Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3-L1 cells.
Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.
Boney
C M
CM
Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, Rhode Island 02903, USA. Charlotte_Boney@brown.edu
Sekimoto
H
H
Gruppuso
P A
PA
Frackelton
A R
AR
D016475:3T3 Cells; D000818:Animals; D002455:Cell Division / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D011720:Pyrazoles; D011743:Pyrimidines; D010566:Virulence Factors, Bordetella; D007334:Insulin-Like Growth Factor I; D004247:DNA; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases