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Intracellular adaptor molecules and AR signalling in the tumour microenvironment
Cellular Signalling 23 (2011) 1017–1021
Contents lists available at ScienceDirect
Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Review
Intracellular adaptor molecules and AR signalling in the tumour microenvironment Vikash Reebye, Andrea Frilling, Nagy A. Habib, Paul J. Mintz ⁎ Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, W12 0NN, UK
a r t i c l e
i n f o
Article history: Received 3 November 2010 Accepted 25 November 2010 Available online 3 December 2010 Keywords: Androgen receptor CRKL Casodex Intracellular adaptor molecules
a b s t r a c t Androgen deprivation therapy is the mainstay for treating advanced prostate cancer. A better understanding in the complexity of the androgen receptor (AR) signalling pathway has highlighted that this form of treatment is not sufficient. Since Huggins and Hodges made their crucial observations on the benefits of castration for prostate cancer, significant progress has been achieved in understanding the importance of the cross-talk between the hormone signalling pathway and the kinase signalling network. We now know that preventing androgen production or ligand binding to the AR does not necessarily mark the end of the road for prostate tumour growth. Emerging evidence suggests that there exists a complex set of compensatory mechanisms which allows growth factors to push the transformed cells into a ‘survival adaptation mode’ within the tumour microenvironment. An increase in autocrine and paracrine cascades of growth factor are the most commonly reported events to correlate with progression of androgen-dependent disease to a disseminated androgen independent state. The mechanism of how growth factors can sustain AR activation when cells are deprived of androgens is unknown. This is due to the lack of information about the critical factors linking the intracellular signalling molecules associated with the downstream AR signalling events triggered by growth factors. The aim of this mini review is to highlight a potentially new insight into how intracellular adaptor molecules activated by growth factors may influence and act as a molecular switch to allow the continuation of AR activity in the presence of therapeutic anti-androgens following chemical or surgical castration. © 2010 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . 2. Growth factor signalling in prostate cancer . . . . . . . 3. The role of intracellular adaptor proteins in growth factor 4. The intracellular adaptor protein CRKL in prostate cancer. References . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . signalling . . . . . . . . . .
1. Introduction The androgen receptor (AR) is a member of a large family of nuclear ligand-activated transcription factors crucial for the development and maintenance of the prostate gland but also responsible for progression of prostate cancer [1,2]. In the normal prostate, the AR is expressed mainly in the cytoplasm of epithelial cells where it specifically responds to stimulation from the androgens testosterone or its hydrolysed variant dihydrotestosterone (DHT). It is then phosphorylated and translocated
⁎ Corresponding author. Imperial College London Department of Surgery and CancerHammersmith Hospital 1st Floor, B BlockDu Cane RoadLondon, W12 0NN United Kingdom. E-mail address: [email protected] (P.J. Mintz). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.11.019
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into the nucleus in order to activate the necessary genes responsible for normal homeostasis [3,4]. Although androgens are mainly responsible for activating the AR, it is recognised that growth factors and cytokines can also manipulate activity of this receptor in the tumour microenvironment [5]. It is well established that deregulated growth factors secreted by stromal cells within the prostrate tumour microenvironment play a critical role in tumour growth and angiogenesis of epithelial cells [6,7]. The secreted growth factors may trigger paracrine and autocrine pathways in the tumour milieu and as a result, may directly support prostate cancer growth and promote hormone resistant disease. Growth factors involved in the receptor tyrosine kinase pathways in prostate cancer have been extensively investigated. It is clear that the MAPK (mitogenic activated protein kinase) and AKT (protein kinase B) pathways are important in the downstream signalling event activated
