Identification of 12-lipoxygenase interaction with cellular proteins by yeast two-hybrid screening

The platelet isoform of 12-lipoxygenase (12-LOX) is expressed in a variety of human tumors. 12-LOX metabolizes arachidonic acid to 12(S)-hydroxyeicosa teraenoic acid (12(S)-HETE), which induces a number of cellular responses a ssociated with tumor progression and metastasis. Little is known about 12-L OX regulation and no direct regulators of 12-LOX activity have been identif ied. To identify potential regulators of 12-LOX, we isolated cDNAs encoding 12-LOX interacting proteins using the yeast two-hybrid system. We screened a yeast two-hybrid interaction library from human epidermoid carcinoma A43 1 cells and identified four cellular proteins that interact specifically wi th 12-LOX. We identified type II keratin 5, lamin A, the cytoplasmic domain of integrin beta 4 subunit and a phosphoprotein C8FW as 12-LOX interacting proteins. Here, we demonstrated that keratin 5, a 58 kD protein required f or formation of 8 nm intermediate filaments, binds to 12-LOX in human tumor cells and may contribute to the regulated trafficking of 12-LOX. We also s howed that lamin A binds 12-LOX in human tumor cells. These proteins provid e the first candidate regulators of 12-LOX.