- Email: [email protected]
Molecular scaffold protein and cellular responses
184
Research Update
directly to clinical studies; indeed, the addition of chemopreventive and antitelomerase agents to the culture medium of breast epithelial cells derived from patients with Li-Fraumeni syndrome resulted in significant reductions in the spontaneous in vitro immortalization of these cells5. It is hoped that these models of aging epithelium will, therefore, provide a unique system for the development of novel pharmacophores aimed at preventing the molecular changes that contribute to the pathogenesis of human breast cancer.
TRENDS in Endocrinology & Metabolism Vol.12 No.5 July 2001
Acknowledgement
I would like to thank V. Weaver for thoughtful discussion. References 1 Romanov, S.R. et al. (2001) Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409, 633–637 2 Schmeichel, K.L. et al. (1998) Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial phenotype. J. Mammary Gland Biol. Neoplasia 3, 201–213 3 Clevenger, C.V. and LiVolsi, V.A. (1998) The breast. In Functional Endocrine Pathology (2nd edn) (Kovacs, K. and Asa, S.L., eds), pp. 715–732, Blackwell Science
4 Rha, S.Y. et al. (1999) Changes of telomerase and telomere lengths in paried normal and cancer tissues of breast. Int. J. Oncol. 15, 839–845 5 Herbert, B.S. et al. (2001) Effects of chemopreventive and antitelomerase agents on the spontaneous immortalization of breast epithelial cells. J. Nat. Cancer Inst. 93, 39–45
Charles V. Clevenger Dept of Pathology & Laboratory Medicine, University of Pennsylvania, 513 StellarChance Labs, 422 Curie Blvd, Philadelphia, PA 19104, USA. e-mail: [email protected]
Molecular scaffold protein and cellular responses Susan M. Keenan and Joseph J. Baldassare Mitogen-activated kinases (MAPK) regulate many diverse cellular processes, including growth, differentiation and responses to stress. The organization of MAPKs through the use of scaffolding proteins is crucial for the selective activation of these kinases by different stimuli. Recent studies identify β-arrestins as members of the family of MAPK scaffold proteins. β-Arrestins not only shut off signaling by uncoupling G-protein-coupled receptors (GPCRs) from their heterotrimeric G proteins, but also contribute to the specificity of GPCRs signaling by recruiting and activating selective MAPKs.
Mitogen-activated protein kinases (MAPKs) are recognized for their essential role in intracellular signaling pathways. The kinases comprise a group of highly conserved protein Ser/Thr kinases, with homologs identified in yeast and mammalian cells1,2. Each MAPK is activated through a protein kinase cascade (Fig. 1). The cascades include three successive kinase-dependent activations: a MAPK kinase kinase (MKKK) is activated and phosphorylates a MAPK kinase (MKK), which in turn phosphorylates the terminal member of the cascade, a MAPK. The targets for the terminal MAPKs are both cytosolic and nuclear. Activation of the MAPK cascades by terminal kinases regulates many aspects of cell function, including cell proliferation, survival and differentiation1,2. There are three mammalian MAPK cascades – the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK)
and the p38 families. Components of each cascade are encoded by more than one gene and in some members (e.g. of the JNK family), genes are alternatively spliced, resulting in many isoforms. The MAPK cascades regulate different cellular responses. ERK is activated by growth factors and is thought to be important for cell proliferation, JNK is stimulated by cytokines and by environmental stress and p38 is stimulated by mediators of stress and is important for interleukin-1 induction. Although each of these cascades is Growth factor
MKKK
MKK
MK
activated by an extracellular signal, the protein kinases are rather nonspecific and inappropriate intracellular responses can occur. The localization of these kinases within the same cellular compartment further complicates the regulation of selective cellular functions. How do such cascades elicit widely varying responses from the many potential stimuli? An idea that is gaining acceptance within the signaling community is that these cascades are segregated from each other by the assembling of the cascades within large protein complexes2. The complexes Differentiation TAK1, ASK1, MLK3
Stress
Raf-1, A-Raf, B-Raf, Mos
MEKK4, DLK
MEK1, MEK2
MKK4, MKK7
MKK3, MKK6
ERK1, ERK2
JNK1, JNK2, JNK3
p38α, p38β, p38γ, p38δ
Growth, differentiation
Survival, apoptosis, growth, differentiation
Apoptosis, cytokine production
PAK
TRENDS in Endocrinology & Metabolism
Fig. 1. MAPK cascades. Each cascade consists of a MAPK, a MKK and a MKKK. These cascades respond to a variety of extracellular stimuli. Whereas the MKK and MKKK activate downsteam members of their specific cascades, the MAPK phosphorylates both cytosolic and nuclear targets, including transcription factors. Abbreviations: ASK, apotosis signal-regulatory kinase; DLK, DAP-like kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MEKK, MEK kinase; MKK, MAPK kinase; MKKK, MKK kinase; MLK, mixed linear kinase; PAK, p21-activating kinase; TAK, TFG-β activated kinase.
