http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11381 Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55α, B56α, B56γ, and B56δ). Overexpression of PP2A-Aα in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT⁺ cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT–mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT⁺ cancers. 2012-08-27T06:12:34.327Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10824 Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated. 2012-05-21T06:37:30.612Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10155 Asthma is characterised by antigen-mediated mast cell degranulation resulting in secretion of inflammatory mediators. Protein phosphatase 2A (PP2A) is a serine/threonine protein phosphatase composed of a catalytic (PP2A-C) subunit together with a core scaffold (PP2A-A) subunit and a variable, regulatory (PP2A-B) subunit. Previous studies utilising pharmacological inhibition of protein phosphatases have suggested a positive regulatory role for PP2A in mast cell degranulation. In support of this we find that a high okadaic acid concentration (1 μM) inhibits mast cell degranulation. Strikingly, we now show that a low concentration of okadaic acid (0.1 μM) has the opposite effect, resulting in enhanced degranulation. Selective downregulation of the PP2A-Cα subunit by short hairpin RNA also enhanced degranulation of RBL-2H3 mast cells, suggesting that the primary role of PP2A is to negatively regulate degranulation. PP2A-B subunits are responsible for substrate specificity, and carboxymethylation of the PP2A-C subunit alters B subunit binding. We show here that carboxymethylation of PP2A-C is dynamically altered during degranulation and inhibition of methylation decreases degranulation. Moreover downregulation of the PP2A-Bα subunit resulted in decreased MK2 phosphorylation and degranulation, whilst downregulation of the PP2A-B′δ subunit enhanced p38 MAPK phosphorylation and degranulation. Taken together these data show that PP2A is both a positive and negative regulator of mast cell degranulation, and this differential role is regulated by carboxymethylation and specific PP2A-B subunit binding. 2012-02-24T02:20:04.795Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1293 Mast cells play both effector and modulatory roles in a range of allergic and immune responses. The principal function of these cells is the release of inflammatory mediators from mast cells by degranulation, which involves a complex interplay of signalling molecules. Understanding the molecular architecture underlying mast cell signalling has attracted renewed interest as the capacity for therapeutic intervention through controlling mast cell degranulation is now accepted as a viable proposition. The dynamic regulation of signalling by protein phosphorylation is a well-established phenomenon and many of the early events involved in mast cell activation are well understood. Less well understood however are the events further downstream of receptor activation that allow movement of granules through the cytoskeletal barrier and docking and fusion of granules with the plasma membrane. Whilst a potential role for the protein phosphatase family of signalling enzymes in mast cell function has been accepted for some time, the evidence has largely been derived from the use of broad specificity pharmacological inhibitors and results often depend upon the experimental conditions, leading to conflicting views. In this review, we present and discuss the pharmacological and recent molecular evidence that protein phosphatases, and in particular the protein phosphatase serine/threonine phosphatase type 2A (PP2A), have major regulatory roles to play and may be potential targets for the design of new therapeutic agents. 2010-04-27T06:54:36.129Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:992 Background: Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized γ-actin expression in antimicrotubule drug–resistant leukemia and examined the effect of altered γ-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. Methods: Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLB100 cells) and desoxyepothilone B (CCRF-CEM/dEpoB140 cells). Fluorescence-based cycle sequencing was used to detect gene mutations. Site-directed mutagenesis was used to generate mutant γ-actin expression plasmids, which were used to transfect mouse NIH/3T3 cells. Clonogenic analysis was used for drug sensitivity studies. A small interfering RNA (siRNA) was used to block γ-actin gene expression in human neuroblastoma SH-EP cells. Expression of γ-actin (normalized to that of β2-microglobulin [β₂M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription–polymerase chain reaction. Statistical significance of changes in the ratio of γ-actin to β₂M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. Results: We identified novel mutant forms of γ-actin and the concomitant loss of wild-type γ-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant γ-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with γ-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No γ-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). γ-Actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean γ-actin/β₂M = 0.53) than in samples collected at diagnosis (n = 44; mean ��-actin/������M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). Conclusions: These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between γ-actin and microtubules. 2010-04-27T06:43:19.443Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1006 Intrinsic or acquired resistance to vincristine (VCR), an antimicrotubule agent used in the treatment of childhood acute lymphoblastic leukemia (ALL), is a major clinical problem. Using a clinically relevant NOD/SCID mouse xenograft model of ALL, we established that alterations in the actin and tubulin cytoskeleton are involved in in vivo VCR resistance. Altered protein expression between VCR-sensitive ALL xenografts, and xenografts with intrinsic or acquired VCR resistance, was identified using 2-D DIGE coupled with MS. Of the 19 proteins displaying altered expression, 11 are associated with the actin cytoskeleton. Altered expression of the actin- and/or tubulin-binding proteins gelsolin, moesin, ezrin, tropomyosin, CAP-G, HSP27, HSP70, TCP-1, and stathmin were associated with in vivo VCR resistance. The actin-regulating protein gelsolin was increased in both acquired and resistant leukemia as confirmed by immunoblotting and gene expression. The major cytoskeletal protein, γ-actin, was down-regulated in the VCR-resistant leukemia xenografts; in contrast, there was no significant change in β-actin expression. This study provides the first evidence for a role of the actin cytoskeleton in intrinsic and acquired in vivo antimicrotubule drug resistance in childhood leukemia and highlights the power of 2-D DIGE for the discovery of resistance markers, pharmacoproteomics, and signaling pathways in cancer. 2010-04-27T06:42:43.945Z ]]>