1.     Molecular Regulation of Cellular Signaling and Trafficking of Src Mediated by Thrombin Receptor.


Thrombin, a multifunctional serine protease generated at sites of vascular injury, plays a critical role in hemostasis and thrombosis and perhaps in inflammatory and proliferative responses.  Cellular responses to thrombin are mediated at least in part by a family of G protein-coupled protease-activated receptors (PARs).  PAR1, the prototypical member of this family, is irreversibly activated upon thrombin cleavage.  Src is an important downstream effector of PAR1-mediated cellular signaling.  However, the regulation of Src kinase activity after PAR1 activation is very complex.  In addition to being activated and de-acitivated, Src was found to be degraded and re-expressed after PAR1 activation.  The degradation of Src induced by PAR1 was promoted by b-arrestin2 (Kao et al., 2006, Cell Signal. 18:1914-1923).  Our preliminary results show that Src is targeted to lysosomes for degradation after PAR1 activation.  To investigate the mechanisms by which PAR1 induces degradation of Src in lysosome, we could like to address the following questions.  Is ubiquitin involved in PAR1-induced degradation of Src?  Do ubiquitin E3 ligases and/or lysosomal sorting machinery involved in this event?  How does b-arrestin2 promote this event?  Since Src was re-expressed after its degradation in response to PAR1 activation.  We will elucidate the cellular signaling events involved in PAR1-induced re-expression of Src and characterize whether it is under transcriptional or translational regulation.  The re-expression of Src might regulate PAR1-indcued gene expression, which may be related to PAR1-promoted metastatsis of some cancer cells.  We will also try to explore such possibility.


2.    Involvement of Protease-activated Receptors in Migration and Proliferation of Lung Cancer Cells.


  Protease-activated receptors (PARs), a subfamily of G-protein couple receptor are activated by proteolytic cleavage at specific sites of the extracellular domains by serine proteases.  The newly exposed amino terminus serves as a tethered ligand, which binds to the second extracellular loop of the receptor, to initiate signal transduction.  Today, four subtypes of PARs have been identified.  They are PAR1, PAR2, PAR3, and PAR4.  PARs have been shown to play important roles in coagulation, proliferation, survival, inflammation and tumorigenesis.  The expression of PAR1, PAR2 and PAR4 has been reported to be elevated in lung cancer. The increased expression of PAR1 and PAR2 might be related to the migration and proliferation of lung cancer cells.  Here, we could like to determine whether PAR1 and PAR2 induce migration and proliferation of lung cancer cells and the underlined molecular mechanisms.  The possible PARs-mediated signal transduction pathways leading to proliferation and migration in lung cancer cells are shown in Fig. 1.  The involvement of G proteins and mitogen-activated protein kinases (MAPKs) in PAR1 and PAR2-induced migration and proliferation of lung cancer cells will be examined.  Whether cancer cell secretes unconventional proteases to activate PARs on its cell membrane will be elucidated.  Also, whether PARs transactivate other membrane receptors, such as epidermal growth factor receptor (EGFR), to enhance cellular responses will be investigated.


Fig. 1. Activation of protease-activated receptors involved in migration and proliferation of lung cancer. Protease-activated receptors (PARs) are activated by serine proteases; subsequently the activated PARs initiate the MAPK signaling to elicit cellular responses including migration and proliferation. In addition, transactivation of membrane receptors such as EGFR by PARs enhances PARs-induced cellular responses. Lung cancer cells may also secrete serine proteases and create an autocrine loop to prolong PARs activation.


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