Figure 1

PKA induced phosphorylation of TG2 at Ser ²¹⁶ facilitates downregulation of PTEN in MEF ^tg2-/- cells. (A) Immunoblot showing Ser²¹⁶ in TG2 as a predominant phosphorylation site in response to PKA activation by cAMP. MEF^tg2-/- cells were transfected with Myc tagged wild type TG2 and mutant-TG2 (lacking Ser²¹⁶ phosphorylation site) constructs. 48 h post-transfection, cells were serum starved and incubated with or without db-cAMP (100 μM) in the presence or absence of a PKA inhibitor H89 (100 nM). Cells were harvested and cell lysates were analyzed by western immunoblotting using anti-phospho-serine and anti-Myc antibodies. Representative immunoblots of three different experiments are shown. (B) MEF^tg2-/- cells were transfected with various TG2 constructs. 48 h post-transfection, cells were serum starved and incubated with or without db-cAMP (100 μM) in the presence or absence of a PKA inhibitor H89 (100 nM). Cells were harvested and cell lysates were analyzed by western immunoblotting using protein and phospho-specific antibodies. Anti-Myc immunoblot showing expression levels of wild-type TG2 and m-TG2 is shown as a control. Representative immunoblots of four different experiments are shown. (C) Histograms showing relative quantification of protein/phospho-proteins as shown in left panel. (D) Histogram showing pNF-κB-MetLuc2-reporter gene activity in MEF^tg-/- cells transfected with TG2 and m-TG2 constructs. Data are represented as mean ± SEM, n = 4. * P < 0.05 and ** P < 0.01 (TG2 vs. empty vector control); # P < 0.05 (m-TG2 vs. TG2).