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First published online 12 September 2006
doi: 10.1242/jcs.03097

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Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation

Olga Göransson^1,*,, Maria Deak¹, Stephan Wullschleger¹, Nick A. Morrice¹, Alan R. Prescott² and Dario R. Alessi¹

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

View larger version (25K): [in a new window]   Fig. 1. (A) Identification of GST-MARK3 binding partners. Wild-type GST-MARK3 was expressed in HEK 293 cells, purified on glutathione-Sepharose, and subjected to SDS-PAGE. The gel was stained with colloidal Coomassie Blue, and protein bands were excised, washed and digested with trypsin. The identity of the interacting proteins was determined by mass spectrometry, as described previously (Al-Hakim et al., 2005). eIF1 (eukaryotic elongation factor 1) is a non-specific binding-protein, observed to interact with most GST-fusion proteins expressed in HEK293 cells. (B) Analysis of various MARK mutants with regards to 14-3-3 binding and in vitro kinase activity. Wild-type (wt) or indicated mutant forms of GST-MARK3 (ki, kinase inactive D196A mutant; kd+UBA, kinase domain with ubiquitin-associated domain, residues 1-382), were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blot with regards to total protein (GST), T-loop phosphorylation (p-T211) and 14-3-3 binding (14-3-3). In vitro kinase activity was determined using the Cdc25c protein or AMARA peptide as substrate. The results are presented as the mean of a triplicate sample +s.d., and are representative of at least three experiments.

View larger version (40K): [in a new window]   Fig. 2. (A) Phosphatase treatment of MARK3. Wild-type GST-MARK3 was expressed in HEK 293 cells, purified on glutathione-Sepharose and treated with PP1 in the absence or presence of microcystin-LR. The GST-MARK3, which also carried an HA-tag, was subsequently immunoprecipitated using HA antibodies, washed and analysed by western blot with regards to total protein (GST), T-loop phosphorylation (p-T211) and 14-3-3 binding (14-3-3 or overlay assay with yeast 14-3-3). (B) Peptide elution of 14-3-3 from the GST-MARK3/14-3-3 complex. Wild-type GST-MARK3 was expressed in HEK 293 cells, purified on glutathione-Sepharose and, while still coupled to the resin, washed with a phospho-(phospho-Raf) or dephospho-peptide (dephospho-Raf) encompassing the S259 14-3-3 binding site in Raf. The GST-MARK3 was subsequently analysed by western blot with regards to total protein (GST) and 14-3-3 binding (14-3-3). In vitro kinase activity was determined using the Cdc25c protein or the AMARA peptide as a substrate. The results are presented as the mean of a triplicate sample + s.d., and are representative of at least three experiments. (C) Binding of various 14-3-3 mutants to endogenous MARK3, Raf and QSK. (Upper left panel) X-ray crystallographic structure of the 14-3-3 dimer in complex with a Raf peptide, as adapted from (Rittinger et al., 1999). The amphipathic cleft is formed by helix 3 (turquoise), helix 5 (green), helix 7 (red) and helix 9 (orange). (Upper right panel) Enlarged view of the amphipathic cleft with mutated residues indicated. (Bottom panel) Wild-type and indicated mutant forms of GST-14-3-3, were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blotting with regards to total protein (GST), and binding to endogenous MARK3 (MARK3), Raf (Raf) and QSK (QSK). Results shown are representative of two separate experiments. The E180K, F196Y and Y211K mutants studied previously are shown in pink (Benton et al., 2002).

View larger version (43K): [in a new window]   Fig. 3. (A) Mapping of in vivo MARK3 phosphorylation sites. Kinase inactive (D196A) GST-MARK3 purified from HEK 293 cells on glutathione-Sepharose, was excised from a colloidal Coomassie-Blue-stained polyacrylamide gel, and digested with trypsin. One tenth of the digest was subjected to LC-MS with precursor ion scanning and the phosphorylated residues were identified by manual inspection of the acquired MSMS spectra (left panel) as described in Materials and Methods. The identified peaks are listed in the table, with phosphorylated residues in bold. Similar results were obtained in two subsequent experiments. In a further experiment, using wild type MARK3, the same sites were identified, except that T541 and S543 were assigned as the sites of phosphorylation in the ENSTIPDQRTPVASTHSISSAATPDR peptide (marked with an asterisk). (B) Location of phosphorylation sites in MARK3. Schematic view of MARK3, with each phosphopeptide represented by a box, in which phosphorylated residues have been listed. Phosphorylation sites are represented by vertical lines in the MARK3 structure. Phosphorylation of S619, reportedly phosphorylated in xPAR-1 (Kusakabe and Nishida, 2004), was not detected in our analysis but is included in the figure for reference. (C) Conservation of MARK3 phosphorylation sites. Human MARK3 amino acid sequence was aligned using Clustal W, with the indicated MARK homologues (human: hMARK1, hMARK2, hMARK4; Xenopus: xPAR-1; C. elegans: cePAR-1; Drosophila: dPAR-1; S. cerevisiae KIN1: yPAR-1. Conserved residues are indicated by a tick, no conservation by a cross. E, phosphorylation site is Gln; S, phosphorylation site is Ser rather than Thr; T, phosphorylation site is Thr rather than Ser.

