Gotowa bibliografia na temat „RasGAP SH3 domain binding proteins”

ABSTRACT A novel member of the p62 dok family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62 dok bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62 dok , DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62 dok , suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.

Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.

ABSTRACT Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G1-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2α (eIF-2α) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.

ABSTRACT A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3′-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.

Abstract Abstract 3683 Poster Board III-619 Rap1, a small GTPase of the Ras superfamily, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, cell morphology, adherens junction positioning and adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1 mediated in inside-out activation of integrins and cell adhesion. However, significant differences in the structure and interactions of these molecules indicate that they may mediate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP, multiple XPPPP motifs interacting with Profilin and multiple PXXP motifs capable of interacting with SH3 domain containing proteins. Because of these properties RIAM is a regulator of actin polymerization but also interacts with components of the T cell signaling machinery. In contrast, RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region, and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. For this purpose we used RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, had been depleted by siRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. Moreover, RIAM knockdown resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. In contrast, RapL knockdown had no effect on these events compared to control Jurkat T cells. Strikingly, RIAM-KD cells displayed impaired IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon stimulation under the same conditions. These results indicate that although both RIAM and RapL regulate Rap1-dependent LFA-1 activation, these molecules have distinct roles in regulating signaling and functional outcomes of T cell responses after T cell receptor triggering. Disclosures: No relevant conflicts of interest to declare.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

G3BP1 and G3BP2 are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of various RNA binding proteins is common in cancer, and several of these proteins influence tumorigenesis. Therefore, detailed examination of RNA binding proteins, such as G3BPs, may provide insights into the post-transcriptional mechanisms underlying tumorigenesis. Tumours arise as a consequence of genetic mutation or alteration, which often result from stress-induced DNA damage. Cancer progression is facilitated by various epigenetic stress adaptation mechanisms. Stressful stimuli induce transitory translational shut-off, mediated by phosphorylation of eukaryotic initiation factor alpha;(eIF2alpha;). This phosphorylation event leads to formation of discrete cytoplasmic foci known as stress granules (SGs), which are translationally-silent sites of mRNA sorting. It was initially thought that an RNA-binding protein, T-cell internal antigen 1 (TIA-1), was instrumental in both the formation and functioning of SGs, because over-expression of TIA-1 induces spontaneous SGs and concomitantly causes a decrease in reporter gene expression. It is now clear that SG content can change depending on the type of stress, and that various proteins, including G3BP1, can induce spontaneous SGs. In vitro evidence previously implicated both G3BP1 and G3BP2 as endoribonucleases, so it was suggested that G3BPs act to target mRNA for decay at the SG. This project sought to further investigate this proposal, and in this way gain insight into the specific function of G3BPs in post-transcriptional regulation during tumorigenesis. Characterisation of G3BP1 and G3BP2 expression and localisation patterns in human cells and cancer was necessary before functional analyses in human cell systems could be undertaken. Both proteins were found to be over-expressed in breast cancer, irrespective of cancer stage or grade. G3BP1 and G3BP2 were also expressed in all human cell lines tested, despite previously observed tissue-specific expression. These results support the notion that G3BP expression is switched on in parallel with cell proliferation, and as such, may influence tumorigenesis. The results of further analyses suggested that the diverse functions attributed to G3BP1 and G3BP2 may be facilitated by isoform-specific expression, various post-translational modifications and sub-cellular localisation. Despite the absence of a canonical endoribonuclease domain, it was previously reported that site-specific phosphorylation of G3BP1 enables the protein to degrade a synthetic c-myc RNA substrate in vitro. This finding implicated G3BP in the specific regulation of a proto-oncogene. Tailored reporter assays were thus designed in order to address the in vivo consequences of G3BP's putative endoribonuclease activity. Contrary