The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/ RAFT-1/FRAP is the target through which the rapamycin⅐FKBP12 complex acts to dephosphorylate/ deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin⅐FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivation in situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner.Rapamycin is an immunosurpressive macrolide whose major cellular receptor is the cytosolic 12-kDa FK506-binding protein (FKBP12) 1 (1, 2). Rapamycin binds to FKBP12 at a single site, identical to the site bound by the structurally related drug FK506, and both agents, acting in situ as a drug-protein complex, are immunosurpressive through inhibition of T-cell proliferation. Despite these similarities, the two drugs operate through distinct mechanisms. The FK506⅐FKBP12 complex blocks T-cell receptor signal transduction by directly inhibiting protein phosphatase 2B/calcineurin. The rapamycin⅐FKBP12 complex does not inhibit T-cell receptor signal transduction or calcineurin activity but rather inhibits interleukin-2-stimulated signal transduction, concomitant with a potent (Ͼ95%) and selective inhibition in situ of the p70 S6 kinase (3, 4), an enzyme critical for the G 1 to S transition, at least in some cells (5, 6). Rapamycin, acting indirectly in situ, causes a partial dephosphorylation and deactivation of p70; the direct target of rapamycin⅐FKBP12 complexes in situ relevant to the rapamycin-inhibition of the p70 S6 kinase is the protein known variously as RAFT-1/FRAP/RAPT-1 or mTOR.The TOR proteins were first identified in Saccharomyces cerevisiae, where rapamycin (but not FK506) is growth-inhibitory. The TOR proteins are the product of one class of mutant genes that confer resistance to rapamycin-induced growth inhibition as a dominant phenotype (7,8). The yeast (7-9) and mammalian TOR proteins (specifically, FRAP (10); RAFT-1 (11); RAPT-1 (12); and mTOR (13)) are Ͼ250-kDa polypeptides that contain at their carboxyl terminus a protein and/or lipid kinase catalytic domain, most closely related to those of the DNA protein kinase and the ATM, MEC1, and Tel1 checkpoint gene products, and somewhat more distantly related to the PI-3 kinases (14). The rapamycin/FKBP12 complex b...

Differentiation of adipocytes is an important aspect of energy homeostasis. Although the transcriptional regulation of adipocyte differentiation is relatively well characterized, the subsequent molecular events remain unclear. The activity of phosphoinositide (PI) 3-kinase precipitated with antibodies to phosphotyrosine has now been shown to increase transiently during adipocyte differentiation of 3T3-F442A and of 3T3-L1 cells. PI 3-kinase activity precipitated with antibodies to insulin receptor substrate 1 (IRS1) and association of subunits of PI 3-kinase with IRS1 were also increased at this stage of differentiation, suggesting that IRS1 contributes to PI 3-kinase activation. Inhibition of the activation of PI 3-kinase by expression of dominant negative mutant subunits of the enzyme prevented adipogenesis, as assessed by lipid accumulation and expression of key adipocyte proteins such as GLUT4, adipsin, and aP2, suggesting that PI 3-kinase activation is essential for adipocyte differentiation. However, these mutant proteins did not affect either the expression of the transcription factor PPAR␥ at the mRNA or protein level or the increase in the abundance of mRNAs encoding the adipocyte marker proteins. These results demonstrate that adipocyte differentiation is regulated at the posttranscriptional level and that activation of PI 3-kinase is required for this regulation.

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Extended IBEB is recommended for middle-aged and older Asian women with moderate to severe dermatochalasis. With precise incision and fine suturing, regrowth alleviates eyebrow reduction and scarring among Asians who do not use makeup (male patients and young female patients).