Global Effects of BCR/ABL and TEL/PDGFRβ Expression on the Proteome and Phosphoproteome

Unwin, Richard D.; Sternberg, David; Lu, Yuning; Pierce, Andrew; Gilliland, D. Gary; Whetton, Anthony D.

doi:10.1074/jbc.m410598200

Cited by 41 publications

(36 citation statements)

References 77 publications

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“…However, the supposition that these isoelectric shifts represented dephosphorylation of numerous mitochondrial proteins was further supported by Pro-Q Diamond staining of additional two-dimensional gels obtained from mitochondrial extracts derived from untreated and rapamycin-treated cells. The intensity of the Pro-Q Diamond stain has been shown previously to correlate with the degree of protein phosphorylation (25,29,30). Mass spectrometry allowed us to again identify a number of individual Pro-Q Diamond-positive proteins, and as before, treatment with rapamycin appeared to reduce the level of phosphorylation (Fig.…”

Section: Resultsmentioning

confidence: 78%

The Mammalian Target of Rapamycin (mTOR) Pathway Regulates Mitochondrial Oxygen Consumption and Oxidative Capacity

Schieke

Phillips

McCoy

et al. 2006

Journal of Biological Chemistry

548

464

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Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms.For nearly a century it has been appreciated that an organism's intrinsic metabolic rate is an important determinant of life span. This theory, initially known as the "rate of living" hypothesis, has merged with another proposed mechanism for aging first enunciated by Denham Harman (1) and often called the "free radical theory of aging." The basis for combining these two hypotheses came from observations demonstrating that mitochondria determine both cellular and organismal metabolic rate and that these organelles also produce a continuous stream of reactive oxygen species. Although both the rate of living and the "free radical" theories of aging remain viable and attractive explanations for determining the rate of aging in a wide range of species, neither hypothesis has been conclusively proven (2-4).Surprisingly relatively little is known regarding what regulates the intrinsic metabolic rate of an organism. Similarly on a cellular level the molecular regulation of mitochondrial activity is incompletely understood. Resting oxygen consumption presumably is set at a point to meet overall energetic demands. Nonetheless for most cells this basal respiration is considerably below the maximum oxidative capacity of the mitochondria. The maximal oxidative or ATP synthetic capacity can be easily assessed by treating the cell with various chemical uncouplers and thereby producing the maximal degree of respiration. It is imp...

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Section: Resultsmentioning

confidence: 78%

The Mammalian Target of Rapamycin (mTOR) Pathway Regulates Mitochondrial Oxygen Consumption and Oxidative Capacity

Schieke

Phillips

McCoy

et al. 2006

Journal of Biological Chemistry

548

464

View full text Add to dashboard Cite

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“…Both agents have a relatively high specificity for r-kinase, 10 and have been employed as r-kinase inhibitors by ourselves, 3 and by others, in cell types including haematopoietic cells. 11 In our earlier studies using cell line Y-27632 (50 mM) was found to have the same in vitro activities as a dominant negative r, further confirming its specificity.…”

Section: Resultsmentioning

confidence: 99%

“…11 In our earlier studies using cell line Y-27632 (50 mM) was found to have the same in vitro activities as a dominant negative r, further confirming its specificity. 3 Since CML progenitors mature in vivo in the presence of exogenous growth factors, and it is believed that altered sensitivity to growth factors may play a role in the abnormal growth of CML cells, 12 we studied the growth of progenitor cells during cytokine-stimulated growth and maturation. Moreover, as r-kinase is only one of a wide range of potential downstream targets activated by BCR/ABL, 13 we compared the effects of r-kinase inhibition with direct inhibition of BCR/ABL using imatinib.…”

Section: Resultsmentioning

confidence: 99%

The ρ-kinase inhibitors Y-27632 and fasudil act synergistically with imatinib to inhibit the expansion of ex vivo CD34+ CML progenitor cells

et al. 2007

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Evidence from cell line-based studies indicates that q-kinase may play a role in the leukaemic transformation of human cells mediated by the BCR/ABL tyrosine kinase, manifest clinically as chronic myeloid leukaemia (CML). We therefore employed two separate inhibitors, Y-27632 and fasudil, to inhibit the activity of q-kinase against ex vivo CD34 þ cells collected from patients with CML. We compared the effects of q-kinase inhibition in those cells with the effects of direct inhibition of BCR/ABL using the specific inhibitor imatinib. We found that inhibition of q-kinase inhibited the effective proliferation, and reduced survival of CML progenitor cells. When combined with imatinib, q-kinase inhibition added to the anti-proliferative and pro-apoptotic effects of the BCR/ABL inhibitor. Our studies may indicate therapeutic benefit in some cases for the combination of q-kinase inhibitors with imatinib.

