scholarly journals Calcium-binding protein S100A6 interaction with VEGF receptors integrates signaling and trafficking pathways

2021 ◽  
Author(s):  
Sreenivasan Ponnambalam ◽  
Leyuan Bao ◽  
Gareth W Fearnley ◽  
Chi-Chuan Lin ◽  
Adam F Odell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Calcium Binding ◽  
Mapk Pathway ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Mitogen Activated Protein Kinase ◽  
Tyrosine Kinase Domain ◽  
Dependent Manner ◽  
Endothelial Cell Function

The mammalian endothelium which lines all blood vessels responds to soluble factors which control vascular development and sprouting. Endothelial cells bind to vascular endothelial growth factor A via two different receptor tyrosine kinases (VEGFR1, VEGFR2) which regulate such cellular responses. The integration of VEGFR signal transduction and membrane trafficking is not well understood. Here, we used a yeast-based membrane protein screen to identify VEGFR-interacting factor(s) which modulate endothelial cell function. By screening a human endothelial cDNA library, we identified a calcium-binding protein, S100A6, which can interact with either VEGFR. We found that S100A6 binds in a calcium-dependent manner to either VEGFR1 or VEGFR2. S100A6 binding was mapped to the VEGFR2 tyrosine kinase domain. Depletion of S100A6 impacts on VEGF-A-regulated signaling through the canonical mitogen-activated protein kinase (MAPK) pathway. Furthermore, S100A6 depletion caused contrasting effects on biosynthetic VEGFR delivery to the plasma membrane. Co-distribution of S100A6 and VEGFRs on tubular profiles suggest the presence of transport carriers that facilitate VEGFR trafficking. We propose a mechanism whereby S100A6 acts as a calcium regulated switch which facilitates biosynthetic VEGFR trafficking from the TGN-to-plasma membrane. VEGFR-S100A6 interactions thus enable integration of signaling and trafficking pathways in controlling the endothelial response to VEGF-A.

scholarly journals Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin

1994 ◽  
Vol 124 (5) ◽  
pp. 757-768 ◽  
Author(s):  
K Takenaga ◽  
Y Nakamura ◽  
S Sakiyama ◽  
Y Hasegawa ◽  
K Sato ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
S100 Protein ◽  
Calcium Binding Protein ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Cell Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.

A novel p53 target gene, S100A9, induces p53-dependent cellular apoptosis and mediates the p53 apoptosis pathway

2009 ◽  
Vol 422 (2) ◽  
pp. 363-372 ◽  
Author(s):  
Chunsun Li ◽  
Hongyan Chen ◽  
Fang Ding ◽  
Yu Zhang ◽  
Aiping Luo ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
Cellular Apoptosis ◽  
Reporter Assays ◽  
P53 Activation ◽  
Acute And Chronic Inflammation

S100A9 (S100 calcium-binding protein A9) is a calcium-binding protein of the S100 family, and its differential expression has been associated with acute and chronic inflammation and several human cancers. Our previous work showed that S100A9 was severely down-regulated in human ESCC (oesophageal squamous cell carcinoma). To further investigate the transcriptional regulation of S100A9, we analysed the S100A9 promoter region and found several putative p53BS (p53-binding sites). Luciferase reporter assays showed that constructs carrying the p53BS exhibited enhanced luciferase activity in response to wild-type p53 activation. Further study demonstrated that S100A9 mRNA and protein expression could be positively regulated in a p53-dependent manner and p53 could bind to p53BS on the S100A9 promoter. Overexpression of S100A9 could induce cellular apoptosis, and this was partly p53-dependent. Knockdown of S100A9 impaired the apoptosis induced by p53. Thus we conclude that a gene down-regulated in ESCC, S100A9, is a novel p53 transcriptional target, induces cellular apoptosis in a partly p53-dependent manner and mediates the p53 apoptosis pathway.

Interaction of the proteasome S5a/Rpn10 multiubiquitin-binding protein and the 8 kDa calcium-binding protein of Schistosoma mansoni

Parasitology ◽  
2003 ◽  
Vol 127 (4) ◽  
pp. 337-347 ◽  
Author(s):  
D. RAM ◽  
E. ZIV ◽  
F. LANTNER ◽  
I. SCHECHTER
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
26S Proteasome ◽  
Dependent Manner ◽  
Terminal Portion ◽  
Binding Interaction ◽  
Western Blots ◽  
Free System ◽  
Amino Terminal

A distinct 8 kDa calcium-binding protein (CaBP) is preferentially expressed at the cercarial stage during the life-cycle of the schistosome. Available data indicate that this CaBP may be associated with tissue/organ remodelling (involving protein degradation and synthesis of new proteins) during transformation of the cercariae from free-living form in water to parasitic life in the vertebrate host. Many CaBP molecules (e.g. calmodulin) show Ca++-dependent interaction with target proteins and thus modulate their activity. Accordingly, the parasite 8 kDa CaBP was used as a probe to clone and identify putative target protein(s) directly by binding interaction. Screening of schistosome λgt11 expression library with radio-iodinated CaBP yielded several overlapping clones showing Ca++-dependent binding of the CaBP. Sequence analyses revealed that these clones encode the S5a/Rpn10 multiubiquitin-binding protein which is a component of the regulatory 19S subunit of the 26S proteasome. The schistosome molecule, designated SmS5a, is 420 amino acids long. The nearly full length molecule (Gln3–Ser420) as well as the amino terminal (N-S5a, Gln3–Gly200) and carboxyl-terminal (C-S5a, Asp225–Ser420) portions were synthesized in bacteria, purified, and antibodies to the parasite SmS5a were prepared. Interaction between SmS5a and the 8 kDa CaBP in a Ca++-dependent manner was found under various experimental conditions: CaBP-Sepharose bound soluble SmS5a, immobilized SmS5a bound soluble CaBP, and complex formation was found when both molecules were in solution. Furthermore, it was shown that the C-terminal portion of SmS5a, but not the N-terminal portion of the molecule, reacted with the CaBP. SmS5a synthesized in a cell-free system and Western blots revealed 2 species, conceivably corresponding to the naked molecule (~50 kDa) and the molecule subjected to post-translational modification (~70 kDa). The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

Calcium-binding protein 1 of Entamoeba histolytica transiently associates with phagocytic cups in a calcium-independent manner

2008 ◽  
Vol 10 (6) ◽  
pp. 1373-1389 ◽  
Author(s):  
Ruchi Jain ◽  
Julien Santi-Rocca ◽  
Narendra Padhan ◽  
Sudha Bhattacharya ◽  
Nancy Guillen ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Independent Manner ◽  
Phagocytic Cups
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