Regulation of LKB1/STRAD localization and function by E-cadherin

Abstract

LKB1 kinase is a tumor suppressor that is causally linked to Peutz-Jeghers syndrome. In complex with the pseudokinase STRAD and the scaffolding protein MO25, LKB1 phosphorylates and activates AMPK family kinases, which mediate many cellular processes. The prototypical family member AMPK regulates cell energy metabolism and epithelial apicobasal polarity. This latter event is also dependent on E-cadherin-mediated adherens junctions (AJs) at lateral borders. Strikingly, overexpression of LKB1/STRAD can also trigger establishment of epithelial polarity in the absence of cell-cell or cell-matrix contacts. However, the upstream factors that normally govern LKB1/STRAD function are unknown. Here we show by immunostaining and fluorescence resonance energy transfer that active LKB1/STRAD kinase complex colocalizes with E-cadherin at AJs. LKB1/STRAD localization and AMPK phosphorylation require E-cadherin-dependent maturation of AJs. However, LKB1/STRAD complex kinase activity is E-cadherin independent. These data suggest that in polarized epithelial cells, E-cadherin regulates AMPK phosphorylation by controlling the localization of the LKB1 complex. The LKB1 complex therefore appears to function downstream of E-cadherin in tumor suppression.

Figure 1. LKB1 localizes at adherens junction level in polarized epithelial cells

(A) Polarized Caco-2 stained for LKB1 (green) and E-cadherin (red). (B) xz projection of polarized Caco-2 cells stained for LKB1 (green) and villin (red) or (C) for LKB1 (green) and ZO-1 (red). (D) Polarized Caco-2 cells were fractionated as described in “experimental procedures” and analyzed for indicated proteins. Results are representative of two independent experiments. (E) MDCK clones stably knocked down for LKB1 (cl) by shRNA or control construct (shLuc) transiently transfected by either GFP alone or GFP-LKB1wt were analysed by Western blotting for indicated proteins. (F) AMPKp-172/total AMPK ratio (black bar) were determined and plotted with endogenous (endo, white bar) and exogenous (exo, grey bar) LKB1, all relative to control. Values are means ± S.D., (n=2). (G) Polarized MDCK shLuc and shLKB1 cl 14 were stained for LKB1 (green), E-cadherin (purple) and villin (red). Bars, 10µm.

Figure 2. STRAD localizes with LKB1 at adherens junction level

(A) Polarized MDCK shLuc or shLKB1 cl 14 cells were stained for LKB1 (green), E-cadherin (purple) and STRAD (red). (B) Polarized Caco-2 clone knocked down for LKB1 (cl A12) or not (shLuc) were fractionated as in figure 1. (C) Membranous shLKB1 cl A12/shLuc ratio for LKB1, STRADα, total or phosphorylated AMPK, normalized to E-cadherin. Values are means ± S.D., (n=3). (D) MDCK cells on fibronectin-coated coverslips were transiently transfected with YFP-STRADα (yellow) plus either CFP-Myr, wild type or SL26 CFP-LKB1 (blue). FRET was measured as described in “Experimental Procedures”. The asterisks mark an unidentified perinuclear compartment. (E) Projections of FRET images from CFP-LKB1-WT and YFP-STRADα. FRET intensity is displayed on a pseudocolor scale in arbitrary units. Bars, 10µm.

Figure 3. E-cadherin is required to localize LKB1 at adherens junctions level

(A) MDCK cells expressing control (shLuc) or E-cadherin (shE-cad) shRNA vectors were analysed by Western blot for the indicating proteins. (B) Cells were stained for LKB1 (green) and E-cadherin (red). Bars, 10µm. (C) MDCK cells described in A were fractionated as described in figure 1, (D) Ratio of membranous shE-cad/shLuc of LKB1, STRADα, total or phosphorylated AMPK, normalized to integrin. Values are means ± S.D., (n=2).

Figure 4. E-cadherin knockdown decrease AMPK activation

(A) MDCK cells were transiently transfected with control (shLuc) or shRNA targetting canine E-cadherin (shE-cad). Cells were extracted at indicated times and analyzed by Western blot for indicated proteins. (B) T172 AMPK phosphorylation and E-cadherin levels were quantified and normalized to total AMPK. Values are percent relative to levels in shLuc or shE-cad cells at 24h (means ± S.D. n=3). pAMPK: white bars; E-cadherin: black bars. (C) The same lysates were immunoprecipitated with anti-LKB1 and kinase activity analyzed in vitro (IVK). (D) Kinase activity was quantified and normalized to total immunoprecipitated LKB1, plotted as described in B (means ± S.D. n=4). Control (shLuc) and E-cadherin-depleted MDCK cells were transiently transfected or not with wild type human E-cadherin (pcDNAhE-cad). After 24h cells, were extracted and treated as in A and C (E, F respectively) and quantified (G). T172 AMPK phosphorylation in cells (white bar), LKB1 IP kinase activity (TK) (black bar), control shLuc cells mock transfected was used as reference (means ± S.D. n=3). (H) Stable control (shLuc) and LKB1 depleted (shLKB cl14) MDCK cells were transiently transfected or not with shRNA targeting canine E-cadherin (shE-cad). After 72h, cells were lysed and analyzed by Western blotting with the specified antibodies (n=2).