Cara Jamieson
University of Sydney
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Publication
Featured researches published by Cara Jamieson.
Journal of Biological Chemistry | 2012
Manisha Sharma; Cara Jamieson; Michael Johnson; Mark P. Molloy; Beric R. Henderson
Background: The nuclear localization of β-catenin is directly linked to its cancer causing activity. Results: Armadillo repeats (10–12) mediate nuclear transport of β-catenin through direct interaction with specific nuclear pore complex proteins. Conclusion: β-Catenin can function like a nuclear transport receptor in its ability to translocate independently through the nuclear pore complex. Significance: β-Catenin may transport specific binding partners between the nucleus and cytoplasm in response to Wnt signaling. β-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. β-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10–12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3–8 of β-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of β-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of β-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10–12, and β-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of β-catenin to a greater extent than that of importin-β. The Arm R10–12 sequence facilitated transport even when β-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated β-catenin transport through direct interaction with the NPC.
Seminars in Cancer Biology | 2014
Cara Jamieson; Manisha Sharma; Beric R. Henderson
The nuclear localization of specific proteins is critical for cellular processes such as cell division, and in recent years perturbation of the nuclear transport cycle of key proteins has been linked to cancer. In particular, specific gene mutations can alter nuclear transport of tumor suppressing and oncogenic proteins, leading to cell transformation or cancer progression. This review will focus on one such factor, β-catenin, a key mediator of the canonical wnt signaling pathway. In response to a wnt stimulus or specific gene mutations, β-catenin is stabilized and translocates to the nucleus where it binds TCF/LEF-1 transcription factors to transactivate genes that drive tumor formation. Moreover, the nuclear import and accumulation of β-catenin correlates with clinical tumor grade. Recent evidence suggests that the primary nuclear transport route of β-catenin is independent of the classical Ran/importin import machinery, and that β-catenin directly contacts the nuclear pore complex to self-regulate its own entry into the nucleus. Here we propose that the β-catenin nuclear import pathway may provide an opportunity for identification of specific drug targets and inhibition of β-catenin nuclear function, much like the current screening of drugs that block binding of β-catenin to LEF-1/TCFs. Here we will discuss the diverse mechanisms regulating nuclear localization of β-catenin and their potential as targets for anticancer agent development.
The International Journal of Biochemistry & Cell Biology | 2012
Cara Jamieson; Manisha Sharma; Beric R. Henderson
β-catenin is the central nuclear effector of the Wnt signaling pathway, and regulates other cellular processes including cell adhesion. Wnt stimulation of cells culminates in the nuclear translocation of β-catenin and transcriptional activation of target genes that function during both normal and malignant development. Constitutive activation of the Wnt pathway leads to inappropriate nuclear accumulation of β-catenin and gene transactivation, an important step in cancer progression. This has generated interest in the mechanisms regulating β-catenin nuclear accumulation and retention. Here we discuss recent advances in understanding feedback loops that trap β-catenin in the nucleus and provide potential insights into Wnt signaling and the development of anti-cancer drugs.
Cellular Signalling | 2009
Michael Johnson; Manisha Sharma; Cara Jamieson; Jasmine M. Henderson; Myth T.S. Mok; Linda J. Bendall; Beric R. Henderson
Beta-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3beta. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of beta-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged beta-catenin, and found that a small mobile pool of beta-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t(0.5) of approximately 30 s. Thus, beta-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of beta-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t(0.5) approximately 5 s) is indicative of high turnover and transient association. In contrast, beta-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t(0.5) approximately 8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of beta-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than beta-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3beta increased beta-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of beta-catenin. In summary, beta-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.
