Construction and Characterization of a Conditionally Active Version of the Serine/Threonine Kinase Akt

Abstract

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.

Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells

“…As also shown in Fig. 2A, in F/B* cells (as in other cells transfected with constitutively active PKB (8,37,38)), p70S6 kinase was activated, evidenced by the shift in its mobility on SDS-PAGE. Rapamycin blocked activation of p70S6 kinase without affecting PKB or PDE3B activity, but did reduce PDE4 activity, which was slightly increased in F/B* cells (Table I; Fig.…”

“…4), wortmannin could inhibit constitutively activated PKB by blocking production of phosphoinositides involved in PKB association with membranes and/or in the activation of specific PDK isoforms. Whether activated PKB contributes to feedback activation of PI3-K or sensitization of PDK to phosphoinositides is not known, but other workers have also reported that wortmannin or LY294002 inhibits constitutively activated PKB (activated by targeting to membranes) in 3T3-L1 adipocytes (38) or HEK293 cells (37).…”

Akt activation induces epidermal hyperplasia and proliferation of epidermal progenitors

“…To analyse the effects of Akt signaling activation on epidermal cells, we used a chimeric protein composed of a myristoylated constitutively active form of Akt and the ligand-binding domain of a mutant estrogen receptor (Mer) (Kohn et al, 1998). The fusion protein can control the timing and duration of Akt signaling activation because Akt-Mer is inactivated in the absence of 4-hydroxytamoxifen (4OHT), but activated rapidly by the addition of 4OHT (Watanabe et al, 2006).…”

General aspects of muscle glucose uptake

“…The PI3-K promotes the catalysis of the inositol ring of phosphoinositides at position 3 resulting in (3,4,5) phosphatidylinositol triphosphate (PIP3) from (4,5) phosphatidylinositol biphosphate (Cavalheira et al 2002, van Dam et al 2005. PIP3 provides the phosphorylation of phosphoinositidedependent protein kinase 1 (PDK1) which in turn activates protein kinase B (PKB) or AKT (Kohn et al 1998) and protein kinase C (PKC) (Shepherd and Kahn 1999). Following its recruitment to the cell surface, AKT is activated resulting in the AKTdependent phosphorylation of many substrates.…”