to expectations, all G3BP family members increased or maintained the expression of a range of reporters, at both the mRNA and protein level, irrespective of the presence of any particular cis-acting element, coding sequence or promoter. These results support the emerging notion that G3BPs positively affect the expression of at least some of their target mRNAs, and may also indirectly promote transcription. In contrast to the theory that G3BPs degrade proto-oncogenic mRNA/s, these findings are consistent with a role for G3BP in promoting cell proliferation and survival. Further analyses showed that G3BP1 and G3BP2 simultaneously increased reporter gene expression and induced SG formation. These findings highlighted the fact that SGs are dynamic sorting stations for mRNAs, and not merely sites of stalled translation. This result also supports the notion that a variety of proteins may be recruited to the SG to facilitate a multitude of mRNA fates. Although the precise role of the SG in stress adapation is not known, it is clear that an appropriate integrated stress response (ISR) is required for cells to survive in sub-optimal conditions. It was found that specific G3BP1 knockdown inhibited SG formation and cell survival, and this appeared to occur downstream of eIF2alpha; phosphorylation. The phosphorylation of eIFalpha; is the only factor known to be necessary for SG formation and cell survival. This data is the first to implicate SG formation itself, downstream of eIF2alpha; phosphorylation, in the survival phase of the ISR. The results also suggest that G3BP1 plays a pivotal role in the post-transcriptional mechanisms underlying stress adaptation. To facilitate future analysis of G3BP roles in the regulation of specific transcripts and in SG biology, a pilot study to identify G3BP RNA ligands was undertaken. Immunoprecipitation of epitope-tagged G3BP1 from stable cell lines facilitated purification and isolation of RNA in association with G3BP1. Specific RNA transcripts were subsequently detected and identified by microarray. Many genes were enriched in the G3BP1 immunoprecipitate. Transcript enrichment in the control immunoprecipitate was comparatively weak and seemingly random, suggesting that several replicates will enable generation of a reliable target list. This work forms a promising basis for further investigations into G3BP functionality, and also provides a platform for broader and more large-scale analyses of the mechanisms of post-transcriptional gene regulation. The work presented in this thesis addressed the potential post-transcriptional mechanisms by which the G3BP family of proteins mediate cell proliferation and survival. Both G3BP1 and G3BP2 were shown to be over-expressed in tumours and each appeared to promote reporter gene expression. G3BP1 was also found to play a pivotal role in stress adaptation. A technique to identify novel RNA ligands was assessed, and it was found that G3BP1 may interact with various mRNA transcripts. It is hypothesised that the G3BP family of proteins, and in particular G3BP1, function to determine the fate of specific RNAs in response to cellular stress and other stimuli. In this way, G3BP proteins may facilitate appropriate responses to extra-cellular stimuli which allow for cell proliferation and survival.

G3BP1 and G3BP2 are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of various RNA binding proteins is common in cancer, and several of these proteins influence tumorigenesis. Therefore, detailed examination of RNA binding proteins, such as G3BPs, may provide insights into the post-transcriptional mechanisms underlying tumorigenesis. Tumours arise as a consequence of genetic mutation or alteration, which often result from stress-induced DNA damage. Cancer progression is facilitated by various epigenetic stress adaptation mechanisms. Stressful stimuli induce transitory translational shut-off, mediated by phosphorylation of eukaryotic initiation factor alpha;(eIF2alpha;). This phosphorylation event leads to formation of discrete cytoplasmic foci known as stress granules (SGs), which are translationally-silent sites of mRNA sorting. It was initially thought that an RNA-binding protein, T-cell internal antigen 1 (TIA-1), was instrumental in both the formation and functioning of SGs, because over-expression of TIA-1 induces spontaneous SGs and concomitantly causes a decrease in reporter gene expression. It is now clear that SG content can change depending on the type of stress, and that various proteins, including G3BP1, can induce spontaneous SGs. In vitro evidence previously implicated both G3BP1 and G3BP2 as endoribonucleases, so it was suggested that G3BPs act to target mRNA for decay at the SG. This project sought to further investigate this proposal, and in this way gain insight into the specific function of G3BPs in post-transcriptional regulation during tumorigenesis. Characterisation of G3BP1 and G3BP2 expression and localisation patterns in human cells and cancer was necessary before functional analyses in human cell systems could be undertaken. Both proteins were found to be over-expressed in breast cancer, irrespective of cancer stage or grade. G3BP1 and G3BP2 were also expressed