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Research Update
are assembled on a scaffold protein, which associates the cascade components into a single, preformed complex or module. These complexes not only guarantee specificity, but also increase signaling efficiency. The ability of scaffold proteins to organize and segregate selective MAPK cascades is important in the regulation of differential cellular responses in the budding yeast Saccharomyces cerevisiae3. In S. cerevisiae, Ste5 and Pbs2 are required for pheromone-mating and highosmolarity responses, respectively. Binding of pheromone to its G-proteincoupled receptor (GPCR) results in Gβγinduced stimulation of a specific MAPK cascade that is crucial for the mating response. With the use of two-hybrid analysis, Ste5 has been shown to bind to the members of the MAPK mating cascade, Ste11, Ste7 and Fus3. Therefore, Ste5 organizes a multicomponent signaling complex that is responsive to Gβγ activation. In the osmolarity pathway, Pbs2 scaffolds Sho1, Ste11 and Hog1. Both the mating and the osmolarity pathways include Ste11. However, there is no crosstalk between the pathways, because Ste5 and Pbs2 selectively segregate the signals of the different pathways. Although mammalian scaffolding proteins with homology to Ste5 have not yet been identified, the function of module scaffolding has been ascribed to a growing family of proteins that includes JNKinteracting protein (JIP), MAPK partner 1 (MP1), JNK/SAPK (stress-activated protein kinase)-activating protein 1 (JSAP1) and kinase suppressor of Ras (KSR) (Refs 1,2). MP1 and KSR interact with and appear to assemble members of the ERK pathway. JIP1 binds to selective members of the JNK pathway and might be important in selective activation of the JNK cascade. These non-enzymatic proteins appear to be able to segregate signals and facilitate precise intracellular interactions, which results in a signalspecific response with both speed and precision. Recent work by MacDonald et al.4 has identified a new mammalian scaffold protein, β-arrestin 2, which assembles members of the JNK pathway, including apoptosis signal-regulating kinase (ASK1; MKKK), MKK4 (a MKK) and JNK3 (a MAPK). The novel binding partners of β-arrestin 2 were identified by yeast two-hybrid screening. After demonstrating http://tem.trends.com
TRENDS in Endocrinology & Metabolism Vol.12 No.5 July 2001
that β-arrestin 2 can assemble the JNK cascade in vivo, the authors deciphered the isoform specificity of the scaffolding protein and found that β-arrestin 2 plays a highly specific role in the pathway. JNK3 only phosphorylates c-Jun when β-arrestin 2 is coexpressed with ASK1. By contrast, the activation of JNK1 is not stimulated by expression of β-arrestin 2. Furthermore, only β-arrestin 2, and not β-arrestin 1, activates JNK3 in this manner. Does β-arrestin 2 play a role in the ability of known activators of JNK to stimulate the JNK pathway? JNK activation is important for the cellular responses of several GPCRs, including the angiotensin II (AngII) receptor, AT1aR (Ref. 4). Addition of AngII to COS-7 cells, in which an epitope-tagged β-arrestin 2 is coexpressed with JNK3 and AT1aR, results in JNK3 stimulation. In the absence of β-arrestin 2, AngII does not stimulate JNK3 activity. In addition, coexpression of the tagged β-arrestin 2 with ASK1 and JNK3 results in marked JNK3 activity and detection of JNK3 and ASK1 in immunocomplexes of the tagged β-arrestin 2. These data suggest that