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by growth factors. However, the link between the kinase signalling to the transactivation of the genes in the nucleus is limited. Studies have shown that intracellular adaptor molecules such as Grb2 (growth factor receptor-bound 2), Nck (non-catalytic region of tyrosine kinase adaptor protein), and Crk (chicken tumour virus number 10 Regulator of Kinase) are critical in the MAPK signalling cascade [8,9]. These intracellular adaptor proteins lack catalytic domains but contain src-homology (SH) domains that are important in protein–protein interactions and assembling large multiprotein complexes. Although the mechanisms by which these events occur in prostate cancer is virtually unknown, it is likely that the cross-talk between growth factor signalling, intracellular adaptor molecules and the AR play an essential role in establishing hormone-refractory prostate cancer. A major challenge would be to better understand mechanistically how intracellular adaptor molecules are interconnected with the downstream AR signalling events triggered by extracellular growth factors in the tumour microenvironment. 2. Growth factor signalling in prostate cancer The AR can adapt to stimulation from either steroids or growth factors and cytokines; as a result this contributes to its unpredictable behaviour in a diseased or transformed environment [10]. In vitro analysis of prostate cancer cell lines have demonstrated that various growth factors such as keratinocyte growth factor (KGF), epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-1) are able to unnecessarily maintain activity of the AR even under castrated conditions in order to propagate cell survival and proliferation [11–15]. Signalling events initiated by growth factors are achieved through phosphorylation and dephosphorylation of crucial tyrosine residues. For example, Guo Z et al. reported that of the numerous tyrosine phosphorylation sites present on the AR, activation of only one tyrosine residue at position 534 was sufficient to promote hormone independent growth tumour [16]. Further in vitro analysis of posphorylated tyrosine 534 on the AR showed that the receptor was able to switch on expression of prostate specific antigen (PSA) under conditions mimicking therapeutic androgen depletion where hormone levels were low but secretion of growth factors was upregulated [16]. In a clinical setting, expression levels of the EGF receptor (EGFR) appear to increase during the development of androgen-independent prostate cancer in patients undergoing androgen deprivation therapy [17]. These studies therefore show evidence that post-translational modifications to the AR in conjunction with activation of at least one growth factor promotes survival and growth of prostate epithelial cells. EGF is interestingly now recognised as the most abundant growth factor present in prostate tissue [18,19]. Since growth factors can activate the signal-transduction pathway through adaptor molecules [20], it is not surprising that a single alteration in a growth factor pathway is sufficient to cause multiple downstream effects on tumourigenic cells including accelerated proliferation, differentiation and metastatic invasion. Despite the established concept that the AR signalling axis can still remain switched on through stimulation from alternative ligands to compensate for loss of circulating hormones, the exact mechanisms by which these cells develop androgen independent signalling remains unclear, although multiple models have been proposed and extensively reviewed [21,22]. These models include amplification and clonal selection of AR mutants which promiscuously accept other steroidal compounds for stimulation. An additional model suggests that AR specific co-activating proteins are upregulated to perpetuate signalling of the receptor along a non-steroidal or ‘outlaw’ pathway [22]. It must be noted that these different models do not necessarily occur independently of each other. A concurrent observation, however, is that growth factor expression appears to play a feature role in promoting the ‘outlaw’ pathway. In light of the recent findings
