The Glioma-associated Protein SETA Interacts with AIP1/Alix and ALG-2 and Modulates Apoptosis in Astrocytes

Chen, Baihua; Borinstein, Scott C.; Gillis, Jennifer; Sykes, Virginia W.; Bögler, Oliver

doi:10.1074/jbc.m908994199

Cited by 110 publications

(133 citation statements)

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“…Alix/AIP1 is a cytoplasmic protein ∼98 kDa in size, containing a proline-rich C-terminal region encompassing several Src homology domain 3 (SH3) binding motifs. This region was shown to interact with SETA (SH3 domain expressed in tumorigenic astrocytes) (8), whereby Alix/ AIP1 could mediate UV light-induced cell death in astrocytes. Alix/AIP1 has also been implicated in multivesicular sorting mediated by the interactions of the N-terminal region of the protein with CHMP4b (9), while the C-terminal region is associated with ALG-2 interactions.…”

mentioning

confidence: 99%

Ca²⁺ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

et al. 2004

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Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca 2+ -binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca 2+ concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca 2+ concentration. However, binding of Ca 2+ to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca 2+ affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca 2+ binding to EF-3, while Ca 2+ binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition. † This work was supported by NIH Grants EY12768 and EY13705 (A.S.P.), EY08061 (K.P.), and EY06603 and EY14239 (J.W.C.) and the Swiss National Science Foundation 31-65071.01 (J.C.), as well as grants from the Retina Research Foundation, Research to Prevent Blindness Inc. (RPB), the University of Wisconsin Comprehensive Cancer Center, and the E. K. Bishop Foundation. L.S. is a recipient of the Cremer Scholarship. A.S.P. is a Jules and Doris Stein RPB Professor. * To whom correspondence should be addressed. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptApoptosis-linked gene-2 (ALG-2) was first identified in a death-trap assay using a mouse T cell hybridoma model (1). These cells when transfected with an antisense ALG-2 expression vector were resistant to T cell receptor-mediated cell death and were partially protected from other agents that normally initiate programmed cell death such as Fas, staurosporine, actinomycin D, and C 2 -ceramide (1). More recent work suggests that ALG-2 might be involved in the apaf1-independent intrinsic apoptotic pathway activated as a result of endoplasmic reticulum (ER) stress (2).ALG-2 contains five putative EF-hand sequences and is a member of the family of PEF 1 (penta-EF-hand) proteins that includes the calpain small subunit, sorcin, and grancalcin (3). In ALG-2, EF-1 and EF-3 are considered the high-affinity Ca 2+ -binding sites, while one low-affinity site...

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Ca²⁺ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

et al. 2004

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“…It has been cloned by other groups and named Cin85 or SETA [12,13]. Based on sequence homology and domain organization, Ruk L was integrated into a new subfamily of adaptor molecules that includes the protein CD2AP, also named CMS.…”

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confidence: 99%

“…Although the precise role of this Ruk/CD2AP family is still unknown, they are involved in the control of apoptosis and the regulation of cytoskeletal architecture [11,13,[15][16][17][18].…”

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Ruk is ubiquitinated but not degraded by the proteasome

Verdier

Valovka

Zhyvoloup

et al. 2002

European Journal of Biochemistry

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The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor‐type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3‐kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3‐kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3‐kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015–4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination.

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“…The retrovirus (pLXSN) without insert yielded a similar titer and was used as a control vector. Supernatants with the amphotropic retroviral vectors 1726Zeo and 1726Zeo.DEGFR(2-7) were kindly provided by O Bo¨gler (Chen et al, 2000).…”

Section: Retroviral Vectors and Plasmidsmentioning

confidence: 99%

INK4a/Arf is required for suppression of EGFR/ΔEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma

Lachat¹,

Diserens²,

Nozaki³

et al. 2004

Oncogene

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Amplification of the epidermal growth factor receptor (EGFR) or expression of its constitutively activated mutant, DEGFR(2-7), in association with the inactivation of the INK4a/Arf gene locus is a frequent alteration in human glioblastoma. The notion of a cooperative effect between these two alterations has been demonstrated in respective mouse brain tumor models including our own. Here, we investigated underlying molecular mechanisms in early passage cortical astrocytes deficient for p16 INK4a / p19 Arf or p53, respectively, with or without ectopic expression of DEGFR(2-7). Targeting these cells with the specific EGFR inhibitor tyrphostin AG1478 revealed that phosphorylation of ERK was only abrogated in the presence of an intact INK4a/Arf gene locus. The sensitivity to inhibit ERK phosphorylation was independent of ectopic expression of DEGFR(2-7) and independent of the TP53 status. This resistance to downregulate the MAPK pathway in the absence of INK4a/Arf was confirmed in cell lines derived from our mouse glioma models with the respective initial genetic alterations. Thus, deletion of INK4a/Arf appears to keep ERK in its active, phosphorylated state insensitive to an upstream inhibitor specifically targeting EGFR/DEGFR(2-7). This resistance may contribute to the cooperative tumorigenic effect selected for in human glioblastoma that may be of crucial clinical relevance for treatments specifically targeting EGFR/DEGFR(2-7) in glioblastoma patients.

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The Glioma-associated Protein SETA Interacts with AIP1/Alix and ALG-2 and Modulates Apoptosis in Astrocytes

Cited by 110 publications

References 13 publications

Ca²⁺ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

Ca²⁺ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

Ruk is ubiquitinated but not degraded by the proteasome

INK4a/Arf is required for suppression of EGFR/ΔEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma

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The Glioma-associated Protein SETA Interacts with AIP1/Alix and ALG-2 and Modulates Apoptosis in Astrocytes

Cited by 110 publications

References 13 publications

Ca2+ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

Ca2+ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

Ruk is ubiquitinated but not degraded by the proteasome

INK4a/Arf is required for suppression of EGFR/ΔEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma

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Ca²⁺ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma

Ca²⁺ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma