Involvement of Raf-1 kinase and protein kinase C zeta in insulin-like growth factor I-induced brown adipocyte mitogenic signaling cascades: inhibition by cyclic adenosine 3',5'-monophosphate.
In the present study we have examined the signaling cascades involved in insulin-like growth factor I (IGF-I)-induced mitogenesis in fetal rat brown adipocyte primary cultures, a model that constitutively expresses a high number of IGF-I receptors, where IGF-I is a complete mitogen at physiological concentrations. IGF-I rapidly stimulated beta-chain IGF-I receptor autophosphorylation, which peaked at a physiological/mitogenic concentration (1.4 nM) and also stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Tyrosine-phosphorylated IRS-1 bound and subsequently activated phosphatidylinositol 3-kinase by 3.5-fold, whereas the tyrosine-phosphorylated IGF-I receptor was not directly associated with the p85 subunit of the phosphatidylinositol 3-kinase. Moreover, mitogenic concentrations of IGF-I enhanced glucose transport by 2.5-fold. In addition, tyrosine phosphorylation of the 46- and 52-kDa SHC proteins was high in the basal state and doubled after IGF-I treatment, whereas IGF-I enhanced by 4-fold tyrosine phosphorylation of the 66-kDa SHC band. Furthermore, a 2-fold increase in the Ras. GTP active form was induced upon IGF-I stimulation. Downstream from Ras, IGF-I increased both Raf kinase and protein kinase C (PKC) zeta activities by 3.5-fold. (Bu)2cAMP, an inhibitor of IGF-I-induced mitogenesis in fetal brown adipocyte primary cultures, did not block the very early steps of the IGF-I-induced mitogenic cascade, such as IGF-I receptor autophosphorylation, IRS-1 or SHC tyrosine phosphorylation, and Ras activation to its GTP active form. However, (Bu)2cAMP disrupted IGF-I-Raf and IGF-I-PKC zeta signaling pathways by preventing IGF-I-induced Raf-1 kinase and PKC zeta enzymatic activities, respectively. Our results show the first characterization in situ of an IGF-I mitogenic signaling cascade that downstream Ras diverges to the nucleus through two different serine/threonine kinases (Raf-1 kinase and PKC zeta) in mammalian fetal primary cells under physiological conditions. Both kinases represent a point of regulation primarily described for IGF-I-induced, cAMP-inhibited mitogenic pathways.
Valverde
A M
AM
Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Teruel
T
T
Lorenzo
M
M
Benito
M
M
D002001:Adipose Tissue, Brown / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007527:Isoenzymes / Q000502:physiology; D008934:Mitogens / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats / Q000196:embryology; D017208:Rats, Wistar; D017451:Receptors, Somatomedin / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D000099221:Protein Kinase C zeta
D000242:Cyclic AMP; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I; D007527:Isoenzymes; D008934:Mitogens; D009004:Monosaccharide Transport Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D017451:Receptors, Somatomedin; D014443:Tyrosine; D000099221:Protein Kinase C zeta; C526296:Irs1 protein, rat