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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02670

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14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK

¹ MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
² Division of Cell Biology and Immunology, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

View larger version (73K): [in a new window]   Fig. 1. TAP-purification of AMPK-related kinases. (A) The pEGFP-C2-TAP vector, depicting the affinity tag added to the N-terminus of AMPK1 and the AMPK-related kinases, consisting of a green fluorescent protein (GFP), protein A and a calmodulin-binding motif (CAM) flanked by TEV and precision protease cleavage sites, prior to multiple cloning sites. (B) Outline of the strategy that was employed to affinity purify the GFP-TAP-tagged kinases. (C) The TAP affinity purified kinases were electrophoresed on a polyacrylamide gel and the protein bands visualized following colloidal Coomassie Blue staining. Each of the major bands observed that we were able to identify by mass spectrometry was numbered and its identity indicated in Table 1. Bands that were not reliably identified are left unlabelled. *The bands identified as the bait kinase.

View larger version (53K): [in a new window]   Fig. 2. Binding of 14-3-3 to QSK and SIK, but not MARK3 is dependent on LKB1. (A) QSK was immunoprecipitated from the quadriceps muscle derived from wild-type mice (LKB1+/+) and muscle-specific LKB1-knockout (LKB1-/-) mice (Sakamoto et al., 2005). As a control, immunoprecipitation from LKB1+/+ muscle was also performed with a preimmune antibody. The immunoprecipitates (IP) were subjected to immunoblot analysis and also assayed using the AMARA peptide. Muscle samples derived from five LKB1-/- and LKB+/+ mice were analysed and similar results obtained for each one. The QSK activity is the mean±s.d. for each muscle sample assayed in duplicate. Pre-imm, pre-immune antibody. (B) Cell lysates (20 µg) derived from 293 cells, control (CT) HeLa cells that lack LKB1 expression, or HeLa cells that stably express wild-type (WT) or kinase-inactive (KI) LKB1, as well as skeletal muscle derived from wild-type (WT) and muscle-specific LKB1-knockout (KO) mice, were immunoblotted with an antibody recognizing LKB1. (C,D,F) Cell lysates derived from control (CT) HeLa cells that lack LKB1 expression or HeLa cells that stably express wild-type (WT) or kinase-inactive (KI) LKB1 were incubated with glutathione-Sepahrose conjugated to 14-3-3 (14-3-3 pull-down). After washing, the beads were immunoblotted with the indicated antibodies. The activity of the indicated AMPK-related kinases towards the AMARA peptide was also measured following immunoprecipitation. The results are presented as the mean±s.d. from two separate experiments each assayed in triplicate. Cell lysates were also subjected to the immunoblot analysis (lysate). SIK was immunoblotted after its immunoprecipitation from the cell lysate. The results are representative of at least two separate experiments. (E) The indicated HeLa cells stably expressing GFP-TAP-14-3-3 were generated, and the GFP-TAP-14-3-3 was affinity purified and analysed by immunoblotting with the indicated antibodies (TAP-eluate). Cell lysates derived from the HeLa cells were also immunoblotted (lysate). As GFP-TAP-14-3-3 migrates to the same position as endogenous MARK3, thereby interfering with immunoblot analysis, the control MARK3 immunoblot blot in cell lysate was performed using the parental HeLa cell line that does not express GFP-TAP-14-3-3. The upper band in 14-3-3 blots of the TAP-eluate represents exogenously expressed 14-3-3 and the lower doublet represents co-purified endogenous 14-3-3 and 14-3-3. The results are representative of three separate experiments.

