EGF-induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

Nagy, Péter; Bene, László; Balázs, Margit; Hyun, William C.; Lockett, Steven J.; Chiang, Nancy Y.; Waldman, Fred; Feuerstein, Burt G.; Damjanovich, Sándor; Szöllősi, János

doi:10.1002/(sici)1097-0320(19980601)32:2<120::aid-cyto7>3.0.co;2-p

Cited by 50 publications

(48 citation statements)

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“…Using IF we show that Copine-III and ErbB2 colocalize to the PM after HRG stimulation. Moreover, in FRET acceptor photobleaching experiments, we measured a FRET efficiency of 8±3 between ErbB2 and Copine-III, which agrees well with the FRET efficiency of 14 ± 3 between ErbB2 receptors (Nagy et al, 1998). Taken together, our analysis of Copine-III suggests that it is a Ca 2 þ -dependent membrane-binding copine that responds to ErbB2 activation and localizes to the PM in proximity to the receptor.…”

Section: Discussionsupporting

confidence: 83%

Copine-III interacts with ErbB2 and promotes tumor cell migration

et al. 2009

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ErbB2 amplification and overexpression in breast cancer correlates with aggressive disease and poor prognosis. To find novel ErbB2-interacting proteins, we used stable isotope labeling of amino acids in cell culture followed by peptide affinity pull-downs and identified specific binders using relative quantification by mass spectrometry. Copine-III, a member of a Ca 2 þ -dependent phospholipid-binding protein family, was identified as binding to phosphorylated Tyr1248 of ErbB2. In breast cancer cells, Copine-III requires Ca 2 þ for binding to the plasma membrane, where it interacts with ErbB2 upon receptor stimulation, an interaction that is dependent on receptor activity. Copine-III also binds receptor of activated C kinase 1 and colocalizes with phosphorylated focal adhesion kinase at the leading edge of migrating cells. Importantly, knockdown of Copine-III in T47D breast cancer cells causes a decrease in Src kinase activation and ErbB2-dependent wound healing. Our data suggest that Copine-III is a novel player in the regulation of ErbB2-dependent cancer cell motility. In primary breast tumors, high CPNE3 RNA levels significantly correlate with ERBB2 amplification. Moreover, in an in situ tissue microarray analysis, we detected differential protein expression of Copine-III in normal versus breast, prostate and ovarian tumors, suggesting a more general role for Copine-III in carcinogenesis.

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Section: Discussionsupporting

confidence: 83%

Copine-III interacts with ErbB2 and promotes tumor cell migration

et al. 2009

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“…ErbB2 constitutively adopts an extended conformation potentiating the formation of heterodimers (8,9) that can be inhibited by pertuzumab, a monoclonal antibody sterically blocking the heterodimerization arm of ErbB2 (10). Although the extracellular domain of ErbB2 has failed to form crystallographic homodimers, molecular biological and fluorescence resonance energy transfer (FRET) experiments have shown that full-length ErbB2 exists in dimers or higher-order aggregates in the plasma membrane (11,12). The implication is that the transmembrane, juxtamembrane and other intracellular domains (5,13,14) act in conjunction with the membrane environment (15) to mediate the dimerization and, thereby, functional states of ErbB proteins.…”

mentioning

confidence: 99%

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

Nagy

Claus

Jovin

et al. 2010

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

164

161

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Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10 5 − 10 6 receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.EGFR | epidermal growth factor | ErbB proteins | receptor clusters | signal transduction E rbB proteins (ErbB1-4, HER1-4) constitute the best characterized family of receptor tyrosine kinases (1). Biochemical analysis has demonstrated that ErbB1, the prototypical member of the family (also known as the epidermal growth factor receptor, EGFR or HER1) undergoes ligand-induced homodimerization as a key step in its activation (2). More recent crystallographic studies reveal that ligand binding induces a transition from a closed conformation of ErbB1 to an extended configuration with the capacity to dimerize via intermolecular interactions mediated by domain II (3, 4). The ultrastructural data also confirm that dimerization of ErbB1 plays a fundamental role in activating the kinase domain by a mechanism resembling that of cyclin dependent kinases (5, 6). The coreceptor ErbB2 has no known ligand but upon transactivation expresses the most potent kinase activity of the ErbB family, thereby increasing the efficiency of signaling mediated by ErbB2-containing heterodimers (7). ErbB2 constitutively adopts an extended conformation potentiating the formation of heterodimers (8, 9) that can be inhibited by pertuzumab, a monoclonal antibody sterically blocking the heterodimerization arm of ErbB2 (10). Although the extracellular domain of ErbB2 has failed to form crystallographic homodimers, molecular biological and fluorescence resonance energy transfer (FRET) experiments have shown that full-length ErbB2 exists in dimers or higher-order aggregates in the plasma membrane (11,12). The implication is that the transmembrane, juxtamembrane and other intracellular domains (5, 13, 14) act in conjunction with the membrane environment (15) to mediate the dimerization and, thereby, functional states of ErbB proteins.Many investigators have sought to determine the distribution of ErbB1 in ...

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“…Unlike biochemical methods, FRET allows the quantitative and time-resolved determination of stable or transient protein-protein interactions in living cells, including the conformational changes associated with these interactions, by the simultaneous use of proper combinations of fluorescent GFP derivatives. It can be measured by both flow cytometry, which ensures high statistical accuracy, and image cytometry, which provides subcellular resolution (104,105).…”

Section: Cytometry Of Bh3 Moleculesmentioning

confidence: 99%

BH3‐only proteins: The death‐puppeteer's wires

2009

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Most cell death in vertebrates proceeds through the intrinsic pathway of apoptosis and results from unregulated increase of mitochondrial membrane permeability. Bcl2-associated X protein (Bax) and Bcl2-antagonist/killer protein (Bak), the effector proapoptotic members of the Bcl-2 family, are, in their active state, the principal accomplices for this permeabilization process. How exactly Bax and Bak are activated has been a matter of major investigation in the last decade, and suitable tools offered by quantitative cytometric methodologies have significantly contributed to the understanding of the function of Bcl-2 family members. Here, we review the most relevant findings in this field and highlight one common trait that has emerged from the diverse new theories: a crucial role in the control of Bax/Bak activation has to be attributed to the BH3-only subset of the Bcl-2 family. BH3-only proteins exert their proapoptotic activity by hierarchical and tightly tuned interactions with other Bcl-2 family members and operate as sensors of intracellular/extracellular death signals and vectors of information to the core apoptotic machinery. Given their essential role in apoptosis, BH3-only molecules are proposed as molecular targets for the cure of diseases associated with abnormal cell death, as in the case with neurodegenerative conditions. As well, they are explored as possible tools for cancer therapy, according to the concept that molecules mimicking the BH3 domain of these proteins could selectively and efficiently cooperate in the cell killing by chemotherapeutic drugs. A few BH3 mimetics are currently being tested in clinical trials of hematologic and solid tumors. Nevertheless, the knowledge about the cellular and molecular mechanisms that regulate responsiveness to BH3 therapy has to be further expanded and will benefit from recent advances in cytometric quantitative technologies. ' 2009 International Society for Advancement of Cytometry

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EGF-induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

Cited by 50 publications

References 46 publications

Copine-III interacts with ErbB2 and promotes tumor cell migration

Copine-III interacts with ErbB2 and promotes tumor cell migration

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

BH3‐only proteins: The death‐puppeteer's wires

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