Traffic | 2011
Cara Jamieson; Manisha Sharma; Beric R. Henderson
Nuclear localization of β‐catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase‐3β (GSK‐3β), causing nuclear accumulation of β‐catenin and transactivation of genes that transform cells. β‐catenin is a shuttling protein; however, the mechanism by which GSK‐3β regulates β‐catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the β‐catenin‐green fluorescent protein dynamics in NIH 3T3 cells before and after GSK‐3β inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in β‐catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing β‐catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks β‐catenin phosphorylation by GSK‐3β, did not alter nuclear retention or transport, implying that GSK‐3β acts through an independent regulator. We compared five nuclear binding partners and identified LEF‐1 as the key mediator of Wnt3a and LiCl‐induced nuclear retention of β‐catenin. Thus, Wnt stimulation triggered a LEF‐1 positive feedback loop to enhance the nuclear chromatin‐retained pool of β‐catenin by 100–300%. These findings shed new light on regulation of β‐catenin nuclear dynamics.
Journal of Biological Chemistry | 2010
Cameron P. Flegg; Manisha Sharma; Cahora Medina-Palazon; Cara Jamieson; Melanie Galea; Mariana G. Brocardo; Kate Mills; Beric R. Henderson
Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.
Journal of Cell Science | 2015
Cara Jamieson; Christina Lui; Mariana G. Brocardo; Estefania Martino-Echarri; Beric R. Henderson
ABSTRACT β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1–β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin–lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin–LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. Summary: Evidence is presented for a novel role of Rac1 in stimulating β-catenin–LEF-1 complex formation.
Advances in Experimental Medicine and Biology | 2014
Manisha Sharma; Michael Johnson; Mariana G. Brocardo; Cara Jamieson; Beric R. Henderson
Several components of the Wnt signaling pathway have in recent years been linked to the nuclear pore complex. β-catenin, the primary transducer of Wnt signals from the plasma membrane to the nucleus, has been shown to transiently associate with different FG-repeat containing nucleoporins (Nups) and to translocate bidirectionally through pores of the nuclear envelope in a manner independent of classical transport receptors and the Ran GTPase. Two key regulators of β-catenin, IQGAP1 and APC, have also been reported to bind specific Nups or to locate at the nuclear pore complex. The interaction between these Wnt signaling proteins and different Nups may have functional implications beyond nuclear transport in cellular processes that include mitotic regulation, centrosome positioning and cell migration, nuclear envelope assembly/disassembly, and the DNA replication checkpoint. The broad implications of interactions between Wnt signaling proteins and Nups will be discussed in the context of cancer.
Experimental Cell Research | 2016
Cara Jamieson; Kate Mills; Christina Lui; Crystal Semaan; Mark P. Molloy; Manisha Sharma; Jade K. Forwood; Beric R. Henderson
Beta-catenin plays a key role in transducing Wnt signals from the plasma membrane to the nucleus. Here we characterize an unusual subcellular distribution of beta-catenin in MCF-7 breast cancer cells, wherein beta-catenin localizes to the cytoplasm and membrane but atypically did not relocate to the nucleus after Wnt treatment. The inability of Wnt or the Wnt agonist LiCl to induce nuclear localization of beta-catenin was not due to defective nuclear transport, as the transport machinery was intact and ectopic GFP-beta-catenin displayed rapid nuclear entry in living cells. The mislocalization is explained by a shift in the retention of beta-catenin from nucleus to cytoplasm. The reduced nuclear retention is caused by unusually low expression of lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors. The reconstitution of LEF-1 or TCF4 expression rescued nuclear localization of beta-catenin in Wnt treated cells. In the cytoplasm, beta-catenin accumulated in recycling endosomes, golgi and beta-COP-positive coatomer complexes. The peripheral association with endosomes diminished after Wnt treatment, potentially releasing β-catenin into the cytoplasm for nuclear entry. We propose that in MCF-7 and perhaps other breast cancer cells, beta-catenin may contribute to cytoplasmic functions such as ER-golgi transport, in addition to its transactivation role in the nucleus.
Journal of Biological Chemistry | 2016
Manisha Sharma; Cara Jamieson; Michael K. Johnson; Mark P. Molloy; Beric R. Henderson
Abstract β-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. β-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10–12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3–8 of β-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of β-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of β-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10–12, and β-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of β-catenin to a greater extent than that of importin-β. The Arm R10–12 sequence facilitated transport even when β-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated β-catenin transport through direct interaction with the NPC.