Figure 1.
In vitro interaction of the SH3 domain of Fyn kinase with the proline-rich domain of LKB1 (A) Purified His-LKB1-WT fusion protein was immobilized onto cobalt beads and incubated either with purified GST protein (control), GST-Fyn-WT or GST-Fyn-W119A fusion protein.
Retained proteins were separated onto SDS-polyacrylamide gel electrophoresis. Binding was detected using anti-GST (detecting Fyn kinase) and anti-His (detecting LKB1) antibodies. Blots are representative of 4 independent experiments. (B) Purified His-LKB1-WT or His-LKB1-P328A mutant fusion protein was immobilized onto cobalt beads and incubated with either purified GST protein (control) or GST-Fyn-WT. Binding was detected using anti-GST (detecting Fyn kinase) and anti-His (detecting LKB1) antibodies. (C) Purified His-LKB1-WT or His-LKB1-P328A mutant were incubated with GST-Fyn-kinase for the indicated time. Phosphorylation of LKB1 was detected by the phospho-tyrosine specific 4G10 antibody. Blots are representative of 3 independent experiments. D. Signal quantification. Identical letters indicate values that are not statistically different from each other (P>0.05).
Figure 2.
Fyn SH3 domain binds to the proline-rich domain of LKB1 and induces LKB1 cytosol localization in vivo (A) Lysates from HeLa cells expressing Flag-LKB1-WT were immobilized onto Flag-conjugated beads.
Protein extracts (same amount) from HeLa cells expressing V5-Fyn-WT or V5-Fyn-W119A or V5-Fyn-R176K constructs were incubated with the Flag-LKB1-WT. Proteins were separated by electrophoresis and immunoblotting was performed using specific V5 antibody (detecting Fyn) and Flag antibody (detecting LKB1). (B) Signal quantification from 3 independent experiments. Identical letters indicate values that are not statistically different from each other (P>0.05). (C) Lysates from HeLa cells expressing either Flag-LKB1-WT or Flag-LKB1-P328A were immobilized onto Flag-conjugated beads. Protein extracts (same amount) from HeLa cells expressing V5-Fyn-WT construct were incubated with either Flag-LKB1-WT or Flag-LKB1-P328A. Proteins were separated by electrophoresis and immunoblotting was performed using specific V5 antibody (detecting Fyn) and Flag antibody (detecting LKB1). (D) Signal quantification from 3 independent experiments. Identical letters indicate values that are not statistically different from each other (P>0.05). Images in (A and C) are representative of 3 independent experiments. (E) Fully differentiated 3T3L1 adipocytes were co-transfected with pcDNA3-Flag-LKB1 or pcDNA-LKB1-P328A. LKB1 subcellular localization was assessed by immunofluorescence using the rabbit Flag polyclonal antibody followed by Alexa Fluor 488 Anti-Rabbit IgG. Images are representative of 3 independent experiments. (E) Percentage of 3T3L1 cells with pcDNA-Flag-LKB1 signal detected in the cytoplasm. Data are representative of 3 independent experiments.
Figure 3.
LKB1 P328A mutation induces AMPK activation in skeletal muscle.
(A) Tibialis anterior of control mice was transfected with either with pcDNA-Flag-LKB1-WT (right leg) or pcDNA-Flag-LKB1-P328A mutant (left leg). Lysates were prepared and proteins separated by electrophoresis. LKB1, AMPK, phospho- T172 AMPK, ACC and phospho S79-ACC expression levels were determined using specific antibodies. Images represent a single experiment (n = 2 mice) that was repeated 3 times (n = 6 mice). (B) Signal quantification of the expression levels of phospho- T172 AMPK, and phospho S79-ACC from 3 independent experiments
Figure 4.
The LKB1-proline-rich domain mimicking peptide (11R-WT) inhibits the endogenous interaction of Fyn with LKB1, LKB1 Fyn-dependent phosphorylation and induces AMPK phosphorylation in C2C12 myotubes.
(A) C2C12 myotubes were transducted with either the 11R-LKB1-proline-rich domain (11R-WT) or the 11R-Scrambled LKB1 peptides (11R-Sca). Cell extracts (Lysates) were immunoblotted for the endogenous Fyn and LKB1. (B) Cell extracts were immunoprecipitated with the LKB1 monoclonal antibody and immunoblotted with Fyn or LKB1 antibodies. (C) Endogenous LKB1 was immunoprecipitated and LKB1 Y261 and Y365 tyrosine phosphorylation was determined using specific LKB1 tyrosine antibodies. Images in (A, B and C) are representative of 3 independent experiments. (D) C2C12 myotubes were transduced with the indicated amount of either 11R-WT or 11R-Sca peptides and total AMPK (t-AMPK), phospho- T172 AMPK and loading control GAPDH protein expression levels were determined using specific antibodies. Image is representative of at least 3 experiments. (E) Signal quantification of the expression levels of phospho- T172 AMPK from 3 independent experiments
Figure 5.
Effects of the LKB1-proline-rich domain mimicking peptide (11R-WT) on AMPK phosphorylation are inhibited in LKB1-deficient C2C12 myotubes.
(A) C2C12 cells were infected with either shRNA encoding for a non-target or LKB1 sequences and differentiated into myotubes before being transduced with either the 11R-WT or 11R-Sca peptides (40nM). LKB1, total AMPK (tAMPK), phospho- T172 AMPK, total ACC (tACC), phospho S79-ACC and loading control GAPDH protein expression levels were determined using specific antibodies. Images are representative of 3 independent experiments. (B) Signal quantification from 3 experiments. Identical letters indicate values that are not statistically different from each other (P>0.05). (C) 3 months old male C57B6/J mice were euthanized and EDL muscles were rapidly removed and incubated for 30 min in oxygenated (95% O2, 5% CO2) DMEM supplemented with 10% fetal bovine serum and 11R-WT (right leg EDL) or 11R-Sca peptides (left leg EDL). AMPK, ACC phosphorylation, total AMPK (t-AMPK) and total ACC (t-ACC) and loading control tubulin alpha expression levels were detected using specific antibodies. Figure displays n = 2 mice and blots are representative of 3 independent experiments (n = 6 mice). (D) Signal quantification of 3 independent experiments. Identical letters indicate values that are not statistically different from each other (P>0.05).