and Kitamura.96,97,116,117

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Ras/Raf/MEK/ERK pathway

The Ras/Raf/MEK/ERK pathway is a central signal transduction pathway, which transmits signals from multiple cell surface receptors to transcription factors in the nucleus.119,120,121 This pathway is frequently referred to as the MAP kinase pathway as MAPK stands for mitogen-activated protein kinase indicating that this pathway can be stimulated by mitogens, cytokines and growth factors. The pathway can be activated by Ras stimulating the membrane translocation of Raf. This pathway also interacts with many different signal transduction pathways including PI3K/Akt and JAK/STAT (see below).

Ras is a small GTP-binding protein, which is the common upstream molecule of several signaling pathways including Raf/MEK/ERK, PI3K/Akt and RalEGF/Ral.119,120,121 The switch regions of the Ras proteins are in part responsible for the switch between the inactive and active states of the protein. Switching between these states has been associated with conformational changes in the switch regions, which allows the binding of GTPase-activating proteins (GAPs, of which the tumor suppressor gene NF-1 is a member) and guanine nucleotide exchange factors (GEFs). When Ras is active GTP is bound, whereas when Ras is inactive GDP is bound. GTPases inactivate the Ras proteins while GEFs activate the Ras proteins by either stimulating the removal of phosphate from GTP or addition of GTP respectively. A diagram of the activation/inactivation of the Ras protein is presented in Figure 5.

Figure 5.
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Ras activation and recruitment of Raf. The activity of the Ras protein is regulated by its conformational state. There are switch regions in the Ras protein, which effect whether Ras is in an active or inactive conformation (see Figure 6). Switching between these states has been associated with conformational changes in the switch regions, which allows the binding of guanine nucleotide exchange factors (GEFs) or GTPase-activating proteins (GAPs, of which the tumor suppressor gene NF-1 is a member). When Ras is active GTP is bound, whereas when Ras is inactive GDP is bound. GTPases inactivate the Ras proteins while GEFs activate the Ras proteins by either stimulating the removal of phosphate from GTP or addition of GTP respectively. Active Ras can then bind Raf on the Ras-binding domain (BD) present in Raf. This results in the translocation of Raf to the cell membrane. Raf is then activated by phosphorylation and dephosphorylation as shown in Figure 8.

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Different mutation frequencies have been observed between Ras genes in human cancer.122,123 An excellent concise summary of the modes of regulation, activities and frequency of Ras mutations is presented in the website of Drs Watzinger and Lion.123 The structural domains of the Ras proteins, the sites of the Ras protein that are mutated, the structures of the naturally occurring Ras retrovirus and a genetically engineered Ras retrovirus124 are presented in Figure 6.

Figure 6.
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Structure of wild-type and activated ras proteins. (a) Structures of the wild-type Ras proteins containing the phosphate-binding motifs, the switch regions, the guanine base-binding domains and the CAAX motifs are presented. The switch regions of the Ras proteins are in part responsible for the switch between the inactive and active states of the protein. The Ras proteins have been divided into four regions: the amino-terminal region which is identical in Ha-Ras, Ki-Ras and N-Ras, which is followed by a 70– 80% conserved region which contains the sequences necessary for binding the guanine bases, which is followed by a hypervariable domain, and finally a CAAX domain which is necessary for interaction with the cell membrane. The amino-acid residues in the CAAX domain are C=cysteine, A=any aliphatic residue and X=any uncharged amino acid. (b) Sites of mutations in the Ras proteins. The sites of mutations detected in Ras are indicated. These mutations occur in codons that are associated with guanine nucleotide-binding sites. These mutations result in reduced GTPase activity (12, 13, 59, 61, 63) or decreased nucleotide affinity (residues 116, 117, 119, 146, 165). These mutations result in locking the Ras protein in an active state, which cannot be inactivated by the GTPase. Deletion of the NF-1 tumor suppressor gene also results in deregulated Ras expression and has been observed in certain childhood leukemias. Much of the information presented in (a,b) was derived from the Watzinger and Lion Ras family website.123 (c) Structures of the naturally occurring HaSV and engineered v-Ha-Ras-encoding retroviruses. The engineered v-Ha-Ras retrovirus allows the introduction of the activated v-Ha-Ras gene into diverse cells. The SV-X-Ha-Ras is described in detail in the work of Dr Witte and colleagues.124

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There are three different Ras family members: Ha-Ras, Ki-Ras and N-Ras. The Ras proteins show varying abilities to activate the Raf/MEK/ERK and PI3K/Akt cascades, as Ki-Ras has been associated with Raf/MEK/ERK while Ha-Ras is associated with PI3K/Akt activation.125 Amplification of ras proto-oncogenes and activating mutations that lead to the expression of constitutively active Ras proteins are observed in approximately 30% of all human cancers.123, 126, 127 The effects of Ras on proliferation and tumorigenesis have been documented in immortal cell lines.128, 129, 130, 131, 132, 133, 134, 135, 136 However, antiproliferative responses of oncogenic Ras have also been observed in nontransformed fibroblasts, primary rat Schwann cells and primary fibroblast cells of human and murine origin.133,134 This premature G1 arrest and subsequent senescence is dependent on the Raf/MEK/ERK pathway and was shown to be mediated by many cell cycle inhibitory molecules including p15Ink4b/p16Ink4a and p21Cip1.135

The Raf protein family consists of A-Raf, B-Raf and Raf-1, which are involved in the regulation of proliferation, differentiation and apoptosis induced after cytokine stimulation.1, 136, 137, 138, 139 The Raf proteins have three distinct functional domains: CR1, CR2 and CR31 (Figure 6). The CR1 domain is necessary for Ras binding and subsequent activation. The CR2 domain is a regulatory domain. CR3 is the kinase domain. Deletion of the CR1 and CR2 domains produces a constitutively active Raf protein due in part to the removal of phosphorylation sites that serve to negatively regulate the kinase in the CR2 domain. An overview of the Raf proteins is presented in Figure 7.

Figure 7.
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Structures of Raf proteins. Depicted are the structures of A-Raf, B-Raf and Raf-1 proteins. These proteins consist of three domains: CR1, CR2 and CR3. CR1 contains the Ras-binding domain (RBD) and the cysteine-rich domain (CRD). CR2 contains the regulatory domain which has sites for B-Raf and Raf-1 phosphorylation by Akt and SGK. CR3 is the kinase domain which also has many serine (S), threonine (T) and tyrosine (Y) residues which are involved in activation/inactivation. More regulatory sites have been identified on the Raf-1 protein than either the B-Raf or A-Raf proteins. This is because Raf-1 has been more intensively studied. The protein sizes, chromosomal locations and estimates of the gene sizes are indicated. Interestingly, each Raf gene is comprised of at least 15 exons and the genes range in size from 11 to 80 kb. Different alternative splicing variants of B-Raf have been detected. In the bottom half of this panel are retroviral constructs encoding activated versions of Raf. 3611-MSV is a naturally occurring Raf retrovirus. Retroviruses encoding activated versions of the Raf proteins have been made by Dr Martin McMahon and colleagues at DNAX (Palo Alto, CA, USA) and the University of California, San Francisco (San Francisco, CA, USA)158, 159, 160, 161 and Dr Hartmund Land (Imperial Cancer Research Fund, London, UK).162 These engineered Raf constructs have the Ras-binding and -regulatory domains deleted.15 The hormone-binding (hb) domains of the estrogen receptor (ER) or the androgen receptor (AR) were inserted to make the constructs conditionally active in that they require either beta-estradiol or testosterone for activation, respectively. The GFPDeltaRaf-1:ER and GFPDeltaRaf-1:AR have the green fluorescent protein (GFP) moiety fused to the amino terminus making them fluorescent upon activation by light of the appropriate wave length.15 Neo=gene encoding G418 (geneticin, G418) resistance, Puro=gene encoding puromycin resistance, Blast=gene encoding blasticidin resistance.

