Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation.
Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyl phosphorylation of the beta-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44mapk and p42mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44mapk and p42mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.
D'Onofrio
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Centre de Recherche/Hôpital Hôtel-Dieu de Montréal, Department of Medicine, Université de Montréal, Qué, Canada.
Le
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Chiasson
J L
JL
Srivastava
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AK
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000187:drug effects* / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*
D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1