β-arrestin 2 can assemble JNK3 module components and cause downstream amplification of the AngII signal. This finding challenges current ideas about the role of arrestins in GPCR signaling. Although high levels of β-arrestins are found in neuronal tissue, they are also expressed, and, therefore, might be important regulators of select GPCRs, in a variety of tissues2,5. Upon activation of selective GPCRs, the released Gβγ subunits activate specific GPCR kinases (GRKs), which phosphorylate the receptor. Phosphorylation of the GPCR is followed by translocation of β-arrestins to the plasma membrane, where they bind to the GPCR. Binding of β-arrestin to the receptor uncouples the GPCR from its interacting G-proteins and targets the receptor to clathrin-coated pits for internalization, desensitization and, in some cases, degradation. β-Arrestins, therefore, play an important role in ‘shutting off’ signals from GPCRs. Although the importance of β-arrestins in ‘shutting off’ GPCR signaling is well appreciated, a potential role in downstream signaling has only recently come to light. β-Arrestins can recruit nonreceptor tyrosine kinase c-Src family members to GPCRs and confer kinase activity to these
185
receptors. Furthermore, inhibition of β-arrestin-dependent internalization of selective GPCRs can block the stimulation of ERK by selective GPCRs. For example, expression of dominant–negative β-arrestin 1 inhibits m2 muscarinic acetylcholine and α2A adrenergic receptormediated activation of ERK (Refs 6,7). Recent data, mainly from Lefkowitz’s laboratory4, suggest a mechanism by which the GPCR internalization process can effect signaling to the MAPKs. In its new role, β-arrestin 2 appears to enhance AngII activation of the JNK3 signal by acting as a scaffold protein for this cascade. The JNK cascade components are preassociated with β-arrestin 2 and localized to the cytoplasm. Binding of AngII to AT1aR results in the translocation of the complex from the cytosol to the plasma membrane. Interestingly, subsequent to AngII stimulation, JNK3 redistributes from the cytoplasm to the nucleus, suggesting that activation initiates the dissociation of JNK3 from the complex and its translocation to the nucleus. After activation by AngII, AT1aR and β-arrestin 2 are found within endosomal vesicles. Thus, β-arrestin 2 has two roles. It continues the signaling process to JNK3 by scaffolding MKKK, MKK and JNK3, and begins the receptor internalization and desensitization process by targeting AT1aR to endosomes. Luttrell et al.8 have recently extended these observations to the ERK cascade. They demonstrated that ERK2 activation by AngII is regulated by β-arrestin 2. As with the JNK cascade, β-arrestin 2 scaffolds c-Raf-1, MEK1 (MAPK–ERK kinase 1) and ERK2. The mechanism by which GPCRs stimulate the MAPKs appears to depend on the specific cellular milieu in which the endogenous GPCR is expressed. Some GPCRs couple to the MAPK pathways through transactivation of a tyrosine kinase receptor or non-receptor tyrosine kinase receptor9. In other cell types or with selective GPCRs, these cascades are activated while associated on β-arrestins. What could be the purpose of this ability of β-arrestins to couple to these cascades by different mechanisms? Activation through different mechanisms might dictate different cellular outcomes. For example, the different mechanisms might localize the MAPKs to different cellular compartments or might activate the MAPKs at, or for, different periods of time.
