Figure 1.
Figure 1. Human CYK4 is a GAP for Rac and Cdc42 but not RhoA. (A) Recombinant human CYK4 was tested against a representative panel of Rho family GTPases, RalA, RalB, Rap1B, Ras, and Rab1. In brief, 100 pmol of each GTPase was incubated in the presence or absence of 0.5 pmol hexahistidine-tagged CYK4 as described in the Materials and methods. The amount of CYK4-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. (B) Recombinant human ARHGAP1 and CYK4 were tested against RhoA, Rac1, Cdc42, Rab1, or a GAP storage buffer blank. The amount of GAP-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. (C) Endogenous MKlp1–CYK4 centralspindlin complexes were immune precipitated using MKlp1 antibodies from synchronized populations of HeLa cells in interphase, metaphase, or anaphase states. Control isolations were performed using GFP antibodies. These complexes were then used for GAP assays with RhoA, Rac1, Cdc42, and Rab1 as a negative control. The amount of centralspindlin-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. Aliquots of the total cell lysate (input) and isolated complexes were Western blotted for MKlp1, CYK4, phosphothreonine Cdk1-phosphorylated CYK4 (pTP), and tubulin. (D) Endogenous MKlp1–CYK4 centralspindlin complexes were immune precipitated using MKlp1 antibodies from synchronized populations of HeLa cells in metaphase or anaphase cells treated with ZM447439 (Aurora B inhibitor) or BI2536 (Plk1 inhibitor). Control isolations were performed using GFP antibodies. These complexes were then used for GAP assays with RhoA, Rac1, Cdc42, and Rab1 as a negative control. The amount of centralspindlin-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. Aliquots of the total cell lysate (input) and isolated complexes were Western blotted for MKlp1, phosphoserine 911 Aurora-phosphorylated MKlp1 (pS911), CYK4, and tubulin. Error bars indicate the standard deviations. IP, immunoprecipitation.

Human CYK4 is a GAP for Rac and Cdc42 but not RhoA. (A) Recombinant human CYK4 was tested against a representative panel of Rho family GTPases, RalA, RalB, Rap1B, Ras, and Rab1. In brief, 100 pmol of each GTPase was incubated in the presence or absence of 0.5 pmol hexahistidine-tagged CYK4 as described in the Materials and methods. The amount of CYK4-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. (B) Recombinant human ARHGAP1 and CYK4 were tested against RhoA, Rac1, Cdc42, Rab1, or a GAP storage buffer blank. The amount of GAP-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. (C) Endogenous MKlp1–CYK4 centralspindlin complexes were immune precipitated using MKlp1 antibodies from synchronized populations of HeLa cells in interphase, metaphase, or anaphase states. Control isolations were performed using GFP antibodies. These complexes were then used for GAP assays with RhoA, Rac1, Cdc42, and Rab1 as a negative control. The amount of centralspindlin-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. Aliquots of the total cell lysate (input) and isolated complexes were Western blotted for MKlp1, CYK4, phosphothreonine Cdk1-phosphorylated CYK4 (pTP), and tubulin. (D) Endogenous MKlp1–CYK4 centralspindlin complexes were immune precipitated using MKlp1 antibodies from synchronized populations of HeLa cells in metaphase or anaphase cells treated with ZM447439 (Aurora B inhibitor) or BI2536 (Plk1 inhibitor). Control isolations were performed using GFP antibodies. These complexes were then used for GAP assays with RhoA, Rac1, Cdc42, and Rab1 as a negative control. The amount of centralspindlin-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. Aliquots of the total cell lysate (input) and isolated complexes were Western blotted for MKlp1, phosphoserine 911 Aurora-phosphorylated MKlp1 (pS911), CYK4, and tubulin. Error bars indicate the standard deviations. IP, immunoprecipitation.

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