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The A-Raf gene is located on X chromosome in humans137 and produces a 68 kDa protein. The highest level of A-Raf expression in the adult is in the urogenital tract. A-Raf deletion results in postnatal lethality, attributed to neurological and gastrointestinal defects.138 The role of A-Raf has remained elusive. A-Raf is the weakest Raf kinase in terms of activation of ERK activity. ERK activation occurs in A-Raf knockout mice, indicating that other Raf isoforms can compensate for this deficiency.139 Some studies have indicated that A-Raf has an important role in stimulating the growth of hematopoietic cells.140 Moreover, we have observed that overexpression of activated A-Raf will abrogate the cytokine dependence of hematopoietic cells (Table 2).


The B-Raf gene is located on chromosome 7q34.141,142 B-Raf encodes a 94 kDa protein. Highest expression of B-Raf is in the testes and neuronal tissue. The B-Raf gene produces splice variants, the physiological roles of which are not yet elucidated.141 Loss of B-Raf expression in mice results in intrauterine death between days 10.5 and 12.5.142 The B-Raf knockout mouse embryo displays enlarged blood vessels and increased apoptosis of differentiated endothelial cells. This is an indication that Raf kinases can regulate apoptosis. B-Raf and Raf-1 activity are negatively regulated by Akt phosphorylation.143,144 Akt also has other effects on the presentation of apoptosis, discussed in more detail later. In contrast, other studies have shown that overexpression of B-Raf in Rat-1 cells results in decreased apoptosis due to inhibition of caspase activity.145 Historically, B-Raf is the strongest Raf in terms of induction of MEK activity as determined by in vitro kinase assays. B-Raf activation is different from either Raf-1 or A-Raf, as its activation requires phosphorylation of one regulatory residue while activation of Raf-1 and A-Raf requires two phosphorylation events.146,147 B-Raf is also regulated by Akt and the serum glucocorticoid-regulated kinase (SGK).148,149 Recently, it has been shown that B-Raf may be required in the activation of Raf-1 and that B-Raf and Raf-1 may be induced at different times following stimulation.149,150 Moreover, the three different Raf proteins may have different subcellular pools, and Raf-1 and A-Raf may be localized in some cases to the mitochondrion.151 The roles of B-Raf in human neoplasia will be discussed later.

The Raf-1 gene is located on chromosome 3p25 and produces a 74 kDa protein.137 The Raf-1 proto-oncogene was the first cloned Raf gene as it is the cellular homolog to v-Raf contained in the naturally occurring acute retrovirus 3611-MSV.152 Raf-1 is ubiquitously expressed in adult tissues, with highest expression in muscle, cerebellum and fetal brain.137 It is the most studied Raf isoform. A DN version of Raf-1 inhibits Ha-Ras-induced transformation and tumor formation.153 Raf-1 has important roles in apoptosis as it phosphorylates and inactivates Bad.154 Raf-1 phosphorylates and co-immunoprecipitates with Bcl-2155,156 as well as regulates Bag and Bad expression, in BCR/ABL-expressing cells.155 Recently, Raf-1 has been proposed to have roles independent of MEK/ERK that are often involved in the regulation of cell cycle progression and apoptosis.156,157 These new roles for Raf-1 will be discussed below.

Conditionally active Raf proteins have been made by Dr Martin McMahon's laboratory first at DNAX (Palo Alto, CA, USA) and then at the University of California (San Francisco, CA, USA).158, 159, 160, 161 These constructs contain the kinase domain (CR3) ligated to the hormone-binding (hb) domain of the estrogen receptor, rendering their activity dependent on the presence of beta-estradiol or the estrogen receptor antagonist 4 hydroxyl tamoxifen (4HT) (Figure 7). Dr McMahon and Dr Hartmund Land (ICRF, London, UK)162 have also made conditional Raf-1 kinases which contain the hb domain of the androgen receptor linked to the DeltaRaf-1, which makes Raf-1 activity dependent on the presence of testosterone. The ability of Raf proteins to phosphorylate MEK1 varies from B-Raf>Raf-1>A-Raf.159,161 The ability of Raf to abrogate cytokine dependency is inversely proportional to their MEK1 activity, with A-Raf>Raf-1>B-Raf.160,161 Stimulation of Raf activates MEK1 and ERK resulting in phosphorylation of transcription factors, proliferation, and inhibition of apoptosis.

The activation of the three Raf isoforms is complex and not totally understood. Ras is known to activate B-Raf independently of Src activity, while Raf-1 and A-Raf require Src for complete activation.146,147 Src phosphorylates Y340, Y341 of Raf-1 and Y301, Y302 on A-Raf, which are not present in B-Raf.163 B-Raf contains aspartic acid (D) at the corresponding residues. Aspartic acid residues are negatively charged and are believed to confer a constitutively active configuration at this site. B-Raf is constitutively phosphorylated on S445, a site equivalent to S338 in Raf-1, which is phosphorylated as a result of Ras activation.163 The Ras-binding domains of B-Raf and Raf-1 have a greater affinity for Ras than A-Raf.164 This suggests that A-Raf may have a primary activator other than Ras. Phosphorylation of the activation loop of Raf-1 and B-Raf is necessary but not sufficient for activation.164 Deletion or mutation of inhibitory phosphorylation sites present at certain residues by site-directed mutagenesis activates Raf proteins.165 Akt (PKB) also phosphorylates Raf-1 at S259, which has been associated with inhibition of Raf-1 activity.142,143 Akt and SGK phosphorylate B-Raf at the equivalent residue.148,149

Several protein kinases are known to regulate Raf-1 activity. CAMP-dependent protein kinase (PKA) inhibits Raf-1. CAMP activates PKA resulting in phosphorylation of Raf-1 on S43 and S621, inhibiting Raf-1 activity.166, 167, 168 This contrasts with B-Raf activation in response to PKA.168, 169, 170 Raf-1 is activated through p21-associated protein (Pak).171 Activation of conventional protein kinase C isoforms (alpha, beta and italic gamma) stimulates Raf-1 activity in a baculovirus system.172 Protein kinase Calt epsilon is associated with c-N-Ras and Raf-1 and is necessary for activation of Raf-1 by phorbol 12 myristate 13 acetate in fibroblasts.173 RKIP, a Raf-1-interacting protein, inhibits Raf-1 activation of MEK1.174 A model for activation of Raf-1 is presented in Figure 8a.

Figure 8.
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Models for Raf activation. (a) Raf-1, (b) B-Raf. These models are adapted from the publications of Dr Walter Kolch174 and Dr Catrin Pritchard.136

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14-3-3 proteins are known to bind Raf. Interaction of 14-3-3 proteins with phosphorylated S259 and S621 inhibits Raf-1.175, 176, 177, 178 Protein phosphorylase 2A can remove the phosphate on S259 allowing Raf to become disassociated from 14-3-3. This allows Raf-1 to assume a conformation in which it can be phosphorylated and activated by other kinases including PAK and Src family kinases.

