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(Received for publication, May 5, 1995; and in revised form, June 6, 1995) 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-I
Volume 270,
Number 31,
Issue of August 04, pp. 18531-18538, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
SYNTHESIS, DEGRADATION, AND PHOSPHORYLATION BY A
RAPAMYCIN-SENSITIVE AND MITOGEN- ACTIVATED PROTEIN KINASE-INDEPENDENT
PATHWAY
eukaryotic
initiation factor-4E (eIF-4E) complex, effects that were maximal within
10 min. With recombinant [H
]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
]PHAS-I when the
[H
]PHAS-IeIF-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-I
eIF-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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