Control of the Translational Regulators PHAS-I and PHAS-II by Insulin and cAMP in 3T3-L1 Adipocytes

Abstract

The eukaryotic initiation factor 4E (eIF-4E)-binding proteins PHAS-I and PHAS-II were found to have overlapping but different patterns of expression in tissues. Both PHAS proteins were expressed in 3T3-L1 adipocytes, in which insulin stimulated their phosphorylation, promoted dissociation of PHAS⅐eIF-4E complexes, and decreased the ability of both to bind exogenous eIF-4E. The effects of insulin were attenuated by rapamycin and wortmannin, two agents that block activation of p70 S6K. Unlike PHAS-I, PHAS-II was readily phosphorylated by cAMP-dependent protein kinase in vitro; however, the effects of insulin on both PHAS proteins were attenuated by agents that increase intracellular cAMP, by cAMP derivatives, and by phosphodiesterase inhibitors. These agents also markedly inhibited the activation of p70 S6K . In summary, our results indicate that PHAS-I and -II are controlled by the mammalian target of rapamycin and p70 S6K signaling pathway and that in 3T3-L1 adipocytes this pathway is inhibited by increased cAMP.

Ser-64 and Ser-111 in PHAS-I Are Dispensable for Insulin-stimulated Dissociation from eIF4E

“…Proteins were transferred to membranes (Immobilon) and immunoblotted with the appropriate antibodies as described previously (19). Bound antibodies were detected by using alkaline phosphatase-conjugated secondary antibodies and CDPStar (Tropix).…”

“…Antibodies-Antibodies to the COOH-terminal regions of PHAS-I and eIF4E (19) and the phosphospecific antibodies, P-Thr-36/45, P-Ser-64, and P-Thr-69 (10), were generated in rabbits and affinity-purified as described previously (10,19). The sequences of amino acids immediately surrounding Thr-36 and Thr-45 are identical.…”

“…Expression Vectors-cDNA encoding rat PHAS-I (12), mouse PHAS-II (19), and PHAS-I proteins having Ser-64 to Ala (3) and Ser-111 to Ala (15) mutations were generated and subcloned into pET-14b (Novagen) as described previously (3,12,15,19). To create vectors for expressing Myc epitope-tagged proteins, PHAS-I (pCMV-Tag 3A PHAS-I , pCMV-Tag 3A A64 PHAS-I , pCMV-Tag 3A A111 PHAS-I ) and PHAS-II (pCMVTag3A PHAS-II ) proteins, cDNAs were excised from pET-14b by using BamHI and inserted into the BamHI site of pCMV-Tag 3A (Stratagene).…”

Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines

“…The 4E-binding proteins are reversibly phosphorylated in response to conditions which stimulate translation (Lin et al, 1994Lin and Lawrence, 1996;Fadden et al, 1997;Lawrence and Abraham, 1997) and this process favours Figure 4 Changes in levels of initiation factors in cycloheximide-treated BJAB cells. Exponentially growing cells were treated with cycloheximide (100 mg/ml) for the times indicated and cytoplasmic extracts prepared as described in Materials and methods.…”

Integration of hormonal and nutrient signals that regulate leptin synthesis and secretion

“…Thus exercise, cold stress, and fasting may decrease leptin production via cAMP signaling. Increasing intracellular cAMP has been shown to decrease mTOR kinase activity and phosphorylation of mTOR downstream targets, 4E-BP1 and S6K, in several cell types including adipocytes (26,45,72). Thus cAMP may decrease overall or specific rates of leptin translation through cross-talk with the mTOR pathway.…”