Raf-1 was originally characterized as phosphorylating MEK1, but B-Raf is the primary MEK1 activator in bovine brain extracts, NIH 3T3 cells, PC12 cells and other cells.179, 180, 181, 182, 183, 184, 185 Raf-dependent activation of p90 ribosomal S6 kinase (p90Rsk) and NF-kappaB is activated by a Raf-1 mutant, which no longer binds or phosphorylates MEK1.186 This suggests that Raf-1 has physiological substrates other than MEK1.

Recently, the roles of Raf-1 in the Raf/MEK/ERK signal transduction pathway have become controversial due to the discovery that B-Raf was the more potent activator of MEK and that many of the 'functions' of Raf-1 still persist in Raf-1 but not B-Raf knockout mice. Raf-1 has been postulated to have important roles in cell cycle progression, activation of the p53 and NF-kappaB transcription factors and the prevention of apoptosis. Raf-1 has been postulated to have nonenzymatic functions and serve as a docking protein. An excellent summary of these non-MEK/ERK-related activities of Raf-1 is presented in the review by Hindley and Kolch.157 Raf-1 has been proposed to have important functions at the mitochondrial membrane. Interestingly, it has been shown that BCR-ABL may interact with Raf-1 to alter the distribution of Bag at the mitochondrial membrane and hence regulate (prevent) apoptosis in hematopoietic cells.155,157 A diagram depicting some of the non-MEK1/ERK postulated effects of Raf-1 is presented in Figure 9.

Figure 9.
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Novel Raf-1 pathways that are independent of MEK1/ERK. Many non-MEK1/ERK-mediated functions of Raf-1 have been postulated. Some of the purported roles (eg, cell cycle regulatory, antiapoptotic effects on NF-kappaB activation) may be more likely than others (eg, effects of Raf-1 on p53 transcription). BCR-ABL can activate Raf-1 activity and thus have antiapoptotic and cell cycle effects. Raf can activate the Cdk25 phosphatase, which results in CDK activation. This diagram was adapted from the information presented in the excellent reviews on non-MEK1/ERK-mediated effects on Raf-1 by Hindley and Kolch.157

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Recently, the mechanism of B-Raf regulation has been more intensively investigated.136, 179, 180, 181, 182, 183, 184, 185 A comparison of Raf-1 and B-Raf activation is presented in Figure 8a and b. B-Raf activation may occur through the GTP-binding protein Rap-1, which is in turn is activated by RA-GEF-1 and Src. PKA may activate Src in some cells, which results in Rap-1 activation (Figure 10). There are three regulatory residues on B-Raf: S444, T598 and S601. The S/T kinases which phosphorylate these residues are not known. Activated B-Raf can interact with Ras, which in turn results in the activation of Raf-1.184 B-Raf can activate MEK, which results in ERK and downstream kinases and transcription factors. Rheb (Ras-enriched homolog found in brain) and 14-3-3 proteins can bind and inhibit B-Raf activity.183 B-Raf can also be negatively regulated by phosphorylation by either SGK or Akt. Akt has been shown to phosphorylate B-Raf on three residues: S364, S428 and T439 (see below). However, Akt may have greater affinity for other substrates besides B-Raf. SGK has been proposed to be a more relevant kinase for regulating B-Raf through phosphorylation of S364.185 Potential mechanisms for activation of B-Raf are presented in Figure 10.

Figure 10.
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Potential mechanisms of activation of B-Raf. B-Raf can be activated by interactions with Rap-1. Akt and SGK can also phosphorylate B-Raf, which results in its inactivation. Rheb and 14-3-3 can negatively affect Raf activity. B-Raf may also activate Raf-1 through interaction with Ras. The S/T kinases which activate B-Raf remain controversial.

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B-Raf may also activate Raf-1 via Ras-GTP.184 While Rap-1 activates B-Raf, it may also serve to inactivate Raf-1.186 Thus the pathways for activation and inactivation of B-Raf and Raf-1 are complex and may appear contradictory.

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MEK

MEK proteins are the primary downstream targets of Raf. The MEK family of genes consists of five genes: MEK1, MEK2, MEK3, MEK4 and MEK5. This family of dual-specificity kinases has both S/T and Y kinase activity. The structure of MEK consists of an amino-terminal negative regulatory domain and a carboxy-terminal MAP kinase-binding domain, which is necessary for binding and activation of ERKs.187, 188, 189 Deletion of the regulatory MEK1 domain results in constitutive MEK1 and ERK activation.

MEK1 is a 393-amino-acid protein with a molecular weight of 44 kDa.189 MEK1 is modestly expressed in embryonic development and is elevated in adult tissue with the highest levels detected in brain tissue.189 Knockout of functional MEK1 results in lethality due to placental vascularization problems.190 Mice with DN MEK1 have also been generated; these mice were viable although defects in T-cell development occurred.191 MEK1 requires phosphorylation of S218 and S222 for activation, and substitution of these residues with D or glutamic acid (E) led to an increase in activity and foci formation in NIH3T3 cells.187, 192, 193, 194 Mutated MEK1 constructs demonstrate that activation of ERK does not require a functional MEK1 kinase domain.194 Replacement of the amino terminus of MEK1 with the hormone-binding domain of the estrogen receptor produces a construct with kinase activity responsive to the presence of estrogen analogs.195, 196, 197 This construct is an invaluable tool in research into MEK1 signaling. Studies with this construct demonstrated that activated MEK1 could abrogate cytokine dependency of certain hematopoietic cells.195, 196, 197, 198, 199 Constitutive activity of MEK1 in primary cell culture promotes senescence and induces p53 and p16INK4a, and the opposite was observed in immortalized cells or cells lacking either p53 or p16INK4a.200 Constitutive activity of MEK1 inhibits NF-kappaB transcription by negatively regulating p38MAPK activity.201

Pharmaceutical companies have developed inhibitors of MEK.202, 203, 204 The two most widely used are U0126 and PD98059, as they are commercially available. These two inhibitors have IC50 values of 70 nM and 2 muM, respectively.204 PD98059 inhibits activation, while U0126 inhibits activity. Both inhibitors have a noncompetitive mechanism of inhibiting ERK activity.202, 203, 204

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ERK

The main physiological substrates of MEK are the members of the ERK (extracellular signal-regulated kinase) or MAPK (mitogen activated protein kinase) family of genes. The ERK family consists of four distinct groups of kinases: ERK, Jun amino-terminal kinases (JNK1/2/3), p38MAPK (p38 alpha/beta/italic gamma/delta) and ERK5. In addition, there are ERK3, ERK4, ERK6, ERK7 and ERK8 kinases, which while related to ERK1 and ERK2 have different modes of activation, and their biochemical roles are not as well characterized. The ERK1 and ERK2 proteins are the most studied with regard to Raf signaling in hematopoietic cells.