on CRKL (Crk-like) and its role in rescuing bicalutamide induced inhibition of the AR by growth factor activation [20], it demonstrates that there can be a molecular switch which allows the AR to connect with the ‘outlaw’ pathway as previously proposed [21,22]. This molecular switch may be in the form of intracellular adaptor signalling proteins that are designed to physically bridge tyrosine phosphorylated proteins to multiple intracellular signalling pathways. It is clear that there is a large gap in understanding how intracellular adaptor molecules such as CRKL are associated with the AR signalling machinery, while promoting tumour growth. 3. The role of intracellular adaptor proteins in growth factor signalling Tyrosine kinases are enzyme modules that generally have broad substrate specificities and can be used in various combinations to elicit distinct biological responses [23]. The process by which a specific response is elicited occurs via recruitment of specialised groups of proteins including scaffolds, kinases and intracellular adaptors. These proteins allow a single kinase molecule to couple with an activated receptor and result in activation of several downstream targets and biochemical pathways. The activation of tyrosine residues on target proteins results in a post-translational modification of the three-dimensional structure for that protein. The effect is that new binding sites are exposed on the target proteins which attract adaptor molecules containing specialised docking sites called src homology (SH) domains. These domains are specific protein–protein interaction modules that are designed for directing and regulating phosphorylation signalling [24]. SH containing proteins are known to bind with key phosphorylated tyrosine residues on growth factor receptors as well as other cytoplasmic proteins involved in signal transduction for cell cycle progression, gene expression, protein translocation or DNA repair [25–27]. Once bound to these key phosphorylated motifs, SH containing proteins act as substrates to regulate the activity of Ras/MAP kinases [28], ErbB2/ HER-2, [29–31] Src-kinase [32–34] and EGFR [35–37]. There are two classes of SH domains, the Src-homology 2 (SH2) and Src-homology 3 (SH3) domains. The SH2 domains are a conserved noncatalytic module of about 100 amino acids long. They were the first modular signalling domain from the Src-family of proteins recognised to bind to target peptides in a phosphotyrosine dependent manner [24,26,38,39]. SH2 domains have a unique function in coupling unrelated polypeptides to receptor tyrosine kinases in order to mediate the formation of multiprotein complexes during signalling [25,40]. This complex is maintained in a dynamic state as their affinity to activated tyrosine residue is strong enough to obtain a degree of specificity while allowing sufficient off rate for rapid reverse signalling [38]. In contrast, SH3 motifs are conserved 50–70 amino acid modules that function independently of posttranslational modification. Instead they mediate protein–protein interactions with proline rich sequences (Pro-X-X-Pro) on molecules involved in subcellular trafficking and cytoskeletal structure [41–44]. When both SH2 and SH3 domains are expressed adjacently on a molecule they have a strong influence on each others' ligand binding ability. Both the SH2 and SH3 domains communicate with each other such that the occupancy of SH2 by a specific tyrosine phosphorylated peptide enhances ligand binding of a proline rich ligand to the SH3 domain [45]. There are over 120 proteins with SH2 and SH3 domains in the human proteome; each falls into the main group of scaffold or kinase and adaptor molecules (Fig. 1) [46,47]. The scaffold molecules include Slp-76, Shc and Dapp1. The Kinases molecules include the Src family, Syk, ZAP-70, Fps, Btk and Tec. The intracellular adaptor molecules include Nck1, Nck2, Crk, CRKL, Grb2 and Grap. These molecules are distinct from the other SH containing proteins as they lack a kinase domain (Fig. 1) [48–50]. Nck, CRKL, and Grb2 are the three most characterised adaptor proteins where they are composed entirely of SH2 and SH3 domains and the relative position
V. Reebye et al. / Cellular Signalling 23 (2011) 1017–1021
Intracellular adaptors
Nck1, Nck2
SH3
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PTB SH2
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Fig. 1. SH containing proteins: scaffolds, kinases and intracellular adaptor molecules. There are over 120 proteins with SH2 and SH3 domains thus far characterised in the human proteome. Examples are shown here where the relative position of the SH domains greatly alters the biological function of the proteins. CRKL, chicken tumour virus number 10 regulator of kinase/like protein. Dapp1, Dual adaptor for phosphotyrosine and 3-phosphoinositide. FCH, FES-CIP4 homology domain. FPS, Fps-Fes protooncogene. Grb2, growth factor receptor-bound 2, Grap, Grb2 related adaptor protein. Nck, non catalytic region of tyrosine kinase. PH, pleckstrin homology domain. PTB, phosphotryrosine binding domain.SH, src homology. Shc, SH2 containing sequence. Slp76, SH2 containing leukocyte protein of 76 kDa. Syk, spleen tyrosine kinase. Zap70, zeta associated protein of 70 kDa. Adapted from Waksman et al. [50].
