טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentPreger Ella
SubjectIsolation and Characterization of Novel Inhibitors of
Fibroblast Growth Factors Mediated Signaling
DepartmentDepartment of Biology
Supervisor Professor Dina Ron


Abstract

Fibroblast growth factor (FGF) signaling is essential for many cellular processes including proliferation, differentiation, survival and migration. Recently, a novel inhibitor of FGF-mediated signaling has been identified in zebrafish. This inhibitor, designated Sef, is conserved across vertebrates and encodes a putative type I transmembrane protein. Here we describe the identification and isolation of several isoforms of human Sef: a form that is similar to zebrafish Sef (designated hSef-a), and three novel isoforms (designated hSef-b to hSef-d). These isoforms are generated from a single gene via an alternative splicing mechanism and differ from each other in their amino terminal sequences. Contrary to hSef-a, which is ubiquitously expressed, hSef-b and hSef-c transcripts display both restricted and differential pattern of expression in human tissues. The differences in the amino-terminus of hSef-a and hSef-b confer distinctive characteristics on each of these isoforms and influence their biological activities. Thus, while hSef-a is a membrane associated glycoprotein, hSef-b lacks signal for secretion and is localized in the cytosol. In addition, hSef-b is translated from a CUG codon whereas hSef-a is translated from a conventional AUG codon. In order to study the biological activities of these isoforms in details, we have established NIH/3T3 stable cell lines in which the expression of hSef-a and hSef-b is regulated by tetracycline. In these cells, both hSef-a and hSef-b inhibit entry to S-phase in response to different FGFs. Human Sef-a does not affect the mitogenic activity of PDGF, EGF and insulin, suggesting that this isoform is a specific inhibitor of FGF-mediated signaling. Preliminary results however, indicate that hSef-b has a broader spectrum of specificity as it inhibits. Our results therefore indicate that alternative splicing mechanism generates hSef isoforms that differs from each other in many aspects including biochemical properties, subcellular localization and specificity.