186
Research Update
This could result in the phosphorylation of different substrates. Couple these different mechanisms with the existence of structural scaffolds, such as caveolae and rafts, and the potential for varied responses increases. The recent studies on the role of these scaffold proteins in GPCR signaling suggest that activation of selective MAPK cascades, the subcellular compartmentalization of these cascades, and the strength and duration of the signal are determined, at least in part, by the scaffold protein which the GPCRs employ to stimulate these cascades. References 1 Davis, R.J. (2000) Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252
TRENDS in Endocrinology & Metabolism Vol.12 No.5 July 2001
2 Garrington, T.P. and Johnson, G.L. (1999) Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211–218 3 Widmann, C. et al. (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143–180 4 McDonald, P.H. et al. (2000) β-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science 290, 1574–1577 5 Ferguson, S.S. (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53, 24 6 Pierce, K.L. et al. (2000) Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors. Proc. Natl. Acad. Sci. U. S. A. 97, 1489–1494
7 Vogler, O. et al. (1999) Regulation of muscarinic acetylcholine receptor sequestration and function by β-arrestin. J. Biol. Chem. 274, 12333–12338 8 Luttrell, L.M. et al. (2001) Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds. Proc. Natl. Acad. Sci. U. S. A. 98, 2449–2454 9 Luttrell, L.M. et al. (1999) Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr. Opin. Cell Biol. 11, 177–183
Sally Keenan Joseph J. Baldassare* Dept of Pharmacological and Physiological Sciences, St Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA. *e-mail: [email protected]
Meeting Report
Hormones and cancer: new insights, new challenges Wayne D. Tilley, Christine L. Clarke, Stephen N. Birrell and Nicholas Bruchovsky The Hormones and Cancer 2000 Conference was held from 3–7 November 2000 in Port Douglas, Australia.
The basic premises that underlie hormonal treatment of breast, prostate and endometrial cancers are as relevant today as they were 20 years ago, but are being continually refined by new experimental and clinical information. Manipulation of hormones is used increasingly in prevention, neoadjuvant and adjuvant treatment and for the systemic management of metastatic disease. In advanced disease, hormonal therapies are not curative, and therefore progression to hormone-refractory disease frequently occurs. If the onset of hormone resistance could be delayed or averted, which is not an unreasonable goal considering current technology, the resulting preservation of the hormonedependent state of a tumor might suffice to turn a fatal disease into a chronic one. The Hormones and Cancer 2000 Conference, which began with an overview of the epidemiology of breast and prostate cancers by Ron Ross (Norris Cancer Center, Los Angeles, CA, USA), was the setting for discussions on many of these topics. ER isoforms
Suzanne Fuqua (Baylor College of Medicine, Houston, TX, USA) examined the relative expression of ER isoforms α
and β in a pilot series of 240 breast tumour specimens. Coexpression of ERα and ERβ was found in the majority of cancers, with 76% expressing ERβ. However, ERα, rather than ERβ, was strongly associated with PR expression, and was positively correlated with biological parameters that are indicative of a good prognosis, such as low tumor grade and diploidy. ERβ expression was correlated with aneuploidy and a less favorable prognosis, especially in the absence of the expression of ERα. In keeping with this finding was work by Leigh Murphy (University of Manitoba, Winnipeg, Canada) who demonstrated that ER-positive tumors with a good prognosis tended to have a higher ERα:ERβ ratio, resulting from a significant increase in ERα mRNA expression. Benita Katzenellenbogen (University of Illinois, Urbana, IL, USA) utilized ER subtype-specific ligands to demonstrate that coactivator recruitment by the resultant ER–ligand complexes accurately reflected the agonist or antagonist character of the complex. However, there were distinct differences in recruitment of various coactivators, implying a spectrum of receptor conformations with different agonist and antagonist ligands and differences in their biology. Myles Brown (Dana-Farber Cancer Institute, Boston, MA, USA) presented a detailed examination of the dynamics of
ER-transcription complex assembly on estrogen-responsive promoters. Interestingly, in response to estrogen, ER and several coactivators are engaged by promoter sequences in a cyclic and precise order. Brown also demonstrated that tamoxifen induced the recruitment of corepressors, rather than of coactivators. Ken Korach (NIEHS/NIH, Research Triangle Park, NC, USA) used genetargeting techniques to produce lines of transgenic mice that were homozygous for a disrupted ERα gene (αERKO) and a disrupted ERβ gene (βERKO). Developmental changes were more apparent in the female reproductive tract than in that of the male. In addition, αERKO mice had underdeveloped uteri and βERKO mice showed ovarian subfertility. By contrast, there were no differences in prostate morphology between wild-type mice, αERKO mice, βERKO mice or heterozygote αERKO/βERKO mice. Shuk-mei Ho (University of Massachusetts, Worcester, MA, USA) examined the expression of ERα and ERβ in biopsy specimens of normal and malignant human prostate, using semiquantitative RT–PCR analysis. In epithelial cells from normal tissue, only transcripts of ERβ were present, along with PR and pS2, the products of two estrogen-responsive genes, suggesting that under normal conditions,
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Research Update
directly to clinical studies; indeed, the addition of chemopreventive and antitelomerase agents to the culture medium of breast epithelial cells derived from patients with Li-Fraumeni syndrome resulted in significant reductions in the spontaneous in vitro immortalization of these cells5. It is hoped that these models of aging epithelium will, therefore, provide a unique system for the development of novel pharmacophores aimed at preventing the molecular changes that contribute to the pathogenesis of human breast cancer.
TRENDS in Endocrinology & Metabolism Vol.12 No.5 July 2001
Acknowledgement
I would like to thank V. Weaver for thoughtful discussion. References 1 Romanov, S.R. et al. (2001) Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409, 633–637 2 Schmeichel, K.L. et al. (1998) Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial phenotype. J. Mammary Gland Biol. Neoplasia 3, 201–213 3 Clevenger, C.V. and LiVolsi, V.A. (1998) The breast. In Functional Endocrine Pathology (2nd edn) (Kovacs, K. and Asa, S.L., eds), pp. 715–732, Blackwell Science
4 Rha, S.Y. et al. (1999) Changes of telomerase and telomere lengths in paried normal and cancer tissues of breast. Int. J. Oncol. 15, 839–845 5 Herbert, B.S. et al. (2001) Effects of chemopreventive and antitelomerase agents on the spontaneous immortalization of breast epithelial cells. J. Nat. Cancer Inst. 93, 39–45
Charles V. Clevenger Dept of Pathology & Laboratory Medicine, University of Pennsylvania, 513 StellarChance Labs, 422 Curie Blvd, Philadelphia, PA 19104, USA. e-mail: [email protected]
Molecular scaffold protein and cellular responses Susan M. Keenan and Joseph J. Baldassare Mitogen-activated kinases (MAPK) regulate many diverse cellular processes, including growth, differentiation and responses to stress. The organization of MAPKs through the use of scaffolding proteins is crucial for the selective activation of these kinases by different stimuli. Recent studies identify β-arrestins as members of the family of MAPK scaffold proteins. β-Arrestins not only shut off signaling by uncoupling G-protein-coupled receptors (GPCRs) from their heterotrimeric G proteins, but also contribute to the specificity of GPCRs signaling by recruiting and activating selective MAPKs.
Mitogen-activated protein kinases (MAPKs) are recognized for their essential role in intracellular signaling pathways. The kinases comprise a group of highly conserved protein Ser/Thr kinases, with homologs identified in yeast and mammalian cells1,2. Each MAPK is activated through a protein kinase cascade (Fig. 1). The cascades include three successive kinase-dependent activations: a MAPK kinase kinase (MKKK) is activated and phosphorylates a MAPK kinase (MKK), which in turn phosphorylates the terminal member of the cascade, a MAPK. The targets for the terminal MAPKs are both cytosolic and nuclear. Activation of the MAPK cascades by terminal kinases regulates many aspects of cell function, including cell proliferation, survival and differentiation1,2. There are three mammalian MAPK cascades – the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK)
and the p38 families. Components of each cascade are encoded by more than one gene and in some members (e.g. of the JNK family), genes are alternatively spliced, resulting in many isoforms. The MAPK cascades regulate different cellular responses. ERK is activated by growth factors and is thought to be important for cell proliferation, JNK is stimulated by cytokines and by environmental stress and p38 is stimulated by mediators of stress and is important for interleukin-1 induction. Although each of these cascades is Growth factor
MKKK
MKK
MK
activated by an extracellular signal, the protein kinases are rather nonspecific and inappropriate intracellular responses can occur. The localization of these kinases within the same cellular compartment further complicates the regulation of selective cellular functions. How do such cascades elicit widely varying responses from the many potential stimuli? An idea that is gaining acceptance within the signaling community is that these cascades are segregated from each other by the assembling of the cascades within large protein complexes2. The complexes Differentiation TAK1, ASK1, MLK3
Stress
Raf-1, A-Raf, B-Raf, Mos
MEKK4, DLK
MEK1, MEK2
MKK4, MKK7
MKK3, MKK6
ERK1, ERK2
JNK1, JNK2, JNK3
p38α, p38β, p38γ, p38δ
Growth, differentiation
Survival, apoptosis, growth, differentiation
Apoptosis, cytokine production
PAK
TRENDS in Endocrinology & Metabolism
Fig. 1. MAPK cascades. Each cascade consists of a MAPK, a MKK and a MKKK. These cascades respond to a variety of extracellular stimuli. Whereas the MKK and MKKK activate downsteam members of their specific cascades, the MAPK phosphorylates both cytosolic and nuclear targets, including transcription factors. Abbreviations: ASK, apotosis signal-regulatory kinase; DLK, DAP-like kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MEKK, MEK kinase; MKK, MAPK kinase; MKKK, MKK kinase; MLK, mixed linear kinase; PAK, p21-activating kinase; TAK, TFG-β activated kinase.
