PMC

Fig. 5. STRAD-mediated activation of LKB1 does not affect its substrate specificity. (A) Clockwise, starting upper left. Tryptic peptide map of GST–LKB1 and GST–LKB1 activated by STRAD, and the corresponding Edman degradation diagrams and phospho-amino acid (PAA) analysis (inserts). The increased stoichiometry of LKB1 autophosphorylation upon STRAD activation enabled the identification of two novel autophosphorylation sites: Thr185 and Thr402. (B) Tryptic peptide map of MBP phosphorylated by GST–LKB1 and GST–LKB1/flag-STRAD (left panel), and the corresponding Edman degradation diagram (right panel) and PAA analysis (insert). The LKB1 phosphorylation site on MBP was determined as Thr65.

Fig. 2. (A) Endogenous LKB1 (55 kDa) interacts with STRAD (45–48 kDa) in Rat-2 and HEK-293T cells. Co-immunoprecipitations (IP) were performed on 1 mg of indicated cell lysates with either α-LKB1 polyclonal antibody (sheep) or an irrelevant sheep polyclonal antibody. Protein complexes (right panel) and 20 µg of total lysates (left panel) were immunoblotted (WB) with α-LKB1 or α-STRAD. (B) STRAD specifically interacts with LKB1. GST–LKB1 and several unrelated GST fusion proteins were isolated from HEK-293T cells using GST-affinity chromatography. Protein complexes were immunoblotted (WB) with α-GST or α-STRAD. (C) Amino acids 319–343 of LKB1 are necessary for STRAD binding. Indicated GST–LKB1 deletion constructs were isolated from HEK-293T using GST-affinity chromatography. Protein complexes were immunoblotted (WB) with α-GST or α-STRAD.

Fig. 7. STRAD determines the cytoplasmic subcellular localization of LKB1, which is dependent on the kinase activity of LKB1 and its ability to associate with STRAD. Phosphorylation of STRAD by LKB1 is not essential for this process. (A) Myc-tagged LKB1-WT, -KD, -SL26 and flag-STRAD were transfected into COS cells. The localization of proteins was determined by incubation with α-myc/TRITC–goat α-mouse or α-STRAD–biotin/FITC–streptavidin and visualized by fluorescence microscopy. (B) Co-transfection of STRAD with LKB1-WT results in translocation of the LKB1-WT–STRAD complex to the cytoplasm. The STRAD mutant T329A/T419A is also able to re-localize LKB1-WT. LKB1-KD and LKB1-SL26 are not able to translocate to the cytoplasm upon forced expression of STRAD. Detection was performed as above.

Fig. 6. (A) LKB1 phosphorylates STRAD at Thr329 and Thr419. Tryptic map of GST–STRAD phosphorylated by myc–LKB1-WT (upper panel), and the corresponding Edman degradation diagrams (bottom two panels) and phospho-amino acid (PAA) analysis (inserts). (B) Mutation of Thr329 and Thr419 abolishes STRAD phosphorylation by LKB1, but does not interfere with STRAD’s ability to bind or activate LKB1. Thr329 and/or Thr419 of flag-STRAD were mutated to Ala (T329A, T419A) or Asp (T329D, T419D). The indicated protein complexes were isolated from transfected HEK-293T cells using GST-affinity chromatography and immunoblotted (WB) with α-GST or α-STRAD. In addition, protein complexes were subjected to an in vitro kinase assay (IVK), separated by SDS–PAGE and visualized by autoradiography.

Fig. 8. STRAD is an essential co-factor for LKB1-mediated G₁ cell cycle arrest in G361 cells. (A) Cell cycle profiles of G361 cells, transfected with the indicated plasmids and blocked with nocadozole. Both LKB1 kinase activity and its ability to associate with STRAD are required for its G₁ arrest-inducing potential, as is demonstrated by the failure of LKB1-KD and LKB1-SL26 to induce cell cycle arrest. (B) pS-STRAD4 efficiently knocks-down STRAD expression. The indicated pS-STRAD constructs were co-transfected with flag-STRAD and Renilla luciferase. Cell lysates were immunoblotted with α-flag to analyse STRAD expression. Transfection efficiencies were determined to be equal by measuring Renilla luciferase units (data not shown). (C) Cell cycle profiles of G361 cells, transfected with pS or pS-STRAD4 in the absence or presence of LKB1-WT (left panel). The amount of cells in G₁ was determined and ΔG₁ is represented in a graph (right panel).

