Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells.

Our previous study demonstrated that tyrosine phosphorylation of p145(met)/ß-subunit of hepatocyte growth factor receptor by epidermal growth factor receptor and Src contributes to the anti-apoptotic growth of human bladder carcinoma cell 5637 under serum-starved conditions. Here, we show that some other cell lines of human bladder carcinoma, but not other types of human cancer cells, also exhibit Src-dependent, anti-apoptotic proliferation under serum-starved conditions, and that low-density, detergent-insoluble membrane microdomains (MD) serve as a structural platform for signaling events involving p145(met), EGFR, and Src. As an MD-associated molecule that may contribute to bladder carcinoma-specific cellular function, we identified uroplakin IIIa (UPIIIa), an urothelium-specific protein. Results obtained so far revealed: 1) UPIIIa undergoes partial proteolysis in serum-starved cells; 2) a specific antibody to the extracellular domain of UPIIIa inhibits the proteolysis of UPIIIa and the activation of Src, and promotes apoptosis in serum-starved cells; and 3) knockdown of UPIIIa by short interfering RNA also promotes apoptosis in serum-starved cells. GM6001, a potent inhibitor of matrix metalloproteinase (MMP), inhibits the proteolysis of UPIIIa and promotes apoptosis in serum-starved cells. Furthermore, serum starvation promotes expression and secretion of the heparin-binding EGF-like growth factor in a manner that depends on the functions of MMP, Src, and UPIIIa. These results highlight a hitherto unknown signaling network involving a subset of MD-associated molecules in the anti-apoptotic mechanisms of human bladder carcinoma cells.

Amyloid beta peptide specifically promotes phosphorylation and nuclear translocation of the extracellular signal-regulated kinase in cultured rat cortical astrocytes.

To explore cellular signaling molecules that respond to amyloid beta protein (A beta), we investigated the effect of A beta on tyrosine phosphorylation of cellular proteins in cultured rat cortical astrocytes. Western blotting with the phosphotyrosine-specific monoclonal antibody 4G10 demonstrated that exposure of cultured rat cortical astrocytes to 20 microM A beta 1-40 or A beta 25-35 for 24 h resulted in a prominent increase in the phosphotyrosine content of 44-kDa protein. The A beta-induced increase in tyrosine phosphorylation of 44-kDa protein was blocked by U0126, a specific inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK. Western blotting with anti-phospho-ERK1/2 antibody and anti-ERK1/2 antibody demonstrated that A beta 1-40 or A beta 25-35 induced an increase in the dually (tyrosine and threonine) phosphorylated form of ERK1 and ERK2, with no change in total ERK1/2 level. In addition, immunofluorescent staining with anti-ERK1/2 antibody revealed that A beta induced a significant increase in the number of cells expressing ERK1/2 mainly in the nucleus. These results suggest that A beta specifically promotes tyrosine phosphorylation and nuclear translocation of ERK in astrocytes.