Rat mast cell protease I alters cell metabolism.
Rat connective tissue mast cells are known to store significant amounts of mast cell protease I (RMCP I), which suppresses normal cell growth and mediates cytotoxicity against tumor cell lines, including the fibrosarcoma cell line FL. To better define its effects on FL cells, RMCP I was added to FL cultures for 30 min. Analysis of de novo nuclear protein synthesis revealed that RMCP I suppressed the expression of three proteins (41, 46, and 69 kD) and enhanced the expression of two other proteins (25 and 32 kD). Treatment of FL cells with diisopropylfluorophosphate (DFP)-inactivated RMCP I proved that these effects were largely independent of the protease catalytic site. Western blot hybridization, using a monoclonal antibody to phosphotyrosine-containing proteins, revealed that RMCP I inhibited phosphorylation of a nuclear and a cytoplasmic 81-kD tyrosylprotein. Inhibition of nuclear tyrosine kinase activity by RMCP I appeared to be catalytic site dependent, whereas cytoplasmic tyrosine kinase inhibition was independent of RMCP I proteolytic activity. Biotinylated RMCP I was used to identify potential surface-binding proteins. Three specific binding complexes (130, 150, and 210 kD) were detected. The binding of biotinylated RMCP I to these surface proteins was inhibited by excess unlabeled RMCP I, but not by trypsin or chymotrypsin. We speculate that the binding proteins may be critical in initiating RMCP I-induced metabolic changes on FL cells. The ability of RMCP I to alter the metabolism of cells suggests that it may have an important role in regulating their functions.
Department of Dermatology, University of Pittsburgh, Pennsylvania 15213.
I J
IJ
Chan
M D
MD
Tharp
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005354:Fibrosarcoma / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D008970:Molecular Weight; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D011505:Protein-Tyrosine Kinases; D012697:Serine Endopeptidases; D053818:Chymases