Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways

Adams, Anthony G.; Thorn, Judith M.; Yamabhai, Montarop; Kay, Brian K.; O’Bryan, John P.

doi:10.1074/jbc.m004810200

Cited by 51 publications

(77 citation statements)

References 39 publications

(18 reference statements)

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“…This plasmid described previously (20) as CMV·(His) 6 N·hTTP contains DNA sequences encoding the six consecutive histidine residues inserted between the first methionine and the second aspartic acid residues of full-length hTTP. Plasmid pEF-X EH was used as a negative control for expressing a His-tagged Xenopus Eps15 homology (EH) domain of intersectin (26) in plasmid vector pEF6/V5-His-TOPO (Invitrogen, Gaithersburg, MD).…”

Section: Methodsmentioning

confidence: 99%

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Cao

2004

Biochemistry

122

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Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AUrich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor α mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 μM Zn 2+ . The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zincdependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex. Tristetraprolin (TTP)1 (also known as ZFP36, Nup475, TIS11, and G0S24) is a prototypical member of a small family of mammalian proteins with tandem CCCH (CX 8 CX 5 CX 3 H) zinc finger motifs separated by 18 amino acid residues (1). Similar zinc finger sequences are found in at least 19 species in the GenBank database, including human, cow, mouse, rat, sheep,

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Section: Methodsmentioning

confidence: 99%

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Cao

2004

Biochemistry

122

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“…Intersectin has been shown to regulate the Ras/mitogen-activated protein (MAP) kinase pathway and activate Elk-1 transcription factor, resulting in oncogenic transformation of rodent cells. [38][39][40] Overexpression of a dominant negative mutant with SH3 domains of intersectin repressed the activation of Ras and Elk-1 39 and failed to induce transformation. 40 In contrast to intersectin, the expression For personal use only.…”

Section: Discussionmentioning

confidence: 99%

“…[38][39][40] Overexpression of a dominant negative mutant with SH3 domains of intersectin repressed the activation of Ras and Elk-1 39 and failed to induce transformation. 40 In contrast to intersectin, the expression For personal use only. on April 6, 2019. by guest www.bloodjournal.org From of EBP not only suppressed the transcriptional activation of Elk-1 but also Ras-induced oncogenic transformation (Figure 7).…”

Section: Discussionmentioning

confidence: 99%

“…Because EBP displays high structural similarity with the multiple SH3 domains in the C-terminal of intersectin, which has been reported to interact with Sos and activate Elk-1 activation, 39,40 we also investigated the effect of EBP on the downstream signaling of Ras by performing luciferase assay using Gal4-Elk-1 transreporter. Expression of RasQ61L, a constitutively active mutant, enhanced the activation of Elk-1 transreporter by 6-fold (P Ͻ .001, ANOVA) ( Figure 7C).…”

Section: Expression Of Ebp and Een Inhibits Ras Transformation Potentmentioning

confidence: 99%

“…Rat 6 (R6) cells 33 40]). Lysed cells were cleared by centrifugation, and supernatants were collected.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

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Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways

Cited by 51 publications

References 39 publications

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Identification and characterization of EBP, a novel EEN binding protein that inhibits Ras signaling and is recruited into the nucleus by the MLL-EEN fusion protein

EH Domains and Their Ligands

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Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways

Cited by 51 publications

References 39 publications

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

Identification and characterization of EBP, a novel EEN binding protein that inhibits Ras signaling and is recruited into the nucleus by the MLL-EEN fusion protein

EH Domains and Their Ligands

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Product

Resources

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Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,