of these domains significantly alters the biological function of the protein. The SH2 domain targets specific phosphorylated tyrosine motifs on activated receptors and docking proteins, while their SH3 domains associate with proline rich regions of the same receptor or docking proteins. Together these modules provide an appropriate platform to facilitate growth factor signalling, cytoskeletal organisation and transcriptional regulation [24,51,52]. An example of this is seen with Grb2 where its centrally located SH2 domain binds to phosphorylated tyrosine on cell surface receptor tyrosine kinases; its N-terminal SH3 domain binds to Ras-guanine nucleotide exchange factor SOS and its C-terminal SH3 domain binds to the gab proteins [53]. Collectively the three SH modules of Grb2 recruit other intracellular signalling factors in order to promote cell survival and proliferation [54]. Similarly, the related adaptor protein Nck has also been well characterised and shows comparable and sometimes overlapping roles with Grb2 [55]. 4. The intracellular adaptor protein CRKL in prostate cancer Chicken tumour virus number 10 Regulator of Kinase-Like protein (CRKL) is a mutli-functional intracellular adaptor cell signalling protein originally isolated from a spontaneous tumour in an adult chicken [56]. It is one of the best studied intracellular adaptor molecules. To date, CRKL and Crk proteins have been shown to participate in numerous biological roles including cell proliferation, adhesion, migration and regulation of gene expression [57]. Immunohistochemical analysis of human cancers highlight that CRKL and Crk are potentially important determinants in the progression of adenocarcinoma of the prostate, breast, lung, ovary and stomach as well as sarcomas [58–61]. These studies show the importance of CRKL in not only increasing the responsiveness of cells to growth and proliferation within a transformed tumour microenvironment through intracellular signal transduction; but also in regulating the amplitude at which cells respond to this change. Mechanistically the input signal to CRKL occurs through its SH2 domain, where it specifically binds to activated phosphotyrosine containing proteins. Its output signal occurs via the two SH3 domains to mediate
subsequent protein–protein interactions for signal transduction events (similar to Grb2). Activation of the input signal is dependent on the availability of extracellular triggers including hormones and growth factors. This is also dependent on the state of the microenvironment in which the cells reside in. A change in proliferation, differentiation, or metabolic constituent of the cell causes a rapid response of CRKL to fluctuate between different signal transduction pathways [57]. Since the progression of advance prostate cancer reflects a significantly enhanced crossover of signalling events between the epithelial and stromal layers, it is not surprising that an intracellular adaptor protein such as CRKL is involved in changing the behaviour of the AR [20]. Numerous studies have attempted to uncover the mechanism(s) by which the AR progresses towards an androgen independent state. Although several models have been proposed as mentioned previously, no single study has been able to explain how the AR can bypass its dependency on steroids while maintaining its transcriptional activity. It is likely that intracellular adaptor molecules such as CRKL are part of a dynamic signalling process which promotes the assembly and compartmentalisation of accessory proteins through its SH2 and SH3 domains to physically bridge this to the AR complex. These protein–protein interactions can further amplify AR signalling in an androgen depleted environment thus leading to continuous transactivation of genes involved in cell survival and tumour growth (Fig. 2). This model is a modified version of the outlaw pathway as proposed by Pienta and Bradley [22] which incorporates the recent finding by Reebye et al., [20] and suggests that intracellular adaptor molecules are responsible for assembling and relaying downstream signalling events from the MAPK pathway to the AR transcriptional complex in the nucleus. The advantage of having intracellular adaptor proteins is that it can adapt to a dynamically variable environment where stromally derived growth factors and cytokines are constantly changing as androgen independent disease progresses (Fig. 2). The role that adaptor proteins play in steroid receptor signalling has been largely neglected and thus the recent finding by Reebye et al. [20] and the model proposed in this review provides new insight into the importance of intracellular adaptor
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Fig. 2. Intracellular adaptor proteins are responsible for relaying downstream signalling events from MAPK pathway to the AR transcriptional complex. Signal transduction derived from the tumour microenvironment including secretion of growth factors activates the SH2 domain of adaptor proteins via growth factor receptors or receptor tyrosinse kinases. This in turn recruits intracellular adaptor proteins such as CRKL to interact with phosphorylated tyrosine motifs or proline rich regions on the target protein such as the AR. The SH3 domain of the adaptor protein is then thought to mediate subsequent protein–protein interactions for transcriptional activity of the AR. As a result, adaptor proteins are thought to be responsible for physically bridging intracellular events directly to the AR for its continual transcriptional activity to maintain growth and survival of prostrate epithelial cells.
