Activation of tissue plasminogen activator by metastasis-inducing S100P protein

S100P protein in human breast cancer cells is associated with reduced patient survival and, in a model system of metastasis, it confers a metastatic phenotype upon benign mammary tumour cells. S100P protein possesses a C-terminal lysine residue. Using a multiwell in vitro assay, S100P is now shown for the first time to exhibit a strong, C-terminal lysine-dependent activation of tissue plasminogen activator (tPA), but not of urokinase-catalysed plasminogen activation. The presence of 10 μM calcium ions stimulates tPA activation of plasminogen 2-fold in an S100P-dependent manner. S100P physically interacts with both plasminogen and tPA in vitro, but not with urokinase. Cells constitutively expressing S100P exhibit detectable S100P protein on the cell surface, and S100P-containing cells show enhanced activation of plasminogen compared with S100P-negative control cells. S100P shows C-terminal lysine-dependent enhancement of cell invasion. An S100P antibody, when added to the culture medium, reduced the rate of invasion of wild-type S100P-expressing cells, but not of cells expressing mutant S100P proteins lacking the C-terminal lysine, suggesting that S100P functions outside the cell. The protease inhibitors, aprotinin or α-2-antiplasmin, reduced the invasion of S100P-expressing cells, but not of S100P-negative control cells, nor cells expressing S100P protein lacking the C-terminal lysine. It is proposed that activation of tPA via the C-terminal lysine of S100P contributes to the enhancement of cell invasion by S100P and thus potentially to its metastasis-promoting activity

The metastasis associated protein S100A4: role in tumour progression and metastasis

The metastasis associated protein S100A4 is a small calcium binding protein that is associated with metastatic tumors and appears to be a molecular marker for clinical prognosis. Below we discuss its biochemical properties and possible cellular functions in metastasis including cell motility, invasion, apoptosis, angiogenesis and differentiation

Phosphoproteomic analysis of Her2/neu signaling and inhibition

Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20–30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and δ-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein–protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins

Simultaneous targeting of requiem & Alg-2 in Chinese hamster ovary cells for improved recombinant protein production

10.1007/s12033-010-9304-3Molecular Biotechnology463301-30