in all human cell lines tested, despite previously observed tissue-specific expression. These results support the notion that G3BP expression is switched on in parallel with cell proliferation, and as such, may influence tumorigenesis. The results of further analyses suggested that the diverse functions attributed to G3BP1 and G3BP2 may be facilitated by isoform-specific expression, various post-translational modifications and sub-cellular localisation. Despite the absence of a canonical endoribonuclease domain, it was previously reported that site-specific phosphorylation of G3BP1 enables the protein to degrade a synthetic c-myc RNA substrate in vitro. This finding implicated G3BP in the specific regulation of a proto-oncogene. Tailored reporter assays were thus designed in order to address the in vivo consequences of G3BP's putative endoribonuclease activity. Contrary to expectations, all G3BP family members increased or maintained the expression of a range of reporters, at both the mRNA and protein level, irrespective of the presence of any particular cis-acting element, coding sequence or promoter. These results support the emerging notion that G3BPs positively affect the expression of at least some of their target mRNAs, and may also indirectly promote transcription. In contrast to the theory that G3BPs degrade proto-oncogenic mRNA/s, these findings are consistent with a role for G3BP in promoting cell proliferation and survival. Further analyses showed that G3BP1 and G3BP2 simultaneously increased reporter gene expression and induced SG formation. These findings highlighted the fact that SGs are dynamic sorting stations for mRNAs, and not merely sites of stalled translation. This result also supports the notion that a variety of proteins may be recruited to the SG to facilitate a multitude of mRNA fates. Although the precise role of the SG in stress adapation is not known, it is clear that an appropriate integrated stress response (ISR) is required for cells to survive in sub-optimal conditions. It was found that specific G3BP1 knockdown inhibited SG formation and cell survival, and this appeared to occur downstream of eIF2alpha; phosphorylation. The phosphorylation of eIFalpha; is the only factor known to be necessary for SG formation and cell survival. This data is the first to implicate SG formation itself, downstream of eIF2alpha; phosphorylation, in the survival phase of the ISR. The results also suggest that G3BP1 plays a pivotal role in the post-transcriptional mechanisms underlying stress adaptation. To facilitate future analysis of G3BP roles in the regulation of specific transcripts and in SG biology, a pilot study to identify G3BP RNA ligands was undertaken. Immunoprecipitation of epitope-tagged G3BP1 from stable cell lines facilitated purification and isolation of RNA in association with G3BP1. Specific RNA transcripts were subsequently detected and identified by microarray. Many genes were enriched in the G3BP1 immunoprecipitate. Transcript enrichment in the control immunoprecipitate was comparatively weak and seemingly random, suggesting that several replicates will enable generation of a reliable target list. This work forms a promising basis for further investigations into G3BP functionality, and also provides a platform for broader and more large-scale analyses of the mechanisms of post-transcriptional gene regulation. The work presented in this thesis addressed the potential post-transcriptional mechanisms by which the G3BP family of proteins mediate cell proliferation and survival. Both G3BP1 and G3BP2 were shown to be over-expressed in tumours and each appeared to promote reporter gene expression. G3BP1 was also found to play a pivotal role in stress adaptation. A technique to identify novel RNA ligands was assessed, and it was found that G3BP1 may interact with various mRNA transcripts. It is hypothesised that the G3BP family of proteins, and in particular G3BP1, function to determine the fate of specific RNAs in response to cellular stress and other stimuli. In this way, G3BP proteins may facilitate appropriate responses to extra-cellular stimuli which allow for cell proliferation and survival.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

The RasGAP—SH3-domain Binding Protein (G3BP1) is an endoribonuclease that links Ras-signalling pathways and mRNA stability. Though implicated in many cellular roles, a well defined function for G3BP1 has yet to be determined. The aim of the following dissertation is to further implicate G3BP1 in mammalian cellular responses to environmental stress, most notably that of Ultraviolet Radiation. We observe G3BP1 transverse from the cytoplasm to the nucleus upon treatment of HeLa cells with apoptotic levels of UVC. Interestingly, forcing its nuclear accumulation in the absence of stress is sufficient to induce apoptotic characteristics. Preliminary observations suggest that plasma membrane signalling and the resultant activation of stress activated protein kinases, JNK and p38 MAPK, and CASPases might play a role in this process. Thus, we further elaborate G3BP1's role in cellular responses to stress and suggest a new function for the protein, that being the promotion of apoptosis.