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“…Interestingly, Src was also found to coprecipitate with RhoGDI2. Recent reports identified RhoGDI1 (8,9) and RhoGDI2 (10) as tyrosinephosphorylated proteins in cancer cells. Furthermore, Src regulates RhoGDI1 function through phosphorylation on a tyrosine that is conserved in RhoGDI2 (11).…”

Section: Mass Spectroscopic Identification Of Rhogdi2 Immunocomplex Pmentioning

confidence: 99%

Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function

Moissoglu

Wang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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RhoGDI2 is a suppressor of metastasis in human bladder cancer. Although diminished RhoGDI2 expression in tumors is associated with decreased patient survival, normal expression in some metastatic tumors led us to wonder whether other mechanisms regulate RhoGDI2 function. Protein interaction analysis identified Src as a novel RhoGDI2 interaction partner. Gene expression profiling and immunohistochemistry of human tumors revealed that Src levels diminish as a function of bladder cancer stage. In addition, diminished Src levels and RhoGDI2 levels appear mutually exclusive in individual tumors, indicating that both genes are likely involved in the same signaling pathway leading to metastasis suppression. Studies confirmed that activated Src kinase binds and phosphorylates RhoGDI2 in vitro and vivo. Mutagenesis revealed that Tyr-153 and, to a lesser degree, Tyr-24 were the primary Src phosphorylation sites. Phosphorylation decreased the amount of Rac1 in RhoGDI2 complexes and increased RhoGDI2 association with cell membranes. Stable expression of phosphomimetic Tyr-153 RhoGDI2 in metastatic human bladder cancer cell lines had no effect on primary tumor growth but suppressed metastasis more potently than WT RhoGDI2. These data suggest that phosphorylation by Src enhances RhoGDI2 metastasis suppression and that loss of Src relieves metastasis suppression in tumor cells that maintain RhoGDI2 expression. Our findings also suggest caution in using Src inhibitors in the hope of delaying progression in patients with bladder cancer.bladder neoplasms ͉ guanine nucleotide dissociation inhibitors ͉ neoplasm metastasis ͉ src-family kinases B ladder cancer is common in the United States (1). Most deaths from this disease are due to metastases, commonly to lung and liver. RhoGDI2 (LyGDI/D4GDI) has been shown to be a metastasis suppressor in animal models of bladder cancer and to be lost in many metastatic human tumors (2, 3). RhoGDI2 belongs to a family of related proteins that includes RhoGDI1 and RhoGDI3 (reviewed in ref. 4). RhoGDI1 is the most widely expressed and the most intensely studied. Previously, it was thought that RhoGDI2 expression was confined to cells of hematopoietic origin, but our studies showed it is expressed in a wider range of tissues and cell types (5). Much less is known about RhoGDI3. Its expression is highest in brain, lung, and testis and, in contrast to RhoGDI1 and RhoGDI2, it is not found in the cytoplasm, but is associated with vesicular membranes. RhoGDIs are thought to inhibit the activation of small GTPases of the Rho family by sequestering the GTPases in the cytosol and blocking activation by guanine nucleotide exchange factors (GEFs) (4). Previous work identified RhoA, Rac1, and Rac2 as targets of RhoGDI2-mediated inactivation (6, 7), suggesting that RhoGDI2's function overlaps that of RhoGDI1.Although RhoGDI2 expression is reduced in many metastatic bladder cancers, Ϸ35% of patients with moderate or high levels of RhoGDI2 protein still developed metastatic disease. Although factors unrelated to...

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Global Effects of BCR/ABL and TEL/PDGFRβ Expression on the Proteome and Phosphoproteome

Cited by 41 publications

References 77 publications

The Mammalian Target of Rapamycin (mTOR) Pathway Regulates Mitochondrial Oxygen Consumption and Oxidative Capacity

The Mammalian Target of Rapamycin (mTOR) Pathway Regulates Mitochondrial Oxygen Consumption and Oxidative Capacity

The ρ-kinase inhibitors Y-27632 and fasudil act synergistically with imatinib to inhibit the expansion of ex vivo CD34+ CML progenitor cells

Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function

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