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Contents lists available at ScienceDirect
Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Review
Intracellular adaptor molecules and AR signalling in the tumour microenvironment Vikash Reebye, Andrea Frilling, Nagy A. Habib, Paul J. Mintz ⁎ Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, W12 0NN, UK
a r t i c l e
i n f o
Article history: Received 3 November 2010 Accepted 25 November 2010 Available online 3 December 2010 Keywords: Androgen receptor CRKL Casodex Intracellular adaptor molecules
a b s t r a c t Androgen deprivation therapy is the mainstay for treating advanced prostate cancer. A better understanding in the complexity of the androgen receptor (AR) signalling pathway has highlighted that this form of treatment is not sufficient. Since Huggins and Hodges made their crucial observations on the benefits of castration for prostate cancer, significant progress has been achieved in understanding the importance of the cross-talk between the hormone signalling pathway and the kinase signalling network. We now know that preventing androgen production or ligand binding to the AR does not necessarily mark the end of the road for prostate tumour growth. Emerging evidence suggests that there exists a complex set of compensatory mechanisms which allows growth factors to push the transformed cells into a ‘survival adaptation mode’ within the tumour microenvironment. An increase in autocrine and paracrine cascades of growth factor are the most commonly reported events to correlate with progression of androgen-dependent disease to a disseminated androgen independent state. The mechanism of how growth factors can sustain AR activation when cells are deprived of androgens is unknown. This is due to the lack of information about the critical factors linking the intracellular signalling molecules associated with the downstream AR signalling events triggered by growth factors. The aim of this mini review is to highlight a potentially new insight into how intracellular adaptor molecules activated by growth factors may influence and act as a molecular switch to allow the continuation of AR activity in the presence of therapeutic anti-androgens following chemical or surgical castration. © 2010 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . 2. Growth factor signalling in prostate cancer . . . . . . . 3. The role of intracellular adaptor proteins in growth factor 4. The intracellular adaptor protein CRKL in prostate cancer. References . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction The androgen receptor (AR) is a member of a large family of nuclear ligand-activated transcription factors crucial for the development and maintenance of the prostate gland but also responsible for progression of prostate cancer [1,2]. In the normal prostate, the AR is expressed mainly in the cytoplasm of epithelial cells where it specifically responds to stimulation from the androgens testosterone or its hydrolysed variant dihydrotestosterone (DHT). It is then phosphorylated and translocated
⁎ Corresponding author. Imperial College London Department of Surgery and CancerHammersmith Hospital 1st Floor, B BlockDu Cane RoadLondon, W12 0NN United Kingdom. E-mail address: [email protected] (P.J. Mintz). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.11.019
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into the nucleus in order to activate the necessary genes responsible for normal homeostasis [3,4]. Although androgens are mainly responsible for activating the AR, it is recognised that growth factors and cytokines can also manipulate activity of this receptor in the tumour microenvironment [5]. It is well established that deregulated growth factors secreted by stromal cells within the prostrate tumour microenvironment play a critical role in tumour growth and angiogenesis of epithelial cells [6,7]. The secreted growth factors may trigger paracrine and autocrine pathways in the tumour milieu and as a result, may directly support prostate cancer growth and promote hormone resistant disease. Growth factors involved in the receptor tyrosine kinase pathways in prostate cancer have been extensively investigated. It is clear that the MAPK (mitogenic activated protein kinase) and AKT (protein kinase B) pathways are important in the downstream signalling event activated