View larger version (36K): [in a new window]   Fig. 4. Analysis of single MARK3 phosphorylation-site mutants. (A,B) Wild-type (wt) and indicated phosphorylation-site-mutant forms of GST-MARK3, were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blot with regards to total MARK3 protein (GST), T-loop phosphorylation (p-T211) and 14-3-3 binding (14-3-3). In vitro kinase activity was determined using the AMARA peptide substrate. S619 was not identified in our phosphopeptide mapping analysis but was mutated, because the equivalent site on xPAR-1 was reported to be phosphorylated by aPKC (Kusakabe and Nishida, 2004). The results are presented as the mean of a duplicate sample +s.d., and are representative of two experiments.

View larger version (29K): [in a new window]   Fig. 5. Analysis of a total MARK3 phosphorylation-site mutant. (A,B) Wild-type (wt) and indicated mutant forms of GST-MARK3 (ki, kinase inactive D196A mutant), were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blot with regards to total MARK3 protein (GST), T-loop phosphorylation (p-T211) and 14-3-3 binding (14-3-3). In the 17A-MARK3 mutant, the 17 phosphorylation sites, indicated in Fig. 3 (S42, S45, T61, T211, S378, S396, S400, S419, S469, T491, T507, T536, T549, T564, T576, S583, S619), were converted to alanine residues. S619 was not identified in our phosphopeptide mapping analysis but was mutated, because the equivalent site on xPAR-1 was reported to be phosphorylated by aPKC (Kusakabe and Nishida, 2004). The 17A+T211-MARK3 mutant is identical to the 17A mutant, except that it has an intact T211 in the T-loop. In vitro kinase activity was determined using the protein substrate Cdc25c or the AMARA peptide substrate. The results are presented as the mean of a triplicate (AMARA) or duplicate (Cdc25c) sample + s.d., and are representative of at least three experiments.

View larger version (77K): [in a new window]   Fig. 6. Subcellular localisation of MARK3. (A) Wild-type (wt) or indicated mutant forms of GST-MARK3 (see Fig. 5) were expressed in HEK 293 cells, which were fractionated into a cytosolic and a membrane fraction. Subcellular fractions, including solubilised portions of the homogenates, were subsequently analysed by western blot with regards to MARK3 (GST), MARK3 T-loop phosphorylation (p-T211), the cytosolic marker GAPDH (GAPDH) and the plasma membrane marker Na-K-ATPase (Na-K-ATPase). The results are representative of three experiments. (B) Wild-type (wt) or indicated mutant forms of GFP-MARK3 (see Fig. 5) were expressed in HEK 293 cells, which were fixed in 3% paraformaldehyde 24 hours post transfection. The GFP-fluorescence was analysed by confocal fluorescence microscopy. The cells shown are representative of images obtained in two separate experiments. Bars, 10 µm.

View larger version (45K): [in a new window]   Fig. 7. (A) 14-3-3 binding to truncated MARK3. Wild-type (wt) or indicated truncated forms of GST-MARK3 (CT, residues 327-729; CTKA, residues 327-630) were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blot with regards to total protein (GST) and 14-3-3 binding (14-3-3). (B) Subcellular localisation of truncated MARK3. Wild-type (wt) or indicated truncated forms of GFP-MARK3 were expressed in HEK 293 cells, which were fixed in 3% paraformaldehyde 24 hours post transfection. The GFP-fluorescence was analysed by confocal fluorescence microscopy. The cells shown are representative of images obtained in two separate experiments. Bars, 10 µm.

View larger version (64K): [in a new window]   Fig. 8. (A) Binding of 14-3-3 to MARK2 and MARK3 after PKC co-expression. Wild-type (wt) or indicated mutant forms of GST-MARK isoforms were expressed in HEK 293 cells, in the absence or presence of kinase inactive (D394/A, ki) or wild-type (wt) Flag-PKC, purified on glutathione-Sepharose and analysed by western blot with regards to total protein (GST) and 14-3-3 binding (14-3-3). Expression of Flag-PKC was monitored by western blotting of the lysates (Flag). (B-E) Subcellular localisation of MARK2 and MARK3 after PKC co-expression. (B,D) As in A, except cells were fractionated into a cytosolic and a membrane fraction. Subcellular fractions, including solubilised portions of the homogenates were analysed by western blot with regards to MARK isoforms (GST), PKC (Flag), the cytosolic marker GAPDH (GAPDH) and the plasma membrane marker Na-K-ATPase (Na-K-ATPase). (C,E) As in A, except that GFP fusions of MARK isoforms were employed and cells were fixed in 3% paraformaldehyde 24 hours post transfection. The cells were subsequently permeabilised and stained with anti-Flag and Alexa Fluor 594-labelled anti-mouse antibody. The fluorescence was analysed by confocal fluorescence microscopy. The cells shown are representative of images obtained in two separate experiments. Bars, 10 µm.

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