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MECHANISMS OF UROKINASE MEDIATED ACTIVATION OF THE MAMMALIAN TARGET OF RAPAMYCIN (mTOR)
Author(s) -
Davies Mark G,
Roztocil Elisa,
Nicholl Suzanne
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a192-c
Subject(s) - pi3k/akt/mtor pathway , mtorc2 , wortmannin , chemistry , rptor , ly294002 , phosphorylation , microbiology and biotechnology , pertussis toxin , cancer research , sirolimus , rheb , signal transduction , biology , mtorc1 , g protein , biochemistry
Background: Vascular smooth muscle cell (VSMC) migration is an important component of the development of intimal hyperplasia and uPA is a key serine protease in this process. We have shown that rapamycin an antifungal and immunosuppressant that inhibits mTOR, inhibits VSMC migration and that uPA activates mTOR. Purpose: To examine the signaling pathways that lead to the activation of mammalian target of rapamycin (mTOR) by urokinase (uPA) Methods: Rat VSMCs were cultured in vitro. Western blotting was performed for phosphorylated and total mTOR and its downstream kinase p70S6K after stimulation with uPA (10nM) with and without inhibitors of Gαi (pertussis toxin and siRNA against Gαi2 and Gαi3) and Gβγ(adenovirus with βARKCT), the EGFR inhibitor AG1478, PI3Kinase (wortmannin and LY294002), and src (PP2) and rapamycin, a mTOR inhibitor. Results: uPA stimulated phosphorylation of mTOR and p70S6K (2‐fold increase over control for both, p<0.05). These responses were inhibited the Gαi inhibitor pertussis toxin and by siRNA to Gαi2 and Gαi3. Gβγ was also required for activation of mTOR Phosphorylation of mTOR and p70S6K by uPA was dependent on EGFR and PI3K (p<0.01) but was src independent. PI3K activation was dependent on EGFR and Gβγ. Rapamycin blocked mTOR and p70S6K activation without affecting ATF activation of EGFR or PI3K. Conclusions: uPA‐induced mTOR activation and subsequent p70S6K is Gαi and Gβγ dependent and is modulated through a EGFR mediated pathway leading to PI3K activation of mTOR. Understanding the basic mechanisms of cell migration will allow therapeutic intervention for restenosis.