Spermine, a molecular switch regulating EGFR, integrin β3, Src, and FAK scaffolding.
Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.
Department of Physiology, The University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163, USA. rray3@uthsc.edu
Ramesh M
RM
Ray
Chunying
C
Li
Sujoy
S
Bhattacharya
Anjaparavanda P
AP
Naren
Leonard R
LR
Johnson
D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism*; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000235:genetics*; D013096:Spermine / Q000494:pharmacology*; D015533:Transcriptional Activation; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D039661:Integrin beta3; D013096:Spermine; C512478:EGFR protein, human; D066246:ErbB Receptors; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases; D009955:Ornithine Decarboxylase