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Volume 270, Number 31, Issue of August 04, pp. 18531-18538, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Control of PHAS-I by Insulin in 3T3-L1 Adipocytes
SYNTHESIS, DEGRADATION, AND PHOSPHORYLATION BY A RAPAMYCIN-SENSITIVE AND MITOGEN- ACTIVATED PROTEIN KINASE-INDEPENDENT PATHWAY

(Received for publication, May 5, 1995; and in revised form, June 6, 1995)

Tai-An Lin Xianming Kong Alan R. Saltiel Perry J. Blackshear John C. Lawrence , Jr.

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-Ibulleteukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H^6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H^6]PHAS-I when the [H^6]PHAS-IbulleteIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-IbulleteIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.




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