"RasGAP—SH3-domain Binding Protein (G3BP)" est un endoribonucléase qui associe la voie de signalisation de Ras et la stabilité d'ARN messager. On ne connaît pas une fonction bien défini pour cette protéine, même si c'est impliquée dans plusieurs rôles cellulaire. Le but de cette dissertation est d'appuyer l'implication du G3BP1 aux réponses cellulaire mammalien au stress environmental, particulièrement à la radiation ultraviolet. On observe le G3BP1 transverse du cytoplasm au noyau au traitement des cellules HeLa avec des doses UV apoptotique. La chose fascinante est quand on introduit s'accumulation du noyau en l'absence du stress est suffisant de provoquer les charactéristiques apoptotique. Les observations au premier degré suggèrent que les signalisations d'origine de membrane plasmique et l'activation resultant du JNK, p38 MAPK, et CASPases peuvent jouer un rôle en ce processus. Ainsi, on a élaboré le rôle du G3BP1 en réponse cellulaire au stress et suggère une nouvelle fonction pour la protéine : la promotion de l'apoptose.

Breast cancer is a complex heterogenous disease with distinct molecular subtypes and metabolic behaviour, disparate responses to therapies, and considerable differences in the overall survival of the patients. Insights into the biological heterogeneity of the disease has led to the development of therapeutic strategies for the effective treatment of breast cancer. The current treatment options include hormone therapy for estrogen positive (ER-positive); anti-HER2 antibody (trastuzumab) therapy for human epidermal growth factor receptor 2 (HER2)-amplified, and general chemotherapy for triple-negative (TN) breast cancers. Unfortunately, therapeutic outcomes remain poor due to inherent or acquired resistance of the cells to the treatments and the toxicity associated with therapy. Metabolic adaptation of breast cancer cells is considered to play a crucial role in enabling the cells to become resistant to therapy and acquire metastatic potential. Bioenergetic alteration (the Warburg effect) is fundamental to all forms of cancer and is regarded as one of the hallmarks. The Warburg effect is characterized by the presence of glycolytic phenotypes within the tumor tissue. Like most cancers, an increase in lactate production observed in breast cancers may be related to the upregulation of lactate dehydrogenase (LDH) enzyme that catalyses the conversion of pyruvate to lactate. Recently, human RasGAP -SH3 domain binding protein (G3BP), an RNA binding protein, was shown to interact with the mRNA of mitochondrial H+-ATP synthase subunit β (β-F1-ATPase), an enzyme that the mediates ATP production through oxidative phosphorylation in mitochondria. That study implicated the role of G3BP in the glycolytic phenotype observed in cancers. Indeed, G3BP is overexpressed in all forms of cancers including breast cancer. The focus of this study was to investigate the potential relevance of G3BP-1 as a breast cancer biomarker and targeting G3BP-1 by lapatinib (Lap) to inhibit the morphological adaptation and cell migration of SKBR3 breast cancer cell line. Whilst, much of the signaling mechanisms have been elucidated, the role of G3BP in the glycolytic switch remains elusive. The first part of this study investigated the potential role of G3BP in the regulation of lactate dehydrogenase A (LDH-A), a key regulator of the glycolytic shift in cancer cells. Thus far, research has not addressed this regulation. In order to achieve this, small interfering RNA (siRNA)-mediated knockdown of G3BP-1 and G3BP-2 (isoforms of G3BP) was performed in MDA-MB-435 breast cancer cell line. Following the knockdown of G3BP, changes in the expression of LDH-A at the transcriptional and translational level were evaluated. The results showed that G3BP-1 (but not G3BP-2) was implicated in the translational regulation of LDH-A. Whilst, depletion of endogenous G3BP-1 resulted in a significant downregulation of LDH-A protein, silencing of G3BP-2 had no effect on the translational regulation of LDH-A. Furthermore, depletion of G3BP-1 and G3BP-2 had no effect on the endogenous mRNA levels of LDH-A. These findings suggest the role of G3BP-1 (but not G3BP-2) in regulating the bioenergetic phenotype of MDA-MB-435 breast cancer cells. Furthermore, the effect of Lap on the protein levels of G3BP-1 and LDH-A was explored in a panel of breast cancer cell lines including SKBR3 (HER2-positive), MDA-MB-231 (triple-negative), and