Figure 3

Phosphoprotein enriched in astrocytes 15 kDa (PEA15) is a novel AMP-activated protein kinase (AMPK) substrate. (A) Primary human mammary epithelial cells (HMECs) cultured in ultra-low (UL) plates were transfected with control small interfering RNA (siRNA) or siRNA targeting PEA15. Two days posttransfection, a quarter of the cells were harvested and subjected to immunoblot analysis for the specified proteins (i), while the rest were scored for total number of mammospheres (MS) formed at the end of a week (ii). Error bars represent standard error of the mean (SEM); n = 4. (B) Primary HMECs cultured in UL plates were infected with control (empty vector) CSCG lentivirus, or those encoding for PEA15-WT or PEA15-S116A mutant with similar infection efficiencies (as scored by GFP-positive cells). Two days following infection, 1 × 10⁵ transduced cells were seeded in methylcellulose and total number of GFP-positive mammospheres formed was counted at the end of a week. Error bars represent standard error of the mean (SEM); n = 3. (C) Primary HMECs seeded in UL plates were treated with AMPK activator A769962 (100 μM) for 0 min, 30 min, 1 hr and 2 hrs, and subjected to immunoblot analysis for the specified proteins; n = 3. (D) Primary HMECs seeded in UL plates were subjected to immunoprecipitation with total AMPK antibody followed by immunoblotting for specified proteins. Rabbit immunoglobulin G (IgG)-treated lysate and resin alone served as controls, n = 3. (E) Commercially procured heterotrimeric AMPK and pure PEA15 were incubated in an in vitro kinase assay buffer in the presence of 50 μM adenosine triphosphate (ATP) and 0.1 mM AMP for 30 min, and then subjected to immunoblot analyses with commercially available antibodies that specifically recognize PEA15 Ser¹¹⁶ phosphorylation; total PEA15 antibody served as a loading control. (F) Autoradiography of in vitro kinase assay using pure AMPK and PEA15 proteins performed in the presence of γ³²P-labeled ATP (0.3 μCi) for 30 min; n = 3.