Ta. Lin et Jc. Lawrence, CONTROL OF PHAS-I PHOSPHORYLATION IN 3T3-L1 ADIPOCYTES - EFFECTS OF INHIBITING PROTEIN PHOSPHATASES AND THE P70(S6K) SIGNALING PATHWAY, Diabetologia, 40, 1997, pp. 18-24
PHAS-I is a recently discovered regulator of translation initiation. N
on-phosphorylated PHAS-I binds and inhibits eukaryotic initiation fact
or-4E, the mRNA cap-binding protein that mediates a rate-limiting step
in translation initiation. When PHAS-I is phosphorylated in response
to insulin! the PHAS-I/eukaryotic initiation factor-4E complex dissoci
ates. The present study was conducted to investigate mechanisms involv
ed in the control of PHAS-I. Phosphorylation of PHAS-I was monitored b
y immunoblotting after subjecting extracts to polyacrylamide gel elect
rophoresis in the presence of sodium dodecyl sulphate. This was possib
le because phosphorylation markedly decreases the electrophoretic mobi
lity of PHAS-I. Incubating 3T3-L1 adipocytes with rapamycin and wortma
nnin inhibited insulin-stimulated phosphorylation of PHAS-I at concent
rations similar to those that inhibited activation of p70(S6K). Both a
gents increased the amount of PHAS-I that co-purified with eukaryotic
initiation factor-4E when extracts were fractionated using a cap affin
ity resin, indicating that PHAS-I binding to the initiation factor was
increased. Incubating adipocytes with the protein phosphatase inhibit
ors, calyculin A and okadaic acid, increased PHAS-I phosphorylation an
d opposed the effects of rapamycin on decreasing PHAS-I phosphorylatio
n. However, neither okadaic acid nor calyculin A abolished the effects
of rapamycin on PHAS-I. These results suggest that: the phosphorylati
on of PHAS-I in response to insulin occurs via the p70(S6K) Signalling
pathway. By regulating eukaryotic initiation factor-4E, PHAS-I may ha
ve important roles in the control of both protein synthesis and mitoge
nesis.