CONTROL OF PHAS-I PHOSPHORYLATION IN 3T3-L1 ADIPOCYTES - EFFECTS OF INHIBITING PROTEIN PHOSPHATASES AND THE P70(S6K) SIGNALING PATHWAY

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.