T47D (ER-positive/PR-positive). The data indicated that Lap significantly downregulated the protein levels of G3BP-1 and LDH-A, independent of HER2 status. There was a significant reduction of G3BP-1 and LDH-A protein levels in Lap-sensitive SKBR3, and Lap-insensitive MDA-MB-231 and T47D cell lines. Furthermore, the findings also indicate that irrespective of the breast cancer subtypes (based on the receptor status), G3BP-1 regulates the protein levels of LDH-A. This argument supports the data obtained from the translational regulation of LDH-A upon G3BP-1 knockdown in MDA-MB-435 breast cancer cell line. Hence these findings have updated the role of G3BP-1 in the glycolytic shift observed in cancers, in this context by regulating the expression of LDH-A protein in a panel of breast cancer cell lines. On a side note, the endogenous levels of G3BP-1 and LDH-A were found to be significantly higher in the breast cancer cell lines used in the study. This observation may justify the potential relevance of G3BP-1 and LDH-A as breast cancer biomarkers. Since its initial identification as a RasGAP binding protein, several studies have implicated the role of G3BP in signaling pathways including Ras signaling, HER2 signaling, NF-κB signaling, and c-myc mRNA turnover. Indeed, these pathways are often derailed in cancer. In addition, findings in this study have reported significantly higher levels of endogenous G3BP-1 in breast cancer cell lines. Based on these evidences, G3BP-1 may be a relevant breast cancer biomarker. Therefore, strategies to target G3BP may provide therapeutic advantages over currently available standard treatment modalities to induce sensitivity of resistant breast cancers to anti-G3BP therapy. In the past, the mechanism of action of some anticancer drugs, including resveratrol and epigallocatechin gallate (EGCG), was elucidated by their direct interaction with the recombinant G3BP. Hence, the strategy of recombinant G3BP production was adopted in this study to identify potential anticancer agents that interact with G3BP. Since, Lap downregulated the protein levels of G3BP-1 in breast cancer cell lines (as reported in this study), protein thermal shift assays (PTS) were performed to investigate a potential interaction of recombinant G3BP-1 with Lap. The first part of study in Chapter 4 concerns construct design, expression, and purification of the full length G3BP-1 and G3BP-2 respectively. This was achieved by inserting the respective gene sequences into the bacterial pRSETC vector with N-terminal histidine His(6)-tag, transformation into competent BL21 (DE3) E.coli cells, isopropyl-β-D-thiogalactoside (IPTG) induction, and nickel-nitrilotriacetic acid (Ni-NTA) purification of His-tagged recombinant G3BPs. The results showed that purified full length G3BP-1 and G3BP-2 corresponded to molecular weights of 65-kDa and 68-kDa respectively. The identity of the proteins was also confirmed by immunoblot analysis using protein-specific antibodies for G3BP-1 and G3BP-2. Upon testing for the expression as well as the recovery of the protein in the soluble fraction, it was observed that G3BP-1 was more efficiently recovered at 25oC compared to G3BP-2. The observed difference in the protein recovery may be attributed to the divergent primary structure of these related, yet distinct proteins. Differences in the composition of the catalytic domains between G3BP-1 and G3BP-2 may have an impact on the recovery of the proteins in the soluble fraction. Nevertheless, his(6)-G3BP-1 was purified using Ni-NTA resin with high purity and a yield of 6mg/l culture. In the next step, the effect of Lap on thermal stabilization of purified G3BP-1 protein was studied using PTS assay. The results showed that the melting temperature (tm) of G3BP-1 in the presence of Lap was higher than for the control. Such thermal stabilization was greatest at the 4:1 stochiometric ratio of Lap to G3BP-1, with an observed thermal shift (Δtm) of ~≤1oC at 15μM of G3BP-1. To distinguish the effect of Lap on thermal stabilization of G3BP-1, Lap was compared to 5-flourouracil (5-FU), a non-ATP-interacting thymidylate synthase inhibitor. G3BP-1 thermal stability in the presence of 5-FU was measured under the same experimental conditions of 4:1 stochiometric ratio of the compound to the protein. In contrast to the earlier observation, the results showed that 5-FU did not induce thermal stabilization of G3BP-1. These findings