Methionine Aminopeptidase 2 Is a New Target for the Metastasis-associated Protein, S100A4

Hideya Endo,Keizo Takenaga,Takayuki Kanno,Hitoshi Satoh,Shigeo Mori

doi:10.1074/jbc.m202244200

Hideya Endo, Keizo Takenaga + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m202244200

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Abstract

S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.

Highlights

S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins
In Vitro Interaction of S100A4 with methionine aminopeptidase 2 (MetAP2)—We examined whether the interaction between S100A4 and the polypeptide encoded by the J6-2 cDNA, hereafter called MetAP2-(1– 229), takes place in vitro as well
In the present study we demonstrated for the first time that the metastasis-associated gene product, S100A4, interacts with an angiogenesis-related protein, MetAP2

Summary

The abbreviations used are

MetAP2, methionine aminopeptidase 2; ␣-MEM, ␣-minimal essential medium; FBS, fetal bovine serum; bFGF, basic fibroblast growth factor; HA, hemagglutinin; GST, glutathione S-transferase; DPBS, Dulbecco’s phosphate-buffered saline; POEP; protection of eIF2␣ phosphorylation; TRITC, tetramethylrhodamine isothiocyanate. Ingly, the growth of endothelial cells is much more sensitive to this compound than that of tumor cells [31, 32]. MetAP2 is thought to concern endothelial cell proliferation through N-. Terminal methionine processing of as yet unknown proteins essential for endothelial cell growth [30]. In an effort to identify additional target proteins for S100A4, we employed the yeast two-hybrid system and screened a cDNA library with S100A4 cDNA as bait. MetAP2 is a new target for S100A4

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION