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1.
Fig. 2.

Fig. 2. From: Characterization of the Mechanisms Controlling Greatwall Activity .

Gwl belongs to the AGC family of kinases. (A) HA-Wt and G825A, K830A, W840A and F863A mutants were used to measure kinase activity as described in the legend to Fig. 1B. (B) Similar to Fig. 1C except for the overexpression of the G825A, K830A, W840A, and F863A mutants. (C) Kinase activity of the HA-Wt, the P849A P852A double mutant (AxxA), and the P849A and P852A simple mutants of hGwl was measured as described in the legend to Fig. 1B. (D) The levels of expression and the phosphorylation of Cdc27 and cyclin B-Cdc2 substrates in these mutants were also analyzed as described in the legend to Fig. 1C.

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.
2.
Fig. 4.

Fig. 4. From: Characterization of the Mechanisms Controlling Greatwall Activity .

The binding of the tail/linker phosphate to the tail/linker binding sites at the N terminus is essential for Gwl activity. (A) Model of active Gwl is shown as a ribbon representation with the side chains of selected residues. The kinase domain is shown in green, the C tail is shown in red, the K residues predicted to bind phosphates of S875 and T860 are shown in violet, and the glycine-rich domain is shown in orange. (B) Kinase activity of the K48A, K65A, K48A K65A, and K39A mutants. (C) The levels of expression and the capacity to maintain mitosis of the Wt and each mutant are measured.

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.
3.
Fig. 3.

Fig. 3. From: Characterization of the Mechanisms Controlling Greatwall Activity .

Gwl contains a functional hydrophobic pocket and a long insert between catalytic domains VII and VIII. (A) The activity of the R82A and Y98A mutants of the hydrophobic pocket were analyzed and compared to the Wt form. (B) The functionality of the different mutants was studied by measuring the phosphorylation of Cdc27 and the different cyclin B-Cdc2 substrates in xGwl-depleted CSF extracts. (C) Depiction of the hGwl sequence in which the position of the central insert is shown. Different deletions and percentage of kinase activity of each mutant are indicated. (D) The expression and the capacity of these mutants to maintain mitosis are shown.

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.
4.
Fig. 5.

Fig. 5. From: Characterization of the Mechanisms Controlling Greatwall Activity .

Phosphorylation of S875 is essential to maintain Gwl activity. (A) The electrophoretic mobility of xGwl was analyzed in interphase (INT) and mitotic (CSF) egg extracts by Western blotting. xGwl was immunoprecipitated from interphase and mitotic egg extracts, and the kinase activities of the immunoprecipitates were analyzed. (B) Activity of the indicated phosphorylation mutants. (C) The levels of overexpression of the different HA-hGwl forms and their capacity to maintain mitosis were analyzed. (D) Inactive GST-hGwl was incubated for 15 min with a synthetic peptide containing 50 μM of an N-terminal sequence of hGwl (residues G44 to M66). Subsequently, 40 units of purified cyclin B-Cdc2 (Ozyme) was added to the mix to activate hGwl. A total of 15 min later, active hGwl was recovered by immunoprecipitation with anti-GST antibodies, and the immunoprecipitate was used to measure hGwl activity. For Gwl assays, Gwl was incubated with cyclin B-Cdc2 with or without the N-terminal binding peptide (50 μM), with MBP used as the substrate. Kinase activity of hGwl is represented as the percentage of the activity of the nontreated kinase.

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.
5.
Fig. 6.

Fig. 6. From: Characterization of the Mechanisms Controlling Greatwall Activity .

The interaction of the tail/linker phosphate with the tail/linker binding sites and the presence of the PxxP motif are essential to maintain Gwl kinase activity. (A) A total of 1.5 μl of nontranslated interphase or mitotic extracts and of mitotic overexpressed Wt HA-hGwl and the S875A mutant were submitted to Western blotting by using anti-hGwl antibodies or anti-phospho serine 875 antibodies. GST-hGwl was incubated or not with 40 units of purified cyclin B-Cdc2 (Ozyme). The mix was divided into two equal fractions. In one fraction, the levels of GST-hGwl protein and the phosphorylation of S875 were analyzed by Western blotting. In the other fraction, GST-hGwl was recovered by immunoprecipitation, and the kinase activity was measured ([γ-33P]MBP). (B) Represented is the kinase activity of a GST-hGwl purified from Sf9 insect cells and a GST-hGwl kinase phosphorylated in vitro by a purified His-Plx1. The levels of GST-hGwl and the phosphorylation of S875 were also analyzed by Western blot. (C) GST-hGwl protein was phosphorylated in vitro by His-Plx1 protein and the phospho-amino acids were identified by mass spectrometry. (D) Represented are the phosphorylation sites found in the central insert in an active xGwl obtained from mitotic egg extracts. Violet square represents sequence from positions 254 to 640 that is not required for Gwl activity. Yellow square indicates deletions performed to eliminate phosphorylation sites T718, Y720, T722, and S725. (E) A total of 1.5 μl of mitotic egg extracts overexpressed with the indicated mutants were analyzed by Western blotting by the use of anti-hGwl antibodies and anti-phospho-S875 antibodies and fluorescent secondary antibodies. (F) The levels of phosphorylation of S875 described in the legend to panel E were measured by using an Odyssey infrared imaging system, corrected by the amount of expression of each ectopic protein obtained by the same system, and represented as a percentage of the phosphorylation compared to that of the Wt form. The kinase activities of these mutants are also represented.

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.
6.
Fig. 1.

Fig. 1. From: Characterization of the Mechanisms Controlling Greatwall Activity .

Greatwall conserves most of the residues distinguishing AGC from other eukaryotic kinases. (A) Alignment showing representative AGC kinases as well as human and Xenopus Gwl (hGw and xGW, respectively). Key regions of the C terminus of AGC kinases are indicated above the alignment. The patterns of conserved residues observed with AGC kinases and with human and xenopus Greatwall are indicated by colored squares. Colors represent different intramolecular interactions and are indicated at the bottom of the figure. Mutants of Gwl are indicated by shapes. (B) A total of 10 μl of CSF extracts was immunodepleted with 2 μg of anti-xGwl antibodies. A total of 1.5 μl of CSF extracts and of the supernatant (SN) was loaded and treated for Western blot analysis to detect endogenous xGwl. (C) Wt and the G44S, K62A, and T741A HA-tagged forms of hGwl were expressed in mitotic egg extracts and subsequently immunoprecipitated with anti (α)-HA antibodies. A total of 20% of this immunoprecipitate (IP) was used to measure the levels of each protein by Western blotting with fluorescent secondary antibodies (HA-Gwl); the remaining 80% was used to measure kinase activity as described in Material and Methods ([γ-33P]MBP). The levels of phosphorylation of MBP in the autoradiography were quantified by densitometry using Image J and corrected by the amount of each ectopic protein present in the immunoprecipitate. The activities are represented as the percentage of kinase activity measured in the Wt hGwl form. Experiments were performed in triplicate. Values are expressed as the mean ± standard deviation. (D) Mitotic extracts overexpressing the HA-Wt and G44S, K62A, and T741A mutants of hGwl were depleted of endogenous xGwl. Supernatants were used to analyze the level of overexpression with anti-HA antibodies by Western blotting. A total of 20 and 40 min after depletion, two aliquots of this supernatant were also analyzed for their capacity to maintain mitosis by measuring the phosphorylation of Cdc27 and of the different cyclin B-Cdc2 substrates by Western blot analysis.

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.
7.
Fig. 7.

Fig. 7. From: Characterization of the Mechanisms Controlling Greatwall Activity .

Gwl activation first requires phosphorylation of S875 and a subsequent binding of the hydrophobic motif of another AGC kinase with the hydrophobic pocket of Gwl. (A) Wild-type GST-hGwl was obtained from Sf9 insect cells and incubated or not with 30 and 50 μM of a synthetic peptide containing the phosphorylated hydrophobic motif of Rsk2 (PIFtide, sequence PGIPPSANLFRGFpSFVA). After a 15-min incubation, kinase activity was directly measured by adding MBP mix and [γ-33P]ATP and represented as the percentage of the nonincubated Wt Gwl. The levels of GST-hGwl in each point are shown by Coomassie blue staining. (B) Similar to experiment depicted in panel A, except that a mutant form of hGwl with a partial activity (K72M) instead of a wild-type Gwl protein was used. (C) Purified Wt or mutant hGwl (MuGwl) was either treated with λ-phosphatase (λPh), nontreated (CT) or prephosphorylated in vitro with purified His-Plx1 (+Plx1) and subsequently submitted to SDS-PAGE and Western blotting with anti-phospho 875 antibodies. (D) Wild-type GST-hGwl purified from Sf9 cells was phosphorylated in vitro with His-Plx1 (WtGwl+Plx1), incubated (+Rsk2 PIFtide) or not with a dose of 10, 30, and 50 μM PIFtide, and used to measure the kinase activity as well as the phosphorylation of S875. Under one condition, WtGwl was directly incubated with 30 μM of PIFtide, and the kinase activity was measured. Control (CT Plx1) corresponds to an in vitro phosphorylation assay in which GST-hGwl was not added and corresponds to the [γ-33P]MBP background promoted by Plx1. The amount of GST-hGwl at each point is shown by Coomassie blue staining. The levels of phosphorylation of MBP in the autoradiography were quantified by densitometry using Image J and corrected by the amount of GST-hGwl at each point. The activities are represented as the percentage of kinase activity measured in Wt hGwl form and the [γ-33P]MBP background levels promoted by Plx1 were deducted. (E) The mutant GST-hGwl form was incubated into interphase (Inter) or mitotic egg extracts (CSF) for 15 min and subsequently recovered by immunoprecipitation. Immunoprecipitates were then used to measure kinase activity or to analyze hGwl levels and phosphorylation of S875 by Western blotting with anti-hGwl and anti-phospho 875 antibodies. Two of these immunoprecipitates were supplemented with 30 or 50 μM PIFtide, and the kinase activity was measured. In one condition, the GST-hGwl mutant was incubated in CSF extracts, immunoprecipitated, and used to measure kinase activity (MuGwl CSF).

Suzanne Vigneron, et al. Mol Cell Biol. 2011 June;31(11):2262-2275.

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