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by growth factors. However, the link between the kinase signalling to the transactivation of the genes in the nucleus is limited. Studies have shown that intracellular adaptor molecules such as Grb2 (growth factor receptor-bound 2), Nck (non-catalytic region of tyrosine kinase adaptor protein), and Crk (chicken tumour virus number 10 Regulator of Kinase) are critical in the MAPK signalling cascade [8,9]. These intracellular adaptor proteins lack catalytic domains but contain src-homology (SH) domains that are important in protein–protein interactions and assembling large multiprotein complexes. Although the mechanisms by which these events occur in prostate cancer is virtually unknown, it is likely that the cross-talk between growth factor signalling, intracellular adaptor molecules and the AR play an essential role in establishing hormone-refractory prostate cancer. A major challenge would be to better understand mechanistically how intracellular adaptor molecules are interconnected with the downstream AR signalling events triggered by extracellular growth factors in the tumour microenvironment. 2. Growth factor signalling in prostate cancer The AR can adapt to stimulation from either steroids or growth factors and cytokines; as a result this contributes to its unpredictable behaviour in a diseased or transformed environment [10]. In vitro analysis of prostate cancer cell lines have demonstrated that various growth factors such as keratinocyte growth factor (KGF), epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-1) are able to unnecessarily maintain activity of the AR even under castrated conditions in order to propagate cell survival and proliferation [11–15]. Signalling events initiated by growth factors are achieved through phosphorylation and dephosphorylation of crucial tyrosine residues. For example, Guo Z et al. reported that of the numerous tyrosine phosphorylation sites present on the AR, activation of only one tyrosine residue at position 534 was sufficient to promote hormone independent growth tumour [16]. Further in vitro analysis of posphorylated tyrosine 534 on the AR showed that the receptor was able to switch on expression of prostate specific antigen (PSA) under conditions mimicking therapeutic androgen depletion where hormone levels were low but secretion of growth factors was upregulated [16]. In a clinical setting, expression levels of the EGF receptor (EGFR) appear to increase during the development of androgen-independent prostate cancer in patients undergoing androgen deprivation therapy [17]. These studies therefore show evidence that post-translational modifications to the AR in conjunction with activation of at least one growth factor promotes survival and growth of prostate epithelial cells. EGF is interestingly now recognised as the most abundant growth factor present in prostate tissue [18,19]. Since growth factors can activate the signal-transduction pathway through adaptor molecules [20], it is not surprising that a single alteration in a growth factor pathway is sufficient to cause multiple downstream effects on tumourigenic cells including accelerated proliferation, differentiation and metastatic invasion. Despite the established concept that the AR signalling axis can still remain switched on through stimulation from alternative ligands to compensate for loss of circulating hormones, the exact mechanisms by which these cells develop androgen independent signalling remains unclear, although multiple models have been proposed and extensively reviewed [21,22]. These models include amplification and clonal selection of AR mutants which promiscuously accept other steroidal compounds for stimulation. An additional model suggests that AR specific co-activating proteins are upregulated to perpetuate signalling of the receptor along a non-steroidal or ‘outlaw’ pathway [22]. It must be noted that these different models do not necessarily occur independently of each other. A concurrent observation, however, is that growth factor expression appears to play a feature role in promoting the ‘outlaw’ pathway. In light of the recent findings
on CRKL (Crk-like) and its role in rescuing bicalutamide induced inhibition of the AR by growth factor activation [20], it demonstrates that there can be a molecular switch which allows the AR to connect with the ‘outlaw’ pathway as previously proposed [21,22]. This molecular switch may be in the form of intracellular adaptor signalling proteins that are designed to physically bridge tyrosine phosphorylated proteins to multiple intracellular signalling pathways. It is clear that there is a large gap in understanding how intracellular adaptor molecules such as CRKL are associated with the AR signalling machinery, while promoting tumour growth. 3. The role of intracellular adaptor proteins in growth factor signalling Tyrosine kinases are enzyme modules that generally have broad substrate specificities and can be used in various combinations to elicit distinct biological responses [23]. The process by which a specific response is elicited occurs via recruitment of specialised groups of proteins including scaffolds, kinases and intracellular adaptors. These proteins allow a single kinase molecule to couple with an activated receptor and result in activation of several downstream targets and biochemical pathways. The activation of tyrosine residues on target proteins results in a post-translational modification of the three-dimensional structure for that protein. The effect is that new binding sites are exposed on the target proteins which attract adaptor molecules containing specialised docking sites called src homology (SH) domains. These domains are specific protein–protein interaction modules that are designed for directing and regulating phosphorylation signalling [24]. SH containing proteins are known to bind with key phosphorylated tyrosine residues on growth factor receptors as well as other cytoplasmic proteins involved in signal transduction for cell cycle progression, gene expression, protein translocation or DNA repair [25–27]. Once bound to these key phosphorylated motifs, SH containing proteins act as substrates to regulate the activity of Ras/MAP kinases [28], ErbB2/ HER-2, [29–31] Src-kinase [32–34] and EGFR [35–37]. There are two classes of SH domains, the Src-homology 2 (SH2) and Src-homology 3 (SH3) domains. The SH2 domains are a conserved noncatalytic module of about 100 amino acids long. They were the first modular signalling domain from the Src-family of proteins recognised to bind to target peptides in a phosphotyrosine dependent manner [24,26,38,39]. SH2 domains have a unique function in coupling unrelated polypeptides to receptor tyrosine kinases in order to mediate the formation of multiprotein complexes during signalling [25,40]. This complex is maintained in a dynamic state as their affinity to activated tyrosine residue is strong enough to obtain a degree of specificity while allowing sufficient off rate for rapid reverse signalling [38]. In contrast, SH3 motifs are conserved 50–70 amino acid modules that function independently of posttranslational modification. Instead they mediate protein–protein interactions with proline rich sequences (Pro-X-X-Pro) on molecules involved in subcellular trafficking and cytoskeletal structure [41–44]. When both SH2 and SH3 domains are expressed adjacently on a molecule they have a strong influence on each others' ligand binding ability. Both the SH2 and SH3 domains communicate with each other such that the occupancy of SH2 by a specific tyrosine phosphorylated peptide enhances ligand binding of a proline rich ligand to the SH3 domain [45]. There are over 120 proteins with SH2 and SH3 domains in the human proteome; each falls into the main group of scaffold or kinase and adaptor molecules (Fig. 1) [46,47]. The scaffold molecules include Slp-76, Shc and Dapp1. The Kinases molecules include the Src family, Syk, ZAP-70, Fps, Btk and Tec. The intracellular adaptor molecules include Nck1, Nck2, Crk, CRKL, Grb2 and Grap. These molecules are distinct from the other SH containing proteins as they lack a kinase domain (Fig. 1) [48–50]. Nck, CRKL, and Grb2 are the three most characterised adaptor proteins where they are composed entirely of SH2 and SH3 domains and the relative position
V. Reebye et al. / Cellular Signalling 23 (2011) 1017–1021
Intracellular adaptors
Nck1, Nck2
SH3
Grb2, Grap
SH3
Crk, CRKL
Src family
Kinases
Syk, Zap-70
SH2
SH2
Tyrosine Kinase
SH2
SAM
Tyrosine Kinase
SH2
Shc Dapp1
Tyrosine Kinase
SH3
FCH
SH2
SH3
SH2
SH2
Sip76
SH3 SH3
SH3
SH3
Fps
Scaffolds
SH3
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PTB SH2
SH2 PH
Fig. 1. SH containing proteins: scaffolds, kinases and intracellular adaptor molecules. There are over 120 proteins with SH2 and SH3 domains thus far characterised in the human proteome. Examples are shown here where the relative position of the SH domains greatly alters the biological function of the proteins. CRKL, chicken tumour virus number 10 regulator of kinase/like protein. Dapp1, Dual adaptor for phosphotyrosine and 3-phosphoinositide. FCH, FES-CIP4 homology domain. FPS, Fps-Fes protooncogene. Grb2, growth factor receptor-bound 2, Grap, Grb2 related adaptor protein. Nck, non catalytic region of tyrosine kinase. PH, pleckstrin homology domain. PTB, phosphotryrosine binding domain.SH, src homology. Shc, SH2 containing sequence. Slp76, SH2 containing leukocyte protein of 76 kDa. Syk, spleen tyrosine kinase. Zap70, zeta associated protein of 70 kDa. Adapted from Waksman et al. [50].
