Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection (original) (raw)
Figure 5
CK2 phosphorylates UPIIIa cytoplasmic domain and is required for FimCH-induced Ca2+ elevation.
(A) UPIIIa cytoplasmic domain is phosphorylated by CK2 in vitro. Recombinant human CK2 (50 U) exhibited autophosphorylation (lane 1) that was blocked by 10 µM of the CK2 inhibitor TBB (lane 2). A fusion protein of the C-terminal domain of uroplakin IIIa with glutathione-S-transferase (UP3C-GST, arrow) served as a concentration-dependent substrate for phosphorylation by CK2 (lanes 3–6 containing 0.01 µg, 0.10 µg, 0.25 µg, or 1.00 µg UP3C-GST, respectively). CK2-mediated phosphorylation of 0.10 µg UP3C-GST was inhibited by TBB (lane 7). (B) Inhibition of CK2 abrogates FimCH-induced calcium elevation. Urothelial cells were loaded with Fura-2 AM and pretreated with 10 µM TBB (or equivalent concentration of vehicle (DMSO)) for 30 minutes, treated with FimCH and followed by imaging. Statistical significance is indicated at *p<0.05 and data are represented as mean±SEM. (C) COS-7 cells were infected with recombinant adenoviruses and FimCH-induced calcium was quantified as in (B) in the presence or absence of TBB. (D) RNA silencing of CK2 inhibits FimCH-induced calcium elevation in urothelial cells. PD07i cells transfected with siCK2 showed significantly reduced elevation in calcium as measured by the change in Fura-2 340/380 ratio compared to transfection with negative control siRNA. All experiments were repeated three or more times, and statistical significance is indicated by *p<0.05, **p<0.01.