confirmed that the observed thermal stabilization of G3BP-1 was specific to Lap. In addition, a slightly higher shift in the tm of G3BP-1 may be indicative of a weak interaction of Lap with the protein. Metastasis is a process that involves migration of cancer cells from the primary tumor site to the distant organs. Approximately 90% of the breast cancer related mortalities have been attributed to the metastatic spread of the disease. Like other cancers, breast cancer metastasis is associated with poor prognosis. Of all the subtypes of breast cancers, HER2-amplified and triple-negative (TN) forms are known to be highly metastatic in nature. Currently, the ability to accurately predict the risk for metastatic potential poses a substantial challenge for clinical management of the disease. Interestingly, adaptive responses are induced in cancer cells following low doses of some chemotherapeutic agents. In chapter 5, it was observed that 0.1μM doxorubicin (Dox) treatment increased the cell viability of SKBR3 breast cancer cell line. Such an increase in cell viability following Dox treatment was not observed in MDA-MB-231, T47D and HMF cell lines. Furthermore, Dox treatment also induced migratory phenotypes in SKBR3 cells. The observations based on the immunofluorescence images of the Dox treated cells revealed that there was a significant increase in the prominent F-actin filaments and the mitochondrial spread as compared to the corresponding controls. In line with these observations, an in vitro scratch wound assay revealed that Dox treated cells showed an enhanced ability to migrate into the scratch wound area as compared to the control cells. However, a combination dose of 0.1μM Dox+5μM Lap not only decreased the viability of SKBR3 cells, but also effectively suppressed the migratory phenotype compared to Dox treated cells. In addition, cells that received the combination therapy showed a significant decrease in the percentage of wound closure compared with the corresponding Dox treated controls. Furthermore, Western blot analysis indicated that SKBR3 cells treated with Lap in combination with Dox showed a significant reduction in the protein levels of G3BP-1 and LDH-A when compared to either vehicle control or Dox treatment alone. Collectively, Lap treatment inhibited Dox-induced migratory phenotypes of SKBR3 breast cancer cell line with a significant downregulation of endogenous G3BP-1 and LDH-A proteins in these cells.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
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The ras-GTPase SH3-domain Binding Proteins (G3BP) are a family of RNA-binding proteins that have been implicated in multiple cellular activities ranging from signal transduction to regulation of messenger RNA (mRNA). G3BPs were named after their interaction with the SH3 domain of Ras-GTPase-activating protein; however recent research did not find this interaction. All three members of the G3BPs family, G3BP1, G3BP2a and G3BP2b, share structural similarities with each other by having four distinct regions (1) the Nuclear Transporting Factor 2, (NTF2) domain at the N-terminal, (2) the acidic and proline-rich domain in the centre, (3) the RNA recognition motif (RRM) and (4) the arginine glycine (RGG)-rich region rich at the C-terminal. The presence of the NTF2 domain in its structure suggests G3BP might play a role in nucleocytoplasmic transportation, which was observed after serum stimulation where G3BP1 was translocated to the nucleus from the cytoplasm. The RNA recognition motif (RRM) region plays a vital role in its interaction with the target RNA. The RGG-rich box is a region rich in arginine and glycine residues, which plays a role assisting RRM in interactions with protein or RNA. G3BP1 is found to be overexpressed in many cancers, including breast cancer, and head and neck tumours, as well as cell lines derived from human lung, prostrate, colon, thyroid and breast cancer. G3BPs have also been implicated in translational control within differentiating neurons, suggesting that G3BP may play several roles in controlling the translational fate of its cargo and that its role may be cell-specific. G3BP1 has also been found in β-integrin- induced adhesion complexes. This information highlights G3BPs as a dynamic protein that is involved in several biological functions.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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