Fig. 3. LKB1 phosphorylates STRAD directly in vitro and in vivo. (A) LKB1-WT (wild type), but not LKB1-KD (kinase dead; D176Y), mediates phosphorylation of STRAD and shows enhanced autophosphorylation upon forced STRAD expression. Myc-tagged LKB1-WT, LKB1-KD and flag-STRAD were transfected into HEK-293T cells as indicated. Protein complexes were co-immunoprecipitated (IP) using either myc-Ab (M) or flag-Ab (F), and immunoblotted (WB) with α-myc or α-STRAD. In addition, the protein complexes were subjected to an in vitro kinase (IVK) assay. Cells transfected with the indicated constructs were metabolically labelled with [³²P]phosphate for 3 h prior to lysing and co-immunoprecipitation. Protein complexes were separated by SDS–PAGE and visualized by autoradiography. (B) The LKB1-SL26 mutant does not bind to STRAD, which prevents STRAD phosphorylation and STRAD-mediated enhanced autophosphorylation of LKB1. Myc-tagged LKB1-WT, LKB1-SL26 and flag-STRAD were transfected into HEK-293T cells as indicated. Protein complexes were co-immunoprecipitated (IP) using either myc-Ab (M) or flag-Ab (F). Subsequently, they were immunoblotted (WB) with α-myc or α-STRAD. In addition, the protein complexes were subjected to an in vitro kinase assay (IVK), separated by SDS–PAGE and visualized by autoradiography. (C) LKB1–STRAD interaction is required for STRAD phosphorylation by LKB1. Anti-myc immunoprecipitated LKB1-WT, LKB1-KD and LKB1-SL26 were subjected to an in vitro kinase assay (IVK) alone, or after mixing with α-flag immunoprecipitated STRAD. Protein complexes were immunoblotted (WB) with α-myc or α-STRAD, and visualized by autoradiography. (D) LKB1 phosphorylates STRAD directly. Bacterially produced GST–LKB1- WT, GST–LKB1-KD and flag-STRAD were subjected to an in vitro kinase assay as indicated. Proteins were separated by SDS–PAGE, and immunoblotted (WB) with α-GST or α-flag. Phosphorylated proteins were visualized by autoradiography.

Fig. 4. (A) STRAD activates LKB1 as visualized by LKB1s enhanced autophosphorylation and its increased ability to phosphorylate MBP. GST–LKB1-WT and GST–LKB1-WT/flag-STRAD protein complexes were isolated from transfected HEK-293T cells using GST-affinity chromatography. Protein complexes were subjected to an in vitro kinase assay (IVK) in the presence or absence of 10 µg of MBP. Protein complexes were immunoblotted (WB) with α-GST or α-flag to determine equal loading. Autophosphorylation of LKB1 was determined by immunoblotting (WB) with phospho-specific antibodies against the LKB1 autophosphorylation sites Thr336 and Thr363. Phosphorylated proteins were separated by SDS–PAGE and visualized by autoradiography. (B) LKB1 autophosphorylation is enhanced 3- to 4-fold upon forced expression of STRAD. A time-course experiment was performed using GST–LKB1-WT, GST–LKB1-WT/flag-STRAD and GST–LKB1-KD isolated from transfected HEK-293T cells. The in vitro kinase assay (IVK) was initiated by adding [γ-³²P]ATP and kinase buffer to the GST proteins, and stopped at the indicated time points by the addition of SDS sample buffer. The protein complexes were separated by SDS–PAGE and immunoblotted (WB) with α-GST. Phosphorylated proteins were visualized by autoradiography. Levels of phosphorylation were quantified and are represented graphically.

Fig. 1. (A) Deduced amino acid sequence of human STRAD aligned with SPAK. Identical residues are represented in black and similar residues in grey. The subdomains of the pseudokinase domain are indicated with Roman numerals. The serine replacement of the essential aspartic acid residue in the catalytic site and the absence of the DFG motif in STRAD compared with SPAK are designated with closed arrows. The STRAD phosphorylation sites Thr329 and Thr419 are designated with open arrows. (B) STRAD does not phosphorylate the exogenous substrates myelin basic protein (MBP), histone 2A, histone 2B and CRE binding protein (CREB). Exogenous proteins (10 µg) were subjected to an in vitro kinase (IVK) assay with either 1 U of cAMP-dependent kinase (PKA) or 1 µg of GST–STRAD. Proteins were separated by SDS–PAGE and immunoblotted (WB) with α-GST or visualized by autoradiography.