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1.
FIGURE 5.

FIGURE 5. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

The presence of PIP2 in the membrane increases the affinity of Pak1 for Cdc42 bound to liposomes. Cosedimentation of Pak1 with liposomes (equimolar PC:PE, 3% DGS-NTA(Ni)) was assessed in the presence or absence of 12% PIP2 by titrating the reactions with increasing concentrations of Cdc42-His. The data were fit in GraphPad Prism to derive apparent affinities (shown). The average ± S.D. of duplicates is shown.

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.
2.
FIGURE 2.

FIGURE 2. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

Pak1 binding is sensitive to the spatial density of PIP2. Pak1 was incubated with 400 μm PC:PE (equimolar) liposomes containing increasing mole fractions of PIP2 (0–20%). Liposomes were sedimented and bound Pak1 was quantified by Western blotting. Results are shown for five replicate experiments (± S.D.) and the data were fit to a sigmoidal dose-response curve using GraphPad Prism to determine the Hill coefficient (nH).

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.
3.
FIGURE 1.

FIGURE 1. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

PI(4,5)P2 selectively enhances Cdc42-dependent Pak1 activation. Left, the experimental system. Synthetic liposomes (double black circle) with bound Cdc42-His (rectangles) that either contained phosphoinositide (small dark circles) or not, were incubated with Pak1 and [γ-32P]ATP to monitor phosphoinositide-dependent Pak1 autophosphorylation. Right, Pak1 autophosphorylation in kinase assays with Cdc42-His-bound liposomes (comprised of equimolar PC:PI and containing 7% (by mole) DGS-NTA(Ni) with or without 6% of the indicated phosphoinositides) is plotted normalized to control reactions with Cdc42-containing liposomes without the test phosphoinositide. Pak1 autophosphorylation was quantified by PhosphorImager analysis of dried electrophoretic gels of the reaction products. Mean results are shown from two independent replicates ± S.D.

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.
4.
FIGURE 7.

FIGURE 7. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

Ack binding and catalytic activity is not enhanced by PIP2. A, cosedimentation of Pak1 and Ack with liposomes (equimolar PC:PE, 3% DGS-NTA(Ni)) was assessed in the presence or absence of 5 μm Cdc42-His and 12% PIP2. Equimolar amounts of Pak1 and Ack were used. B, [γ-32P]ATP kinase assays were conducted with Ack in the presence of the indicated activators and Ack catalytic activity was measured by either Ack autophosphorylation or Ack phosphorylation of a peptide substrate (GST-Acktide). Activators were either PIP2-containing liposomes (PC:PI:PIP2:DGS-NTA(Ni), 43.5:43.5:6:7 mole ratio), or control liposomes (PC:PI:DGS-NTA(Ni), 46.5:46.5:7 molar ratio) with 300 nm Cdc42-His or their combination. These activator concentrations were selected on the basis of their ability to maximally activate Ack when combined. Results are normalized to phosphorylation observed in the reaction containing both activators and are shown for two independent replicates ± S.D.

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.
5.
FIGURE 3.

FIGURE 3. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

A system for rapamycin-inducible recruitment of PIP5K to the Golgi membrane for localized PIP2 synthesis. A, schematic diagram: a fusion protein where the transmembrane region of Tgn38 anchors the FRB domain of mammalian target of rapamycin (labeled with cyan fluorescent protein, CFP) to the Golgi where PI(4)P (purple circles) is present. Rapamycin or its analog 3-methylindole rapamycin (iRap) triggers binding and recruitment of a cytosolic fusion protein containing the FKBP binding domain of FKBP12-rapamycin-associated protein (FRAP) fused to PIP5K (and labeled with monomeric red fluorescent protein, mRFP) to Golgi-localized Tgn38-FRB. Golgi recruitment of PIP5K allows for localized phosphorylation of PI(4)P to PIP2 (blue circles). B, HEK293 cells were cotransfected with plasmids encoding Tgn38-FRB-CFP and PIP5K-FKB-mRFP. Transfected cells were stimulated with iRap for 5 min to induce dimerization. CFP and mRFP were visualized by fluorescence microscopy.

