ORCID Profile
0000-0001-7227-1289
Current Organisation
Monash University
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Publisher: MDPI AG
Date: 17-10-2019
DOI: 10.3390/MOLECULES24203739
Abstract: Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.
Publisher: Springer Science and Business Media LLC
Date: 17-01-2019
DOI: 10.1038/S41467-018-08154-1
Abstract: Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an ex le, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.
Publisher: Elsevier BV
Date: 07-2019
Publisher: Cold Spring Harbor Laboratory
Date: 19-02-2021
DOI: 10.1101/2021.02.17.431740
Abstract: The PEAK family of pseudokinases comprises PEAK1 and PEAK2 as well as the recently-identified PEAK3. PEAK1/2 play fundamental roles in regulating tyrosine kinase signal output and oncogenesis, while PEAK3 remains poorly-characterized. Here, we demonstrate that PEAK3 undergoes homotypic association as well as heterotypic interaction with PEAK1/2. PEAK3 also recruits ASAP1/2, Cbl and PYK2 and the adaptors Grb2 and CrkII, with binding dependent on PEAK3 dimerization. PEAK3 tyrosine phosphorylation on Y24 is also dependent on dimerization as well as Src family kinase activity, and interestingly, is decreased via PTPN12 in response to EGF treatment. Y24 phosphorylation is required for binding of Grb2 and ASAP1. Overexpression of PEAK3 in MDA-MB-231 breast cancer cells enhanced cell elongation and cell motility, while knockdown of endogenous PEAK3 decreased cell migration. In addition, overexpression of PEAK3 in PEAK1/2 compound knock-out MCF-10A breast epithelial cells enhanced acinar growth and invasion in 3D culture, with the latter phenotype dependent on PEAK3 tyrosine phosphorylation and binding of Grb2 and ASAP1. These findings characterize PEAK3 as an integral member of the PEAK family with scaffolding roles that promote cell proliferation, migration and invasion.
Publisher: Research Square Platform LLC
Date: 31-08-2022
DOI: 10.21203/RS.3.RS-1906871/V1
Abstract: PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate new molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.
Publisher: Springer Science and Business Media LLC
Date: 24-01-2023
DOI: 10.1038/S41388-023-02594-W
Abstract: We have determined that expression of the pseudokinase NRBP1 positively associates with poor prognosis in triple negative breast cancer (TNBC) and is required for efficient migration, invasion and proliferation of TNBC cells in culture as well as growth of TNBC orthotopic xenografts and experimental metastasis. Application of BioID/MS profiling identified P-Rex1, a known guanine nucleotide exchange factor for Rac1, as a NRBP1 binding partner. Importantly, NRBP1 overexpression enhanced levels of GTP-bound Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42, suggesting a scaffolding function for this pseudokinase. NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, while constitutively-active Rac1 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. Generation of reactive oxygen species via a NRBP1/P-Rex1 pathway was implicated in these oncogenic roles of NRBP1. Overall, these findings define a new function for NRBP1 and a novel oncogenic signalling pathway in TNBC that may be amenable to therapeutic intervention.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2023
DOI: 10.1038/S41467-023-38869-9
Abstract: PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 22-02-2022
DOI: 10.1126/SCISIGNAL.ABJ3554
Abstract: The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr 24 , likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal- ersifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.
No related grants have been discovered for Xiuquan Ma.