Figure 5

Activated Akt can prevent nucleostemin (NS) loss by focal adhesion kinase (FAK) inhibition; connections to mammalian target of rapamycin (mTOR) as analyzed by pS65 4E-BP1 phosphorylation and rapamycin inhibition. (A) Representative fluorescence confocal microscopy of MDA-MB-231 cells transfected with green fluorescent protein (GFP) or GFP-NS (24 h) (scale 50 μm). (B) Anti-GFP antibody co-immunoprecipitation (IP) of MDA-MB-231 cells transfected with GFP or GFP-NS. Shown is immunoblotting (IB) for GFP, mTOR, FAK, and Akt. (C) Lysates of dimethyl sulfoxide (DMSO)- or PF-271-treated (0.1 μM, 72 h) from adherent cells and immunoblotted for pS473 Akt, total Akt, pS65 4E-BP1, and actin. Reduction in pS473 Akt and pS65 4E-BP1 in PF-271-treated MDA-MB-231 and 4T1L cells (lanes indicated by #). (D) FAK-wild-type (WT) and FAK-kinase-dead (KD) MDA-MB-231 tumor lysates were immunoblotted for pS65 4E-BP1 and quantified by densitometry. Values are means (± SEM) and displayed as the ratio of pS65 4E-BP1 to actin. FAK-WT values were set to 1 (*P <0.05). (E) Stable expression of myristoylated - activated Akt (Akt*) in FAK-WT and FAK-KD MDA-MB-231 cells. Lysates were immunoblotted for pS473 Akt, total Akt, NS, B23, pS65 4E-BP1 and actin. (F) Colony formation of control (CTRL) and Akt*-expressing in FAK-WT and FAK-KD MDA-MB-231 cells. Values are means (± SEM, **P <0.01) of triplicate points. (G) MDA-MB-468 cells treated with PF-271 or rapamycin for 1 h (adherent) and lysates evaluated by pY397 FAK, total FAK, NS, B23, pS473 Akt, total Akt, pS65 4E-BP1, and actin immunoblotting. Blots are from one representative experiment repeated three times.