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Research Update
are assembled on a scaffold protein, which associates the cascade components into a single, preformed complex or module. These complexes not only guarantee specificity, but also increase signaling efficiency. The ability of scaffold proteins to organize and segregate selective MAPK cascades is important in the regulation of differential cellular responses in the budding yeast Saccharomyces cerevisiae3. In S. cerevisiae, Ste5 and Pbs2 are required for pheromone-mating and highosmolarity responses, respectively. Binding of pheromone to its G-proteincoupled receptor (GPCR) results in Gβγinduced stimulation of a specific MAPK cascade that is crucial for the mating response. With the use of two-hybrid analysis, Ste5 has been shown to bind to the members of the MAPK mating cascade, Ste11, Ste7 and Fus3. Therefore, Ste5 organizes a multicomponent signaling complex that is responsive to Gβγ activation. In the osmolarity pathway, Pbs2 scaffolds Sho1, Ste11 and Hog1. Both the mating and the osmolarity pathways include Ste11. However, there is no crosstalk between the pathways, because Ste5 and Pbs2 selectively segregate the signals of the different pathways. Although mammalian scaffolding proteins with homology to Ste5 have not yet been identified, the function of module scaffolding has been ascribed to a growing family of proteins that includes JNKinteracting protein (JIP), MAPK partner 1 (MP1), JNK/SAPK (stress-activated protein kinase)-activating protein 1 (JSAP1) and kinase suppressor of Ras (KSR) (Refs 1,2). MP1 and KSR interact with and appear to assemble members of the ERK pathway. JIP1 binds to selective members of the JNK pathway and might be important in selective activation of the JNK cascade. These non-enzymatic proteins appear to be able to segregate signals and facilitate precise intracellular interactions, which results in a signalspecific response with both speed and precision. Recent work by MacDonald et al.4 has identified a new mammalian scaffold protein, β-arrestin 2, which assembles members of the JNK pathway, including apoptosis signal-regulating kinase (ASK1; MKKK), MKK4 (a MKK) and JNK3 (a MAPK). The novel binding partners of β-arrestin 2 were identified by yeast two-hybrid screening. After demonstrating http://tem.trends.com
TRENDS in Endocrinology & Metabolism Vol.12 No.5 July 2001
that β-arrestin 2 can assemble the JNK cascade in vivo, the authors deciphered the isoform specificity of the scaffolding protein and found that β-arrestin 2 plays a highly specific role in the pathway. JNK3 only phosphorylates c-Jun when β-arrestin 2 is coexpressed with ASK1. By contrast, the activation of JNK1 is not stimulated by expression of β-arrestin 2. Furthermore, only β-arrestin 2, and not β-arrestin 1, activates JNK3 in this manner. Does β-arrestin 2 play a role in the ability of known activators of JNK to stimulate the JNK pathway? JNK activation is important for the cellular responses of several GPCRs, including the angiotensin II (AngII) receptor, AT1aR (Ref. 4). Addition of AngII to COS-7 cells, in which an epitope-tagged β-arrestin 2 is coexpressed with JNK3 and AT1aR, results in JNK3 stimulation. In the absence of β-arrestin 2, AngII does not stimulate JNK3 activity. In addition, coexpression of the tagged β-arrestin 2 with ASK1 and JNK3 results in marked JNK3 activity and detection of JNK3 and ASK1 in immunocomplexes of the tagged β-arrestin 2. These data suggest that β-arrestin 2 can assemble JNK3 module components and cause downstream amplification of the AngII signal. This finding challenges current ideas about the role of arrestins in GPCR signaling. Although high levels of β-arrestins are found in neuronal tissue, they are also expressed, and, therefore, might be important regulators of select GPCRs, in a variety of tissues2,5. Upon activation of selective GPCRs, the released Gβγ subunits activate specific GPCR kinases (GRKs), which phosphorylate the receptor. Phosphorylation of the GPCR is followed by translocation of β-arrestins to the plasma membrane, where they bind to the GPCR. Binding of β-arrestin to the receptor uncouples the GPCR from its interacting G-proteins and targets the receptor to clathrin-coated pits for internalization, desensitization and, in some cases, degradation. β-Arrestins, therefore, play an important role in ‘shutting off’ signals from GPCRs. Although the importance of β-arrestins in ‘shutting off’ GPCR signaling is well appreciated, a potential role in downstream signaling has only recently come to light. β-Arrestins can recruit nonreceptor tyrosine kinase c-Src family members to GPCRs and confer kinase activity to these
