TY - JOUR
T1 - Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins
AU - Doi, Seita
AU - Fujioka, Naoki
AU - Ohtsuka, Satomi
AU - Kondo, Rina
AU - Yamamoto, Maho
AU - Denda, Miwako
AU - Magari, Masaki
AU - Kanayama, Naoki
AU - Hatano, Naoya
AU - Morishita, Ryo
AU - Hasegawa, Takafumi
AU - Tokumitsu, Hiroshi
N1 - Funding Information:
We thank Mami Asakura (Okayama University) for expression and purification of recombinant S100 proteins.
Publisher Copyright:
© 2021 Elsevier Ltd
PY - 2021/6
Y1 - 2021/6
N2 - To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111–160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.
AB - To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111–160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.
KW - Calcium signaling
KW - Dimerization
KW - Genomewide screening
KW - Intracellular calcium signal transduction
KW - Microtubule organization
KW - Multiple system atrophy
KW - Oligodendrocytes
KW - Parkinson's disease
KW - Protein active array
KW - Protein-protein interaction
KW - S100A2
KW - S100A6
KW - S100B
KW - TPPP
KW - Tubulin polymerization-promoting protein
KW - α-Synuclein
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U2 - 10.1016/j.ceca.2021.102404
DO - 10.1016/j.ceca.2021.102404
M3 - Article
C2 - 33831707
AN - SCOPUS:85103710177
VL - 96
JO - Cell Calcium
JF - Cell Calcium
SN - 0143-4160
M1 - 102404
ER -