Stimulation of inorganic phosphate transport by insulin-like growth factor I and vanadate in opossum kidney cells is mediated by distinct protein tyrosine phosphorylation processes.
Insulin-like growth factor I (IGF-I) stimulates sodium-dependent inorganic phosphate (Pi) transport across the apical plasma membrane of confluent opossum kidney (OK) cells. Previous studies indicated that vanadate, at doses known to inhibit protein tyrosine phosphatases, mimicked the effect of IGF-I and suggested the involvement of tyrosine phosphorylation processes in Pi transport regulation. In this study, protein tyrosine phosphorylation and activation of several cellular signaling pathways were investigated in confluent OK cells in response to IGF-I and vanadate. We report that IGF-I and vanadate induced tyrosine phosphorylation of distinct proteins. Tyrosine phosphorylation of p95 (IGF-I receptor beta-subunit) was rapidly and dose dependently increased in response to IGF-I. Associated with phosphorylation of the receptor, the increase in tyrosine phosphorylation of a protein of 50 kDa was observed. Vanadate did not mimic the effect of IGF-I, but increased phosphorylation of seven major proteins of 170, 140, 100, 83, 70-82, 60, and 35 kDa. Among the different tyrosine kinase inhibitors tested, only staurosporine affected Pi transport up-regulation by IGF-I and vanadate, attenuating the effect of IGF-I and completely blocking the response to vanadate. Staurosporine decreased tyrosine phosphorylation of several constitutively phosphorylated proteins and interfered with the increase in tyrosine phosphorylation induced by vanadate. Phosphorylation of p95 in response to IGF-I was not affected. Staurosporine also markedly decreased constitutive association of the adapter protein Nck with tyrosine-phosphorylated proteins and attenuated increases in phosphotyrosine-associated Nck induced by IGF-I and vanadate. In contrast, signaling to other downstream effectors common to IGF-I and vanadate, such as mitogen-activated protein kinase and phosphatidylinositol-3-kinase, was not affected by staurosporine. In conclusion, our results suggest that although IGF-I and vanadate induce distinct protein tyrosine phosphorylation in OK cells, they activate an overlapping set of signaling molecules, among which Nck appears as an interesting candidate to link activation of tyrosine kinases to the stimulation of Pi transport.
Department of Medicine, University of Geneva, Switzerland.
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Palmer
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JP
Bonjour
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Caverzasio
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016227:Benzoquinones; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium; D019833:Genistein; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007529:Isoflavones / Q000494:pharmacology; D007668:Kidney; D047029:Lactams, Macrocyclic; D009281:Naphthalenes / Q000494:pharmacology; D015513:Oncogene Proteins / Q000378:metabolism; D009893:Opossums; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D007328:Insulin; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D009281:Naphthalenes; C078332:Nck protein; D015513:Oncogene Proteins; D010710:Phosphates; D010750:Phosphoproteins; D011809:Quinones; D011994:Recombinant Proteins; D017828:Rifabutin; D019000:Phosphotyrosine; D014638:Vanadates; D007334:Insulin-Like Growth Factor I; C020754:herbimycin; D012964:Sodium; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C