185
receptors. Furthermore, inhibition of β-arrestin-dependent internalization of selective GPCRs can block the stimulation of ERK by selective GPCRs. For example, expression of dominant–negative β-arrestin 1 inhibits m2 muscarinic acetylcholine and α2A adrenergic receptormediated activation of ERK (Refs 6,7). Recent data, mainly from Lefkowitz’s laboratory4, suggest a mechanism by which the GPCR internalization process can effect signaling to the MAPKs. In its new role, β-arrestin 2 appears to enhance AngII activation of the JNK3 signal by acting as a scaffold protein for this cascade. The JNK cascade components are preassociated with β-arrestin 2 and localized to the cytoplasm. Binding of AngII to AT1aR results in the translocation of the complex from the cytosol to the plasma membrane. Interestingly, subsequent to AngII stimulation, JNK3 redistributes from the cytoplasm to the nucleus, suggesting that activation initiates the dissociation of JNK3 from the complex and its translocation to the nucleus. After activation by AngII, AT1aR and β-arrestin 2 are found within endosomal vesicles. Thus, β-arrestin 2 has two roles. It continues the signaling process to JNK3 by scaffolding MKKK, MKK and JNK3, and begins the receptor internalization and desensitization process by targeting AT1aR to endosomes. Luttrell et al.8 have recently extended these observations to the ERK cascade. They demonstrated that ERK2 activation by AngII is regulated by β-arrestin 2. As with the JNK cascade, β-arrestin 2 scaffolds c-Raf-1, MEK1 (MAPK–ERK kinase 1) and ERK2. The mechanism by which GPCRs stimulate the MAPKs appears to depend on the specific cellular milieu in which the endogenous GPCR is expressed. Some GPCRs couple to the MAPK pathways through transactivation of a tyrosine kinase receptor or non-receptor tyrosine kinase receptor9. In other cell types or with selective GPCRs, these cascades are activated while associated on β-arrestins. What could be the purpose of this ability of β-arrestins to couple to these cascades by different mechanisms? Activation through different mechanisms might dictate different cellular outcomes. For example, the different mechanisms might localize the MAPKs to different cellular compartments or might activate the MAPKs at, or for, different periods of time.
186
Research Update
This could result in the phosphorylation of different substrates. Couple these different mechanisms with the existence of structural scaffolds, such as caveolae and rafts, and the potential for varied responses increases. The recent studies on the role of these scaffold proteins in GPCR signaling suggest that activation of selective MAPK cascades, the subcellular compartmentalization of these cascades, and the strength and duration of the signal are determined, at least in part, by the scaffold protein which the GPCRs employ to stimulate these cascades. References 1 Davis, R.J. (2000) Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252
TRENDS in Endocrinology & Metabolism Vol.12 No.5 July 2001
2 Garrington, T.P. and Johnson, G.L. (1999) Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211–218 3 Widmann, C. et al. (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143–180 4 McDonald, P.H. et al. (2000) β-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science 290, 1574–1577 5 Ferguson, S.S. (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53, 24 6 Pierce, K.L. et al. (2000) Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors. Proc. Natl. Acad. Sci. U. S. A. 97, 1489–1494
7 Vogler, O. et al. (1999) Regulation of muscarinic acetylcholine receptor sequestration and function by β-arrestin. J. Biol. Chem. 274, 12333–12338 8 Luttrell, L.M. et al. (2001) Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds. Proc. Natl. Acad. Sci. U. S. A. 98, 2449–2454 9 Luttrell, L.M. et al. (1999) Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr. Opin. Cell Biol. 11, 177–183
Sally Keenan Joseph J. Baldassare* Dept of Pharmacological and Physiological Sciences, St Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA. *e-mail: [email protected]
Meeting Report
Hormones and cancer: new insights, new challenges Wayne D. Tilley, Christine L. Clarke, Stephen N. Birrell and Nicholas Bruchovsky The Hormones and Cancer 2000 Conference was held from 3–7 November 2000 in Port Douglas, Australia.