View larger version (53K): [in a new window]   Fig. 3. Binding of 14-3-3 to the phosphorylated T-loop of QSK and SIK. (A) 293 cell lysates were incubated with the indicated N-terminal biotinylated peptides encompassing the T-loop of QSK. The peptides were affinity purified on streptavidin-Sepharose and immunoblotted with an antibody recognizing 14-3-3 isoforms. The results are representative of at least two separate experiments performed in duplicate. (B) 1 µg of the indicated biotinylated peptides was conjugated to streptavidin-Sepharose and incubated with indicated amounts of GST-14-3-3. Following washing, the beads were subjected to immunoblot analysis with a 14-3-3 antibody. The RAF-phospho peptide LSQRQRSTS(P)TPNVHMV binds 14-3-3 with high affinity (Muslin et al., 1996). (C,E) 293 cells were transfected with constructs encoding GST fusion proteins of wild-type (WT), T-loop mutant (T/A) or kinase-inactive (KI) mutants of the indicated AMPK-related kinases. Thirty-six hours post-transfection, the AMPK-related kinases were affinity purified from the cell lysates using glutathione-Sepharose. Similar amounts of the purified GST-fusion proteins were assayed for AMARA peptide kinase activity (activity) or subjected to immunoblot analysis (GST-pull-down). The QSK, SIK and MARK isoform levels were assessed using anti-GST antibodies. The kinase activities are presented as the mean±s.e.m. for triplicate samples relative to the activity observed for the wild-type kinase (100% activity). The results are representative of at least two separate experiments. (D) Equal amounts of wild-type and T-loop mutant of QSK and SIK GST fusion proteins isolated from 293 cells, as described in panel C, were electrophoresed on a polyacrylamide gel and the protein bands visualized by colloidal Coomassie Blue staining. The identity of the bands indicated with an arrow was determined by mass spectrometry. EF1b2 (elongation factor-1b2) is found as a contaminant in most preparations of GST-fusion derived from 293 cells. (F) 500 ng wild-type and T-loop mutant forms of GST-QSK and GST-SIK proteins, as well as wild-type GST-MARK3, were subjected to polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and subjected to a 14-3-3 overlay assay (upper panel) as described in the Materials and Methods. 50 ng of each of the GST-fusion proteins was also subjected to immunoblot analysis with GST antibody (lower panel).

View larger version (47K): [in a new window]   Fig. 4. Enhancement of QSK and SIK activity by 14-3-3 binding. 293 cells were transfected with constructs encoding GST-QSK (A,C) or GST-SIK (B,D). Thirty-six hours post-transfection, the GST-QSK or GST-SIK was absorbed onto glutathione-Sepharose and the beads were incubated with buffer containing no peptide, non-phosphorylated QSK T-loop peptide TPGQLIKTWCGSPPY (non-phos peptide) or phosphorylated QSK T-loop peptide TPGQLIKT(p)WCGSPPY (phos peptide). The beads were then washed in buffer A and GST-QSK or GST-SIK was eluted with glutathione. Similar amounts of the purified GST-QSK (A) or GST-SIK (C) were subjected to immunoblot analysis or assayed with either TORC2 protein substrate (upper panel), or the AMARA peptide (lower panel). The QSK and SIK levels were assessed in the immunoblot analysis using anti-GST antibodies. (C,D) GST-QSK (C) or GST-SIK (D) was also assayed using the TORC2 substrate (upper panel) or AMARA peptide (lower panel) in the presence (+) or absence (-) of wild-type GST-14-3-3 or mutant GST-14-3-3[E180K]. In addition, the samples were immunoblotted with anti-GST antibodies, to ensure similar amounts of GST-QSK and GST-SIK were present in each assay, and with 14-3-3 antibody, to ensure removal of endogenously associated 14-3-3. The immunoblot analysis is representative of at least three separate experiments performed in duplicate. The kinase activities are presented as the mean±s.e.m. for triplicate samples and the results are representative of at least three separate experiments.

View larger version (40K): [in a new window]   Fig. 5. 14-3-3 and LKB1 cooperate to regulate QSK and SIK localization. Control (CT) HeLa cells that lack LKB1 expression or HeLa cells that stably express wild-type (WT) or kinase-inactive (KI) LKB1 were transfected with the indicated constructs encoding the expression of GFP-QSK (A, panels 1-9) or GFP-SIK (B, panels 1-9). Twenty-four hours post-transfection the cells were fixed in 3% (v/v) paraformaldehyde and GFP localization was visualized directly by observing GFP fluorescence. The cells were viewed using a Zeiss LSM 510 META or Leica Sp2 AOBS confocal microscope. The cells shown are representative images obtained in three separate experiments. The low level of cytoplasmic SIK (cyt) is indicated. Bars, 10 µm.

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