Ub-PDK1 localizes to all cellular compartments, binds phospholipids and mono-ubiquitination is not dependent on kinase activity.

<p><b>A</b>: HEK293T cells were co-transfected with wild type V5-PDK1 (WT) or the indicated lysine mutants and GFP. Whole cell extracts were blotted and probed with anti-V5 and GFP that served as a transfection efficiency control. <b>B</b>: Cell fractionation analysis of HEK293T cells transfected with V5-tagged PDK1 (Cyt = cytoplasmic, Mem = membrane bound, Nuc = nuclear). Ub-PDK1 was detected with a V5 antibody. The HSP90, Histone H1 (H1) and Actin antibodies were used as controls. <b>C</b>: HEK293T cells were transfected as indicated and PDK1 was pulled down using phospholipid-coated (PIP3) or control beads. Pull-downs and input were probed with anti-V5 antibody. <b>D</b>: HEK293T cells were transfected and the catalytically inactive mutant K110R of PDK1 was immunoprecipitated with anti-V5 beads and probed with anti-V5 antibody. The wild type construct (WT, V5-PDK1) was used as a control. <b>E</b>: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) were analyzed with an antibody recognizing phosphorylated PDK1 at the Ser241 site.</p

USP4 and PDK1 interact and co-localize at the plasma membrane.

<p><b>A</b>: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) and input were probed with anti-V5 and MYC antibodies. <b>B</b>: Confocal images from transfected U2OS cells stained with V5-PDK1 (green) and MYC-USP4 (red) antibodies. DNA was visualized by DAPI.</p

USP4 deubiquitinates PDK1 <i>in vivo</i> and <i>in vitro</i>.

<p><b>A</b>: HEK293T cells were co-transfected with V5-tagged PDK1 and a DUB cDNA library. PDK1 was immunoprecipitated with anti-V5 beads and the Ub-PDK1 was detected using a V5 antibody. <b>B</b>: HEK293T cells were co-transfected as indicated. Proteins were immunoprecipitated with anti-V5 beads and immunoblotted with anti-V5 antibody. Whole cell lysates were probed with a MYC antibody to verify the expression of USP4 constructs. <b>C</b>: HEK293T cells were transfected with indicated DUBs. Endogenous PDK1 was immunoprecipitated and immunoblotted with a PDK1 antibody. Whole cell lysates were probed with V5 and FLAG antibodies. <b>D</b>: Quantification of four independent experiments as in C. Indicated are the mean and standard error of the mean and three asterisks indicate T-test p value<0.001. <b>E</b>: HEK293T cells were transfected as indicated and USP4 was immunoprecipitated using a MYC antibody. Immunoprecipitations were incubated <i>in vitro</i> with purified HA-tagged PDK1/Ub-PDK1 as a substrate and immunoblotted with anti-HA antibody. The expression of the USP4 constructs was verified with anti-MYC antibody.</p

The kinase domain of PDK1 is mono-ubiquitinated.

<p><b>A</b>: Overview of the PDK1 constructs used in the figure. PDK1 consists of an amino-terminal kinase domain (KD) and a carboxyl-terminal Pleckstrin Homology (PH) domain. The K-less mutant has all 27 lysine residues (K) mutated to arginines (R). <b>B</b>: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) and input were immunoblotted with anti-V5 antibody. <b>C</b>: HEK293T cells were transfected as indicated and proteins were immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) were immunoblotted with V5 and HA antibodies. <b>D</b>: HEK293T cells were transfected and the lysine-less mutant of PDK1 was immunoprecipitated with anti-V5 beads and probed with anti-V5 antibody. The wild type construct (WT, V5-PDK1) was used as a control.</p

PDK1 is mono-ubiquitinated in a variety of cell lines.

<p><b>A</b>: HEK293T cells were transfected with 6×His-tagged ubiquitin and ubiquitinated proteins were isolated with nickel beads under denaturing conditions. Pull-downs and input were analyzed with an antibody recognizing PDK1. Ub-PDK1 indicates ubiquitin-modified PDK1 species; asterisk indicates unspecific cross-reacting band. <b>B</b>: HEK293T cells were transfected with a V5-tagged PDK1 cDNA and V5-PDK1 was immunoprecipitated. Immunoprecipitations (IP) and input were immunoblotted with a V5 antibody. <b>C</b>: HEK293T cells were co-transfected as indicated with HA-tagged ubiquitin and V5-PDK1. Immunoprecipitation of HA-ubiquitin and input were immunoblotted with a V5 antibody. <b>D</b>: HEK293T cells were co-transfected as indicated and whole cell extracts were probed with anti-V5 or anti-HA antibody. <b>E</b>: HEK293T cells were transfected with V5-tagged PDK1 or a linear PDK1-ubiquitin C-terminal fusion protein. The whole cell extracts were probed with anti-V5 antibody. <b>F</b>: Endogenous PDK1 was immunoprecipitated from HEK293T cells and blotted with anti-PDK1 antibody. The following controls were used: anti-PDK1 antibody only, sepharose beads only, anti-lgG2a control antibody with beads. <b>G</b>: Lysates from the indicated cell lines were subjected to PDK1 immunoprecipitation. Immunoprecipitations were immunoblotted with a PDK1 antibody.</p