The basic premises that underlie hormonal treatment of breast, prostate and endometrial cancers are as relevant today as they were 20 years ago, but are being continually refined by new experimental and clinical information. Manipulation of hormones is used increasingly in prevention, neoadjuvant and adjuvant treatment and for the systemic management of metastatic disease. In advanced disease, hormonal therapies are not curative, and therefore progression to hormone-refractory disease frequently occurs. If the onset of hormone resistance could be delayed or averted, which is not an unreasonable goal considering current technology, the resulting preservation of the hormonedependent state of a tumor might suffice to turn a fatal disease into a chronic one. The Hormones and Cancer 2000 Conference, which began with an overview of the epidemiology of breast and prostate cancers by Ron Ross (Norris Cancer Center, Los Angeles, CA, USA), was the setting for discussions on many of these topics. ER isoforms
Suzanne Fuqua (Baylor College of Medicine, Houston, TX, USA) examined the relative expression of ER isoforms α
and β in a pilot series of 240 breast tumour specimens. Coexpression of ERα and ERβ was found in the majority of cancers, with 76% expressing ERβ. However, ERα, rather than ERβ, was strongly associated with PR expression, and was positively correlated with biological parameters that are indicative of a good prognosis, such as low tumor grade and diploidy. ERβ expression was correlated with aneuploidy and a less favorable prognosis, especially in the absence of the expression of ERα. In keeping with this finding was work by Leigh Murphy (University of Manitoba, Winnipeg, Canada) who demonstrated that ER-positive tumors with a good prognosis tended to have a higher ERα:ERβ ratio, resulting from a significant increase in ERα mRNA expression. Benita Katzenellenbogen (University of Illinois, Urbana, IL, USA) utilized ER subtype-specific ligands to demonstrate that coactivator recruitment by the resultant ER–ligand complexes accurately reflected the agonist or antagonist character of the complex. However, there were distinct differences in recruitment of various coactivators, implying a spectrum of receptor conformations with different agonist and antagonist ligands and differences in their biology. Myles Brown (Dana-Farber Cancer Institute, Boston, MA, USA) presented a detailed examination of the dynamics of
ER-transcription complex assembly on estrogen-responsive promoters. Interestingly, in response to estrogen, ER and several coactivators are engaged by promoter sequences in a cyclic and precise order. Brown also demonstrated that tamoxifen induced the recruitment of corepressors, rather than of coactivators. Ken Korach (NIEHS/NIH, Research Triangle Park, NC, USA) used genetargeting techniques to produce lines of transgenic mice that were homozygous for a disrupted ERα gene (αERKO) and a disrupted ERβ gene (βERKO). Developmental changes were more apparent in the female reproductive tract than in that of the male. In addition, αERKO mice had underdeveloped uteri and βERKO mice showed ovarian subfertility. By contrast, there were no differences in prostate morphology between wild-type mice, αERKO mice, βERKO mice or heterozygote αERKO/βERKO mice. Shuk-mei Ho (University of Massachusetts, Worcester, MA, USA) examined the expression of ERα and ERβ in biopsy specimens of normal and malignant human prostate, using semiquantitative RT–PCR analysis. In epithelial cells from normal tissue, only transcripts of ERβ were present, along with PR and pS2, the products of two estrogen-responsive genes, suggesting that under normal conditions,
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