ERK1 and ERK2 encode 42 and 44 kDa proteins. These proteins were originally isolated by their ability to phosphorylate microtubule-associated protein 2.205 ERKs are activated through dual phosphorylation of T182 and Y184 residues by MEK1 kinases.189 This dual phosphorylation activates ERK; however, ERK activity is downregulated by S/T phosphatases, Y phosphatases or dual-specificity (Y and S/T) phosphatases.206, 207,208, 209, 210, 211 ERK dimerization occurs subsequent to phosphorylation. This dimerization maintains the activation of ERK and promotes nuclear localization of the protein.212 Activated ERK preferentially phosphorylates S/T residues preceding a proline.213

Mice lacking ERK1 have defective T-cell development similar to transgenic mice containing a DN MEK1.214 Recently, different roles have been suggested for ERK1 and ERK2. While these two proteins are often detected at similar levels, their expression patterns may change. ERK1 has been postulated to inhibit ERK2 expression. Increased ERK2 activity is associated with cell proliferation.215 Interestingly, ERK1 has been associated with cognitive brain functions including learning.216

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p90Rsk and downstream transcription factors

Downstream targets of ERK include the p90Rsk kinase and the CREB, c-Myc and other transcription factors. ERK and p90Rsk can enter the nucleus to phosphorylate transcription factors which can lead to their activation. The effects of activation of the Raf/MEK/ERK cascade on downstream transcription factors have been recently reviewed.121

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Roles of the Ras/Raf/MEK/ERK pathway in neoplasia

Mutations at three different Ras codons (12, 13 and 61) convert Ras into a constitutively active protein.217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227 These point mutations can be induced by environmental mutagens.224 Given the high level of mutations that have been detected at Ras, this pathway has always been considered a key target for therapeutic intervention. Approximately 30% of human cancers have Ras mutations. Ras mutations are frequently observed in certain hematopoietic malignancies including myelodysplastic syndromes, juvenile myelomonocytic leukemia and acute myeloid leukemia.122, 225, 226, 227 Ras mutations are often one step in tumor progression, and mutations at other genes (chromosomal translocations, gene amplification and tumor suppressor gene inactivation) have to occur for a complete malignant phenotype to be manifested. Pharmaceutical companies have developed many farnesyl transferase (FT) inhibitors, which suppress the farnesylation of Ras, preventing it from localizing to the cell membrane.228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 The sites of action of FT and other small molecular weight membrane-permeable signal transduction pathway inhibitors are presented in Figure 11.

Figure 11.
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Interactions between the Raf/MEK/ERK and PI3K/Akt pathways and sites of action of small molecular weight signal transduction pathway inhibitors. Interactions between PI3K/Akt and Raf/MEK/ERK pathways are indicated. Established paths of signal transduction pathways are represented by solid lines. Potential sites of interaction between these two pathways are indicated by dotted lines. These pathways interact and crossregulate each other by both direct and indirect mechanisms. These pathways regulate gene expression by transcriptional, post-transcriptional and post-translational mechanisms. Black boxes indicate sites of inhibition of small molecular weight signal transduction inhibitors. Round black ovals represent proteins that serve to inhibit gene expression, induce apoptosis or inactivate proteins.

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As stated previously, there are three different Ras genes: Ki-Ras, Ha-Ras and N-Ras. The biochemical differences between these Ras proteins have remained elusive. Ki-Ras mutations have been more frequently detected in human neoplasia than Ha-Ras mutations. Ras has been shown to activate both the Raf/MEK/ERK and the PI3K/Akt pathways. Thus, mutations at Ras should theoretically activate both pathways simultaneously. Ras mutations have a key role in malignant transformation, as both of these pathways can prevent apoptosis as well as regulate cell cycle progression. Recently, it was shown that there is specificity in terms of the ability of Ki-Ras and Ha-Ras to induce the Raf/MEK/ERK and PI3K/Akt pathways.125, 248, 249 Ki-Ras preferentially activates the Raf/MEK/ERK pathway while Ha-Ras preferentially activates the PI3K/Akt pathway. Therefore, if Ras inhibitors could be developed which would specifically inhibit one particular Ras protein, it might be possible to inhibit one of the downstream pathways. This might under certain circumstances be advantageous. Furthermore, decreases in ERK expression may affect differentiation responses. Thus in certain tumors, one might desire to inhibit the effects Ras has on the PI3K/Akt pathway as opposed to the effects Ras has on Raf. Targeting of Ha-Ras as opposed to Ki-Ras might inhibit apoptosis suppression by Ha-Ras but not the effects Ki-Ras has on cell cycle progression and differentiation. A diagram illustrating the interactions between the Raf/MEK/ERK and PI3K/Akt cascades is presented in Figure 11.

Overexpression of the Raf/MEK/ERK cascade has been frequently observed in human neoplasia.250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270 A prime consequence of this activation may be the increased expression of growth factors that can potentially further activate this cascade by an autocrine loop. Many cytokine and growth factor genes contain transcription factor-binding sites, which are bound by transcription factors (Ets, Elk, Jun, Fos, CREB) whose activities are often activated by the Raf/MEK/ERK cascade. Identification of the mechanisms responsible for activation of this cascade has remained elusive. Genetic mutations at Raf, MEK or ERK were thought to be relatively rare in human neoplasia. For many years, numerous scientists felt that the activation of the Raf/MEK/ERK cascade was mainly due to mutations at Ras and hence studies aimed at elucidating the mechanisms of Ras activation were promulgated.

While mutations at the Raf gene in human neoplasia have been detected, they have not until recently gained the clinical importance that Ras mutations readily achieved. Due to more innovative, high-throughput DNA sequencing, scientists have recently discovered that the B-Raf gene is frequently mutated in certain cancers including hematopoietic tumors.136, 251, 252, 253, 255, 259, 260, 261, 265 Approximately 60% of the melanomas surveyed in one study were observed to have mutations at B-Raf.265 This result provides relevance to investigating signal transduction pathways, as by understanding how B-Raf is activated, one Ras-dependent and one Ras-independent event, scientists could predict why a single missense mutation in B-Raf permitted ligand-independent activation, whereas similar mutation events would not be predicted to result either in Raf-1 or A-Raf activation as they required multiple activation events. Interestingly, in 22 melanomas, colorectal and NSCLC tumors examined, there were 10 with mutations at B-Raf and 10 with mutations at Ras. Two tumors had mutations at B-Raf and Ras and two did not have mutations at either gene. Thus, B-Raf transformation does not appear to require Ras, and many tumors had mutations at one or the other but not both genes. A diagram of sites of mutation detected in the B-Raf protein is presented in Figure 12. Recently, it has been suggested that B-Raf mutations occur during the process of tumor progression as opposed to establishment.263 This was suggested by analysis of B-Raf mutations in different developmental stages of melanomas.

Figure 12.
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Sites of mutation in B-Raf genes. The sites of mutations detected in the B-Raf protein are presented. In all, 90% of B-Raf mutations in melanoma involve V599.136,260 V599E is the most common genetic change in thyroid papillary cancers.252 The residues that are important in B-Raf regulation by AKT are indicated. Mutations at S364, S428 and T439 interfere with Akt phosphorylation of B-Raf, which may negatively affect the induction of apoptosis.260

Full figure and legend (24K)

Raf inhibitors have been developed and some are being used in clinical trials.271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282 Raf-1 has at least 13 regulatory phosphorylation residues. Therefore, inhibition of Raf activity is difficult as certain phosphorylation events stimulate Raf activity while others inhibit Raf activity and promote Raf association with 14-3-3 proteins, which render it inactive and present in the cytoplasm.279, 280, 281, 282 Certain Raf inhibitors were developed that inhibit the Raf kinase activity as determined by assays with purified Raf proteins and substrates (MEK). Some Raf inhibitors may affect a single Raf isoform (eg, Raf-1), others may affect Raf proteins, which are more similar (Raf-1 and A-Raf),278 while other Raf inhibitors may affect all three Raf proteins (Raf-1, A-Raf and B-Raf). We have observed that the L-779,450 inhibitor suppresses the effects of A-Raf and Raf-1 more than the effects of B-Raf.278 Knowledge of the particular Raf gene mutated or overexpressed in certain tumors may provide critical information regarding how to treat the patient. Inhibition of certain Raf genes might prove beneficial while inhibition of other Raf genes under certain circumstances might prove detrimental. Thus the development of unique and broad-spectrum Raf inhibitors may prove useful in human cancer therapy.