of these domains significantly alters the biological function of the protein. The SH2 domain targets specific phosphorylated tyrosine motifs on activated receptors and docking proteins, while their SH3 domains associate with proline rich regions of the same receptor or docking proteins. Together these modules provide an appropriate platform to facilitate growth factor signalling, cytoskeletal organisation and transcriptional regulation [24,51,52]. An example of this is seen with Grb2 where its centrally located SH2 domain binds to phosphorylated tyrosine on cell surface receptor tyrosine kinases; its N-terminal SH3 domain binds to Ras-guanine nucleotide exchange factor SOS and its C-terminal SH3 domain binds to the gab proteins [53]. Collectively the three SH modules of Grb2 recruit other intracellular signalling factors in order to promote cell survival and proliferation [54]. Similarly, the related adaptor protein Nck has also been well characterised and shows comparable and sometimes overlapping roles with Grb2 [55]. 4. The intracellular adaptor protein CRKL in prostate cancer Chicken tumour virus number 10 Regulator of Kinase-Like protein (CRKL) is a mutli-functional intracellular adaptor cell signalling protein originally isolated from a spontaneous tumour in an adult chicken [56]. It is one of the best studied intracellular adaptor molecules. To date, CRKL and Crk proteins have been shown to participate in numerous biological roles including cell proliferation, adhesion, migration and regulation of gene expression [57]. Immunohistochemical analysis of human cancers highlight that CRKL and Crk are potentially important determinants in the progression of adenocarcinoma of the prostate, breast, lung, ovary and stomach as well as sarcomas [58–61]. These studies show the importance of CRKL in not only increasing the responsiveness of cells to growth and proliferation within a transformed tumour microenvironment through intracellular signal transduction; but also in regulating the amplitude at which cells respond to this change. Mechanistically the input signal to CRKL occurs through its SH2 domain, where it specifically binds to activated phosphotyrosine containing proteins. Its output signal occurs via the two SH3 domains to mediate
subsequent protein–protein interactions for signal transduction events (similar to Grb2). Activation of the input signal is dependent on the availability of extracellular triggers including hormones and growth factors. This is also dependent on the state of the microenvironment in which the cells reside in. A change in proliferation, differentiation, or metabolic constituent of the cell causes a rapid response of CRKL to fluctuate between different signal transduction pathways [57]. Since the progression of advance prostate cancer reflects a significantly enhanced crossover of signalling events between the epithelial and stromal layers, it is not surprising that an intracellular adaptor protein such as CRKL is involved in changing the behaviour of the AR [20]. Numerous studies have attempted to uncover the mechanism(s) by which the AR progresses towards an androgen independent state. Although several models have been proposed as mentioned previously, no single study has been able to explain how the AR can bypass its dependency on steroids while maintaining its transcriptional activity. It is likely that intracellular adaptor molecules such as CRKL are part of a dynamic signalling process which promotes the assembly and compartmentalisation of accessory proteins through its SH2 and SH3 domains to physically bridge this to the AR complex. These protein–protein interactions can further amplify AR signalling in an androgen depleted environment thus leading to continuous transactivation of genes involved in cell survival and tumour growth (Fig. 2). This model is a modified version of the outlaw pathway as proposed by Pienta and Bradley [22] which incorporates the recent finding by Reebye et al., [20] and suggests that intracellular adaptor molecules are responsible for assembling and relaying downstream signalling events from the MAPK pathway to the AR transcriptional complex in the nucleus. The advantage of having intracellular adaptor proteins is that it can adapt to a dynamically variable environment where stromally derived growth factors and cytokines are constantly changing as androgen independent disease progresses (Fig. 2). The role that adaptor proteins play in steroid receptor signalling has been largely neglected and thus the recent finding by Reebye et al. [20] and the model proposed in this review provides new insight into the importance of intracellular adaptor
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Fig. 2. Intracellular adaptor proteins are responsible for relaying downstream signalling events from MAPK pathway to the AR transcriptional complex. Signal transduction derived from the tumour microenvironment including secretion of growth factors activates the SH2 domain of adaptor proteins via growth factor receptors or receptor tyrosinse kinases. This in turn recruits intracellular adaptor proteins such as CRKL to interact with phosphorylated tyrosine motifs or proline rich regions on the target protein such as the AR. The SH3 domain of the adaptor protein is then thought to mediate subsequent protein–protein interactions for transcriptional activity of the AR. As a result, adaptor proteins are thought to be responsible for physically bridging intracellular events directly to the AR for its continual transcriptional activity to maintain growth and survival of prostrate epithelial cells.
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