Generation and characterization of a novel, permanently active S100P mutant

Abstract

S100 proteins function as Ca2+ signal transducers by regulating cellular targets in their Ca2+ bound conformation. S100P is a member of the S100 protein family that can activate the membrane and F-actin binding protein ezrin in a Ca2+ dependent manner at least in vitro. Here we generated a novel tool to elucidate directly the S100P-ezrin interaction in vivo. This was achieved by constructing a S100P derivative that contained mutations in the two EF hand loops predicted to lock the protein in a permanently active state. The resulting S100P mutant, termed here S100P pa, could be purified as a soluble protein and showed biochemical properties displayed by wild-type S100P only in the presence of Ca2+. Importantly, S100P pa bound to the N-terminal domain of ezrin in the absence of Ca2+ showing an affinity only slightly reduced as compared to that of Ca2+-bound WT S100P. In line with this permanent complex formation, S100P pa colocalized with ezrin to plasma membrane protrusions of epithelial cells even in the absence of intracellular Ca2+ transients. Thus, S100P pa is a novel type of S100 protein mutant locked in a permanently active state that shows an unregulated complex formation with its cellular target ezrin.

Different roles of S100P Overexpression in Intrahepatic Cholangiocarcinoma

“…Although extracellular S100P stimulates cell proliferation by RAGE, 5 Austermann et al 7,8 have shown S100Pmediated activation of ezrin and suggested that the interaction between S100P and ezrin promotes a migration of tumor cells and metastatic potential. Our study has shown that S100P expression is significantly correlated with ezrin expression, not with RAGE expression, in ICC tissue.…”

Ca2+/S100 proteins inhibit the interaction of FKBP38 with Bcl-2 and Hsp90

“…To overcome this problem, 'permanently active' S100P (S100P-PA) was used and the use of ionophores or thapsigargin was avoided. Following the report of Austermann et al [28], we constructed S100P-PA that contained mutations in the two EF-hand loops predicted to lock the protein in a permanently active state. When S100P-PA was transfected into Huh-7 with Bcl-2, a significant decrease in the protein level of Bcl-2 was observed along with elevation of the cellular levels of S100P-PA ( Figure 6D).…”

“…The Bcl-2 transmembrane deletion mutant (residues 219-239; Bcl-2 C21) was subcloned into pMAL-c2x. pME18S-permanently active S100P (S100P-PA) was prepared as described previously [28].…”

S100 Proteins Modulate Protein Phosphatase 5 Function

“…The construction of the pET11a-S100 and pME18S-S100 plasmids was reported previously (18). A permanently active S100P (S100P-PA) was prepared as described (19). To create a pcDNA3-Tau plasmid, the human Tau441 cDNA was purchased from Addgene and cloned into the pcDNA3 vector.…”

“…To overcome this problem, "permanently active S100P" (S100P-PA) was used, and the use of ionophore or thapsigargin was avoided. Followed the report of Austermann et al (19), we constructed S100P-PA that contained mutations in the two EF-hand loops predicted to lock the protein in a permanently active state. Similar to S100A1, S100A2, S100A6, and S100B (Fig.…”