Activation of Raf is complex and requires numerous phosphorylation and dephosphorylation events.119,120,121 Prevention of Raf activation by targeting kinases (eg, Src, PKC, PKA, PAK or Akt) and phosphatases (eg, PP2A) involved in Raf activation may be a mechanism to inhibit/regulate Raf activity.282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310 It is worth noting that some of these kinases normally inhibit Raf activation (Akt, PKA). A major limitation of this approach would be the specificities of these kinases and phosphatases. Inhibiting these kinases/phosphatases could result in activation or inactivation of other proteins and would have other effects on cell physiology.

Dimerization of Raf proteins is critical for their activity. We often think of a single Raf protein carrying out its biochemical activity. However, Raf proteins dimerize with themselves and other Raf isoforms to become active. Drugs such as coumermycin, which inhibit Raf dimerization, and others such as geldanamycin, which prevent interaction of Raf with 14-3-3 proteins, suppress Raf activity.279, 280, 281, 282 Various Raf isoforms may dimerize and result in chimeric molecules, which may have different biochemical activities. Little is known about the heterodimerization of Raf proteins.

Downstream of Raf lies MEK. Currently, it is believed that MEK1 is not frequently mutated in human cancer. However, aberrant expression of MEK1 has been detected in many different types of cancer, and mutated forms of MEK1 will transform fibroblast, hematopoietic and other cell types. Useful inhibitors to MEK have been developed that display high degrees of specificity. The successful development of MEK inhibitors may be due to the relatively few phosphorylation sites on MEK involved in activation/inactivation. MEK inhibitors are in clinical trials.119

Downstream of MEK lies ERK. To our knowledge no small molecular weight ERK inhibitors have been developed yet; however, inhibitors to ERK could prove very useful as ERK can phosphorylate many targets (Rsk, c-Myc, Elk, etc) that have growth-promoting effects. There are at least two ERK molecules regulated by the Raf/MEK/ERK cascade: ERK1 and ERK2. Little is known about the different in vivo targets of ERK1 and ERK2. However, ERK2 has been postulated to have pro-proliferative effects while ERK1 has antiproliferative effects.311, 312, 313 Development of specific inhibitors to ERK1 and ERK2 might eventually prove useful in the treatment of certain diseases.

The MAP kinase phosphatase-1 (MKP-1) removes the phosphates from ERK.314 MKP-1 is mutated in certain tumors and could be considered a tumor suppressor gene.314,315 An inhibitor to this phosphatase has been developed (Ro-31-8220).315

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PI3K/Akt pathway

Activation of the IL-3 receptor also stimulates the PI3K/Akt pathway. PI3K is a family of proteins that catalyze transfer of the italic gamma-phosphate of ATP to the D3 position of phosphoinositides. This protein family can be divided into three classes. Class I PI3K is a multisubunit protein consisting of an approximately 110 kDa catalytic subunit and a regulatory subunit of 50–100 kDa. Class I PI3K is regulated by growth factor receptor activation. The preferred in vivo substrate of class I PI3K is phosphoinositide-(4,5) diphosphate.316,317 Class II PI3K is a single peptide of 170–210 kDa with a carboxy-terminal C2 domain, necessary for phospholipid binding and kinase activity.318,319 Class III PI3K are related to Saccharomyces cerevisiae vacuolar protein sorting mutant.320 Class I PI3K is the most studied class of PI3K and the most interesting with regard to signaling in hematopoietic cells. PI3K activity is associated with cytoskeletal organization, cell division, inhibition of apoptosis and glucose uptake.321,322,323 Deletion of the PI3K p85 regulatory subunit, or the p110 catalytic subunit, results in embryonic lethality.324,325 The deletion of the carboxyl terminus of p85 produces a constitutively active oncogenic PI3K.326,327 Moreover, a constitutively active mutant was made by mutating lysine to glutamic acid (K227E) in the Ras-binding domain.323 A depiction of the structures of the class I PI3K proteins and their retroviral counterparts used to investigate the oncogenic effects of PI3K is presented in Figure 13.

Figure 13.
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Structure of PI3K. Sh2=Src homology 2 region responsible for binding tyrosine-phosphorylated residues on proteins, Sh3=Src homology region 3 region, which is a proline-rich region. BCR – region homologous to the break point cluster region. The structures of the PI3K p85 and p110 subunits are indicated as well as the sizes of the proteins. An illustration of the PI3K-containing constructs made by Dr Jullian Downward and colleagues at ICRF (London, UK), which have been used to investigate the PI3K pathways, is presented in the bottom of the figure.327

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The prototype regulatory subunit p85 consists of an amino-terminal SH3 domain, a break point-cluster-region homology domain (BCR), a proline-rich region, an interSH2 (iSH2) region and an amino-terminal SH2 domain.328,329,330 The BCR domain is highly homologous to the GTPase-activating domain of the break-point-cluster gene product331 and specifically interacts with Cdc42 and Rac1, although no GTPase activity has been found.332,333 The proline-rich regions associate with the SH3 regions of v-Src, Lyn, Fyn, GRB2, Abl and Lck.334,335,336,337,338,339 The interactions between the p85 and p110 subunits occur by the iSH2 region.340,341,342,343,344 Phosphorylation of S608 present in p85 results in a three- to seven-fold decrease in PI3K activity. The catalytic subunit has been implicated as the kinase phosphorylating S608.342,343,344,345,346,347 PI3K is inhibited by the interaction of the proline-rich domain of the adaptor protein Ruk (SH3-domain kinase-binding protein 1) with an SH3 domain of p85. The inhibition of PI3K by Ruk overexpression promotes apoptosis, which is inhibited by expression of a constitutively active p110.348

The p110 catalytic subunit of PI3K has both lipid and S/T protein kinase activity. The p110 PI3K is a kinase domain that has homology to many protein kinases.349,350 L802 present in the kinase domain is involved in the coordination of the beta-phosphate of the ATP, and is covalently modified to a Schiff base by the PI3K inhibitor wortmannin.351,352 LY294002, a quercetin-derived PI3K inhibitor, inhibits ATP binding.353,354,355 The p85 subunit inhibits and stabilizes the catalytic subunit.356

Activation of class I PI3K requires translocation to the plasma membrane and association with an activated receptor tyrosine kinase or its substrates.357,358 The localization of PI3K to the membrane is activated by the IL-3 receptor, Ras or the proline-rich regions of Shc, Lyn, Fyn, Grb2, v-Src, Abl, Lck, Cbl, or dynamin.336, 337,338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 359, 360, 361, 362 These interactions activate PI3K producing phosphoinositide-3-phosphate and/or phosphoinositide-3,4,5-triphosphate. The phosphoinositide-3-phosphate product is degraded by phosphatases, including the PTEN tumor suppressor. The kinase activity of PI3K is inhibited by cAMP.363 The phospholipid products of PI3K activate downstream targets, including PDK, Akt and PKC.364,365,366 PI3K inhibitors that show specificity have been developed. These and their derivative inhibitors show promise in suppressing this pathway in human cancer.

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PDK

Phosphoinositide-dependent kinase (PDK) requires the phospholipid product of PI3K for activation. Originally isolated from skeletal muscle using a PKB glutathione-S-affinity column, PDK is a lipid-requiring kinase.367 It is a 63 kDa S/T kinase and part of the PDK family of genes. There are believed to be two members of the PDK family – PDK1 and PDK2 – although the identity of PDK2 remains elusive. PDK1 consists of two domains: a carboxy-terminal PH domain and an amino-terminal kinase domain. The PH domain has a greater affinity for binding phosphoinositides than the PH domain of Akt.368 PDK1 is ubiquitously expressed in human tissues and is exclusively located in the cytosol.367,369,370,371

In vitro, PDK1 phosphorylates T308 of Akt, but not S473, which is required for full activation of Akt.372 In vivo, activation of PDK1 and phosphorylation of T308 of Akt is independent of PH-dependent lipid association. Association of Akt with phosphoinositides produces a conformational change allowing S473 to be phosphorylated by PDK1.367,373 Alternatively, interaction of PDK1 with the carboxy-terminal PDK1-interacting fragment of PRK2 (protein kinase C-like 2) allows PDK1 to phosphorylate T308 and S473 activating Akt.374 The precise mechanism of Akt S473 phosphorylation remains controversial. It may be phosphorylated by the integrin-linked kinase (ILK).375, 376, 377, 378, 379, 380 Translocation of PDK1 is inhibited by increased cAMP levels in the cell.381

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Akt

The downstream target of PDK1 and likely PDK2 is Akt or protein kinase B (PKB). Activated Akt was originally isolated from cells of the leukemia and lymphoma prone AKR strain of mice.382,383 c-Akt was cloned by several different groups using degenerate PCR with primers for the kinase domains,384,385,386,387 low stringency screening for protein kinase A386 and sequencing of human DNA hybridizing to v-Akt.387 Multiple clonings of the gene resulted in several different names for the Akt family of genes, including Akt, PKB, RAC-PK (related to A and C protein kinase). The Akt family of genes in mammals consists of three genes: Akt1 (PKBalpha), Akt2 (PKBbeta) and Akt3 (PKBitalic gamma). A diagram of these three proteins and certain retroviral constructs encoding Akt counterparts is presented in Figure 14. Akt1 has been mapped to chromosome 14q32, a region frequently involved in translocations in leukemia and lymphoma.382,385,388 Akt2 has been mapped to 19q13.1–q13.2, a chromosome region known to be amplified in ovarian and pancreatic cancers.389,390 Akt2 is amplified in 12.1% of ovarian and 2.8% breast cancers.391 Akt1 and Akt2 are expressed in all tissues, but the highest expression is in brain, thymus, heart, and lung.385,386,387 Akt3 expression is high in brain and testes.391

Figure 14.
Figure 14 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Structure of Akt genes. Depicted is the structure of the Akt proteins and some modified proteins used to investigate signal transduction and apoptotic pathways in hematopoietic cells. Akt consists of three domains: the pleckstrin homology domain (PH), the kinase domain and the regulatory domain. The three Akt isoforms depicted in the top part of this diagram were adapted in part from the publication of Nicholson and Anderson.385 Various naturally occurring and recombinant retroviral Akt constructs are presented in the bottom portion of the figure. The AKT8 retrovirus contains part of Akt fused with the myristylation site of Mo-MuLV retrovirus GAG protein, which targets the protein to the membrane.382,383 DeltaAkt(Myr+) is a modified Akt gene with the PH domain deleted and a v-Src-encoded myristylation site in its place making it constitutively active.399 DeltaAkt:ER* (Myr-) and DeltaAkt:ER* (Myr+) have the PH domain deleted and either lack or contain a v-Src-encoded myristylation site and a mutated hormone-binding domain (*) of the estrogen receptor, making the kinase activity regulated by 4HT.395 The mutated ER* domain binds 4HT 100-fold better than beta-estradiol.395

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Akt proteins contain three domains: PH, S/T kinase and regulatory. The PH interacts with lipids, and targets the protein to the membrane after activation.392,393 The oncogenic ability of Akt was first observed in the original isolation of the v-Akt-containing retrovirus which contained a GAG-Akt fusion protein. This retroviral-GAG protein contains a myristylation site, which alters the normal localization from the cytosol (90%) to the plasma membrane (40%), nucleus (30%) and cytosol (30%).394 Similar targeting has been achieved with c-Akt constructs used by adding a v-src-encoded myristylation domain in place of the normal PH domain. In addition, various activating point mutations were introduced into the modified Akt gene by site-directed mutagenesis. This resulted in a constitutively activated Akt construct (DeltaAkt(Myr+)). Furthermore, an additional construct was made conditional by the addition of a 4-hydroxy-tamoxifen-responsive ER* domain (DeltaAkt:ER* (Myr+)) (Figure 14). As a control, a construct lacking the v-Src myristylation domain (DeltaAkt:ER*(Myr-)) was also made.395

A model of Akt activation requires translocation of activated PI3K to the membrane385 as a result of PIP3 binding to the Akt PH domain. Next, PI3K phosphorylates phosphoinositides resulting in the translocation and activation of PDKs. PDK1 phosphorylates and activates Akt first on Thr (Akt1 T308, Akt2 T309 and Akt3 T305). PDK2 may then phosphorylate Ser (Akt1 S473, Akt2 S474 and Akt3 S472).385 However, the precise identity of PDK2 and mechanism(s) regulating serine phosphorylation of Akt are not fully defined.

The most studied biological activities of Akt are the regulation of glucose transport/metabolism and apoptosis. PI3K is required for glucose transport. Moreover, inhibition of PI3K decreases glucose transport.396,397,398 Elevated Akt activity increases glucose transport.399,400 Akt phosphorylates glycogen synthase kinase-3 (GSK-3) and 6-phosphofructose-2-kinase (PFK2) in vitro,401,402 thus regulating glucose flux to glycogen and glycolysis. Activated Akt increases protein synthesis in the presence of PI3K inhibitors,403,404,405,406,407 presumably through the increased phosphorylation of the inhibitory binding protein PHAS-1.

The antiapoptotic effects of Akt occur through its phosphorylation of a wide variety of targets. The first antiapoptotic target identified was Bad, a member of the Bcl-2 family. Phosphorylation of Bad at S136 by Akt allows phosphorylated Bad to interact with 14-3-3 proteins, promoting cell survival.408,409 Interaction of Bad with 14-3-3 proteins inhibits the ability of Bad to interact with Bcl-2 and Bcl-XL. This allows Bcl-XL to bind to pro-apoptotic Bax molecules and prevent the formation of pro-apoptotic Bax homodimers (Figure 15). However, as the model presented in Figure 15 indicates, Bad is also phosphorylated on different sites by members of the Raf/MEK/ERK (S112) and PKA (S112, S155) pathways. Thus, signal transduction pathways are complicated and intricately control the activity of apoptotic regulatory molecules such as Bad. As stated previously, Raf-1 has many effects on the regulation of apoptosis. Some of these effects occur at the mitochondrial membrane and are independent of MEK and ERK activity.

Figure 15.
Figure 15 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Interaction of Akt with bad. Bad in the unphosphorylated state associates with Bcl-2 or Bcl-XL, promoting apoptosis. Akt phosphorylates Bad on S136. ERK phosphorylate Bad on S112 and PKA phosphorylates Bad on S155. These phosphorylation events cause Bad to associate with 14-3-3 proteins, thus inhibiting apoptosis.

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Bad and Bcl-XL have been shown to be expressed in normal and leukemic hematopoietic precursor cells.409 Immature hematopoietic cells do not express Bcl-2 but do express Bcl-XL. CD34+ cells express Bcl-2, Bcl-XL and Bad. Bcl-2 expression is higher on CD34+ cells than on AML cells. Phosphorylated Bad was expressed in AML.409

In human cells, Akt phosphorylates and inactivates caspase-9.410 Overexpression of Akt inhibits cytochrome c-induced activation of caspase-9.410 Phosphorylation of the forkhead (FOXO) family of transcription factors is also attributed to Akt.411,412,413,414 This phosphorylation results in forkhead transcription factors translocation to the cytoplasm, thus inhibiting transcription of pro-apoptotic genes such as FasL.412 Akt activates transcription of antiapoptotic genes through phosphorylation of IKK and regulation of NF-kappaB.415 Akt also promotes cell survival and cell cycle progression by its ability to phosphorylate MDM2 and GSK-3.416,417 Once phosphorylated by Akt, MDM2 translocates to the nucleus and interacts with p300.417 p300 dissociates from p19ARF, resulting in the degradation of p53 and cell cycle progression.417 Akt phosphorylates GSK-3, inhibiting its activity.418,419 The decreased GSK-3 activity increases stability of beta-catenin and enhances its association with lymphoid enhancer factor–T cell factor (LEF/TCF).416 The beta-catenin–LEF/TCF complex increases transcription of proteins such as cyclin D1 and c-myc, promoting cell cycle progression.416 Akt also phosphorylates the tumor suppressor BRCA1, but the implications are not yet identified.420,421,422 It appears that Akt has many targets, whether all these targets are directly phosphorylated by Akt remains unclear. Clearly, Akt can affect both cell cycle progression and apoptosis. Akt inhibitors have been developed; however, those that are currently commercially available do not show the effectiveness and specificity that PI3K inhibitors do.119,120,121,278

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Regulation of the PI3K pathway by phosphatases

The PI3K pathway is negatively regulated by phosphatases. PTEN ((phosphatase and tensin homolog deleted on chromosome 10) also known as MMAC1, mutated in multiple advanced cancers) is considered a tumor suppressor gene.423,424,425,426,427,428 PTEN is a dual-specificity lipid and protein phosphatase that removes the 3-phosphate from the PI3K lipid product PtdIns(3,4,5)P3 to produce PtdIns(4,5)P2, which prevents Akt activation. PTEN is often mutated in human cancer. Recently, two other phosphatases, SHIP-1 and SHIP-2, have been shown to remove the 5-phosphate from PtdIns(3,4,5)P3 to produce PtdIns(3,4)P2.429,430 DN SHIP1 mutations have been detected in human leukemia.431 This pathway provides proliferative and antiapoptotic signals, and its dysregulation has often been linked with malignant transformation.432, 433, 434, 435, 436, 437, 438, 439, 440, 441 A diagram of the PTEN and SHIP phosphatases is presented in Figure 16.

Figure 16.
Figure 16 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Structures of the phosphatases PTEN and SHIP. In the top portion of the figure, the domain structure of the PTEN protein is presented. The PEST domains are involved in the regulation of protein stabilization. In the bottom of the figure, the structure of the SHIP-1 phosphatase is depicted. In the top panel, the domains present in the PTEN phosphatase are presented. In the bottom panel, the domains present in the SHIP1 protein are presented. The bottom panel was adapted from Rohrschneider et al.428 The two H above the inositol 5'phosphatase domain represent homology regions with other phosphatases. When the two NPXX (asparagine, proline, X=any amino acid) motifs are tyrosine phosphorylated, they have the potential to bind protein tyrosine-binding domain (PTB) or SH2 domains contained on proteins. The PxxP polyproline motifs can bind SH3 domains present in proteins. A rough scale of the amino acid present in the PTEN and SHIP1 protein is presented underneath the proteins.

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Roles of the PI3K/Akt/PTEN pathway in neoplasia

Ras can activate PI3K, and some Ras mutations result in deregulated PI3K and downstream Akt activation.428 PTEN negatively regulates Akt activity; hence mutations that result in PTEN loss may lead to persistently increased levels of Akt activity.424,425,426,427 Mutations and hemizygous deletions of PTEN have been detected in some primary acute leukemias and non-Hodgkin's lymphomas, and many hematopoietic cell lines lack or have low PTEN protein expression.424,425,426,427 Increased Akt expression has also been linked with tumor progression; the Akt-related Akt-2 gene is amplified in some cervical, ovarian and pancreatic cancers as well as non-Hodgkin's lymphomas.441,442,443 In cancers where there are mutations in the PI3K/Akt/PTEN pathway, there are usually mutations at PTEN or Akt and not both. A diagram of the effects of the PTEN and SHIP phosphatases on the regulation of PI3K/Akt pathway is presented in Figure 17.

Figure 17.
Figure 17 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Regulation of apoptosis and leukemia by BCR-ABL, v-Ha-Ras, PI3K, PTEN and SHIP. The effects of activating mutations on gene expression on the regulation of apoptosis and the induction of leukemia are illustrated. Different mechanisms of activation of this pathway by genetic mutations are indicated. Some of the oncogenes may induce or repress these pathways. For example, BCR-ABL suppresses SHIP1 expression, which results in elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, while BCR-ABL also induces Gap2, which interacts with SHIP1.

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Crosstalk between the Raf/MEK/ERK and PI3K/Akt signal transduction pathways

Interactions between the Raf and PI3K/Akt pathways, or crosstalk, is an area of intense research. The first implication of crosstalk between these pathways was observed after treatment of cells with pharmaceutical inhibitors of PI3K, which decreased ERK activity.444,445,446,447 Secondly, PI3K activated through the fibronectin receptor results in increased Raf activity.448 Thirdly, a DN mutant of the p85 subunit of PI3K decreased MEK1 and ERK activities.449 A decrease in proliferation of cells transfected with DN p85 was also observed.449 PI3K/Akt can affect the Raf pathway, through the interaction of Akt with the Raf family of proteins. Zimmerman and Moelling demonstrated that Raf-1 activity is decreased by Akt-mediated phosphorylation of S259.143,144 An increase in Akt activity decreased Raf-1 activity, which was differentiation specific in skeletal muscle culture models.143 Another report examined the ability of Akt activity to regulate B-Raf activity by phosphorylation on S364 and S428, residues not present in Raf-1 or A-Raf.450 The addition of active Akt decreased B-Raf activity in HEK293 cells. Inhibition of Akt by LY294002 increased B-Raf activity. Many interactions between the Raf pathway and PI3K/Akt pathway have been demonstrated.143, 144,445, 446, 447, 448, 449 Recently, it was demonstrated that it is more effective to inhibit the growth of Raf- and MEK1-transformed hematopoietic cells with inhibitors that target both the Raf/MEK/ERK and PI3K/Akt pathways.423

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Effects of activated JAK/STAT, Ras/Raf/MEK/ERK and PI3K/Akt oncogenes on the cytokine dependence of hematopoietic cells

The effects of activated forms of the JAK/STAT, Ras/Raf/MEK/ERK and PI3K/Akt pathways on the cytokine dependence of hematopoietic cells have been investigated extensively in our as well as other laboratories. Abrogation of cytokine dependence is a key step in leukemogenesis. Results that we have obtained regarding the abilities of these oncoproteins to abrogate the cytokine dependence of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells are summarized in Table 2. In order to investigate the comparative ability of these oncoproteins on cytokine dependence, it is important to investigate the effects of these oncogenes in a single laboratory where culture conditions are relatively constant. While activated TEL-JAK, STAT5A and BCR-ABL can efficiently abrogate the cytokine dependence of these cells, activated forms of Ras, Raf, MEK, PI3K and Akt show significant differences in the ability to abrogate cytokine dependence. Activated Ras and Raf will abrogate the cytokine dependence of FDC-P1 and TF-1 cells but not FL.12 cells. In contrast, activated PI3K or Akt did not abrogate the cytokine dependence of any of the cell lines although they did prolong the survival of the cells. However, on addition of an activated Raf or MEK1 oncoprotein, cytokine-independent cells were isolated from PI3K- or Akt-infected cells indicating that activation of both the Raf/MEK/ERK and PI3K/Akt pathways could synergize and lead to abrogation of cytokine dependence in cells that were normally refractory to abrogation of cytokine dependence.