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.
6.
FIGURE 4.

FIGURE 4. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

Pak1 recruitment to the Golgi membrane is enhanced by Golgi-localized PIP2 and Cdc42. A, HEK293 cells were cotransfected with plasmids encoding Tgn38-FRB-CFP (Golgi targeting protein), PIP5K-FKBP-mRFP (PIP5 kinase), and a GFP-tagged PIP2-specific binding protein (GFP-PH(PLCδ)). Transfected cells were stimulated by iRap for 20 min to induce FRB-FKBP dimerization (bottom panels). The white bars indicate 10 μm. B, Pearson correlation coefficients for the cellular colocalization of FRB and PIP5K were calculated for 5 randomly selected cells before (−) or after (+) 20 min of iRap treatment; colocalization of FRB and GFP-PH before (−) and after (+) iRap treatment was analyzed similarly. The iRap-stimulated colocalization of these proteins is highly significant for both FRB/PIP5K (***, p = 0.0007) and FRB/GFP-PH (***, p = 0.0001). The distribution of Pearson coefficients in each case are shown as a box and whisker plot, where whiskers represent the maximum and minimum values, the box represents the 25th to 75th percentiles, and the central line depicts the median. C, HEK293 cells were transfected with Tgn38-FRB-CFP, PIP5K-FKBP-mRFP, and HA-tagged Pak1, 8T-Pak1 (cannot bind PIP2), or LL-Pak1 (cannot bind Cdc42). Cells were treated for 20 min with iRap and then immunostained for Pak1 using a total Pak1 antibody (Invitrogen). The white bars indicate 10 μm. D, for each condition, 18 cells were randomly chosen and Pearson correlation coefficients were determined for colocalization of Pak1 and FRB. The distribution of Pearson correlation coefficients is shown as box and whisker plots as in B. *, p = 0.0148 and ***, p = 0.0009.

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.
7.
FIGURE 6.

FIGURE 6. From: Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases.

Pak1 catalytic activity is synergistically enhanced by Cdc42 and PIP2. A, [γ-32P]ATP kinase assays were conducted with Pak1 in the presence of the indicated activators and Pak1 catalytic activity was measured by either Pak1 autophosphorylation or Pak1 phosphorylation of a peptide substrate (GST-Paktide). Activators were either PIP2-containing liposomes (PC:PI:PIP2:DGS-NTA(Ni), 43.5:43.5:6:7 mole ratio) or control liposomes (PC:PI:DGS-NTA(Ni), 46.5:46.5:7 molar ratio) with 200 nm Cdc42-His or their combination. These activator concentrations were selected on the basis of their ability to maximally activate Pak1 when combined (supplemental Fig. S3A). Results are normalized to phosphorylation observed in the reaction containing both activators and are shown for two independent replicates ± S.D. B, activation for 8T-Pak1 was measured as in A. Note that higher concentrations of PIP2 (500 μm total lipid and 15 μm Cdc42-His) were required to achieve full activation of 8T-Pak1 (supplemental Fig. S3B). C, 50% activation contour for Pak1 autophosphorylation. The concentration of Cdc42-His required to achieve 50% activation of Pak1 was determined at each indicated PIP2 concentration. Horizontal error bars (smaller than the data symbol in most cases) represent the S.D. calculated from two replicates. The curve that can be drawn through these points represents all combinations of PIP2 and Cdc42-His that lead to 50% activation of Pak1. Table 1 presents the data used to generate the plots in C–F. D, Pak1 catalytic activity in the reactions in C was also measured by phosphorylation of a Pak1 substrate peptide included in the reaction and the results are presented as a 50% activation contour. The significant departure from linearity of the contour is indicative of synergistic activation. 8T-Pak1 autophosphorylation (E) and 8T-Pak1 substrate phosphorylation (F) is not enhanced by PIP2. The 50% activation contours were determined as in C and D and the data are summarized in Table 1. The vertical nature of the 50% activation contour demonstrates a lack of sensitivity to PIP2.

Kimberly A. Malecka, et al. J Biol Chem. 2013 March 29;288(13):8887-8897.

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