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Involvement and crosstalk of BCR-ABL between the Ras/Raf/MEK/ERK and PI3K/Akt and JAK/STAT pathways

For almost 20 years now, it has been known that the BCR-ABL chromosomal translocation plays a key role in the pathogenesis of CML.451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464 The BCR-ABL oncoprotein induces a proliferative effect, while full transformation requires additional genetic events (translocation of other oncogenes, for example, AML1/EVI1, AML/ETO, NuP98/HOXA) that block differentiation.451 Over the past few years, it has been discovered that BCR-ABL also has effects on pre-existing cellular signaling circuits. BCR-ABL activates the Ras/Raf/MEK/ERK, JAK/STAT and PI3K/Akt signal transduction pathways, which results in a proliferative effect.451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464 BCR-ABL-transformed cells display hyperactivity of Ras, Raf and JAK/STAT. This could occur by multiple mechanisms, by BCR-ABL activating these pathways directly, or by the induction of autocrine cytokines, which in turn activate these pathways. BCR-ABL lies at a pinnacle position in signal transduction pathways. It can interact with Grb-2, which couples its response to Gab2 and Shc. Moreover, BCR-ABL has negative effects on the SHIP1 phosphatase, which results in an antiapoptotic effect.463 Recently, it was shown that the autophosphorylation site (Y177) present on BCR-ABL binds the Grb2 adaptor protein and is partly responsible for the lineage and severity of experimentally induced disease.464 Mutation of Y177 to Y117F dramatically impaired myeloid leukemogenesis but had less of an effect on lymphoid leukemogenesis. These investigators also determined that Y177 recruits the scaffolding adaptor protein Gab2 via a Grb2/Gab2 complex. In mutant Y177F cells, Gab2 phosphorylation was reduced. Gab2 is important for activation of both the PI3K/Akt and Raf/MEK/ERK pathways. Furthermore, these authors demonstrated that myeloid progenitors from Gab2 knockout mice are resistant to transformation by BCR-ABL as well as activation of the PI3K/Akt and Ras/Raf/MEK/ERK pathways. Thus these studies indicate the role that BCR-ABL oncoprotein has on the PI3K/Akt and Ras/Raf/MEK/ERK pathways and the critical importance of Gab2 in transformation mediated by BCR-ABL. Furthermore, as discussed previously, STAT5 is constitutively phosphorylated and Bcl-XL is overexpressed in BCR-ABL cells.75 An overview of the effects of BCR-ABL on the JAK/STAT, PI3K/Akt and Ras/Raf/MEK/ERK pathways is presented in Figure 18.

Figure 18.
Figure 18 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

BCR-ABL activation of cytokine signal transduction and sites of inhibition. BCR-ABL is a potent oncogene that can affect many downstream signaling pathways including JAK/STAT, PI3K/Akt and Raf/MEK/ERK. Furthermore, BCR-ABL can affect adaptor proteins such as Gab2 and phosphatases such as SHIP1, which further fine-tune these pathways. Many sites of inhibition of cell growth have been discussed in this review and some of them are indicated in the figure. As chemotherapeutic drugs such as imatinib are used more frequently to treat CML, more imatinib resistance will be observed. Potential methods to circumvent this drug resistance include treatment of the cells with inhibitors that target other molecules in the pathway dysregulated by BCR-ABL (eg, Ras, Raf, MEK, PI3K, BCL-2) or affect BCR-ABL function by different mechanism (eg, geldanamycin).

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There has been great success in targeting the BCR-ABL tyrosine kinase with a signal transduction inhibitor developed by Novartis, imatinib (also known as gleevec, STI571). However, upon treatment of some patients with this drug, resistance occurs.465,466,467,468 This has also been observed in in vitro studies with cells transformed to grow in response to BCR-ABL. There are different mechanisms of resistance to imatinib, for example, point mutations in the BCR-ABL kinase domain or gene amplification. Therefore, scientists have searched for additional methods to suppress the growth of these BCR-ABL-transformed, imatinib-resistant cells. One method that is showing success in suppressing the growth of the imatinib-resistant cells is to treat them with inhibitors that target either Ras or PI3K.469 It may also be effective to treat the cells with inhibitors that target the JAK kinases or a cocktail of inhibitors. An additional method to treat imatinib-resistant cells is geldanamycin treatment.470,471,472,473,474 Geldanamycin can target molecules that bind Hsp-90. Geldanamycin will destabilize the BCR-ABL protein and inhibit the growth of imatinib-resistant CML cells.

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Summary

In this review, the effects of three different signal transduction pathways and the BCR-ABL chimeric oncoprotein on normal and malignant hematopoietic cell growth have been summarized. The three pathways are normally activated after cytokine receptor ligation and they can induce growth, cell cycle progression and prevent apoptosis. Their effects on normal cell growth are often kept in check by naturally occurring inhibitors or tumor suppressor proteins. For example, the JAK/STAT pathway has the SOCS/CIS family of proteins, which serve to limit its effects by a negative feedback pathway. Deletion of the SOCS genes can lead to lethality by altering the responses to interferon-italic gamma, erythropoietin and other cytokines. Moreover, deletion of the SOCS gene can result in large mice due to defects in limiting the effects of certain cytokines that serve to limit cell growth. The abnormal TEL-JAK chimeric proteins can contribute to the neoplastic transformation of hematopoietic cells. This occurs in part due to the constitutive, cytokine-independent activation of the JAK kinase activity.

Experimentally induced deletion of Raf and MEK genes can result in embryonic lethality indicating the critical roles that these genes play in life. Mutations in Ras have been known for many years and shown to be important in myelodysplastic syndromes. Recently, mutations at Raf genes, particularly B-Raf, have been shown to play roles in human cancer, especially melanomas. The Raf/MEK/ERK pathway can be negatively regulated by the PI3K/Akt cascade as well as the MKP1 phosphatase, which inactivates phosphorylated ERK.

The PI3K/Akt pathway has the PTEN and SHIP phosphatases, which serve to fine-tune its antiapoptotic effects. Mutations at the PI3K/Akt/PTEN pathway can contribute to leukemogenesis by elevating the expression of the pleiotropic antiapoptotic Akt protein, which can both stimulate antiapoptotic and inhibit pro-apoptotic proteins. There are many different mechanisms by which mutations at the PI3K/PTEN/Akt pathway can contribute to neoplastic transformation, including point mutation (PTEN, SHIP), amplification (Akt) or deletion (PTEN).

As our knowledge of the particular genes mutated in cancers improves, our ability to treat patients afflicted with certain diseases will increase substantially. Hopefully we will be able to target the particular gene(s) in a pathway(s) affected. The genetic mutation may affect multiple signal transduction pathways. This may require development of specific treatments that target more than one pathway. Moreover, targeting multiple pathways may be more efficacious as this approach may suppress or eliminate tumor growth at lower concentrations of the drugs than that required to inhibit growth by targeting a single pathway.

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