Journal:
Article Title: PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase
doi: 10.1038/sj.emboj.7600345
Figure Lengend Snippet: Characterization of PKN3 catalytic activity. (A) Full-length or truncated PKN3 versions were modified with the Myc epitope at the C-terminus and transiently expressed in HeLa cells. Schematic structures of the molecules are shown (top): Presumed functional regions (Mukai, 2003) are represented by white (ACC-finger domains), hatched (C2-like domain) and black (kinase domain) boxes. The fragment lacking the first 287 amino acids from the N-terminus is labeled ΔN. A protein fragment spanning the kinase domain at the C-terminus is labeled KD. KR588 (KR) and TA718 (TA) denote point mutations in the ATP-binding site (lysine to arginine or glutamic acid (KE588) at position 588 behaved identically) and in the activation loop (T-loop) phosphorylation site (threonine to alanine, position 718) of the catalytic domain. The first and last amino acids of each fragment are indicated. Cell extracts were analyzed using anti-PKN3 antiserum or anti-phospho(T-loop)-PKN1/2 antibody (P*-PKN1/2) as indicated. The position of wt and truncated PKN3 molecules is indicated by black arrows at the left of each filter; the position of endogenous phosphorylated PKN1/2 molecules is shown by a white arrowhead. Anti-Myc precipitates were tested for kinase activity in vitro using MBP as a substrate; radiolabeled MBP (32[P]MBP) was detected by autoradiography (bottom). (B) HeLa cells stably expressing a 4-OHT-regulatable version of PKN3, PKN3-ER, and its inactive version, PKN3Δkin-ER (carrying mutation KE588), were stimulated for 30 min with 200 nM 4-OHT in DMSO (D). Cell extracts were immunoblotted and tested for in vitro kinase activity after precipitation with anti-ER antibody. (C) Serum-starved cells were stimulated in a time course with or without insulin (10 μg/ml)±4-OHT. Activation of the PI3K pathway was confirmed with anti-phospho(S473)-Akt antibody (P*-Akt). (D) Quiescent cells were stimulated for 6 h as in (C) ±10 μM LY, 10 μM U0126 (U0) or 20 nM rapamycin (Rap). Inhibitor treatment was monitored by P*-Akt, phospho(T202/Y204)-MAP kinase (P*-MAPK) and phospho(T389)-p70S6K (P*-p70S6K) levels.
Article Snippet: The following antibodies were used: Akt, phospho-Akt (S473), MAP kinase, phospho-MAP kinase (T202/Y204), phospho-PKN1/2 (T778/T816) and phospho-p70S6 kinase (T389) antibody (Cell Signalling Technology); phospho-FOXO3a (T32) antibody (Upstate); p110β (H-239) and H-Ras antibodies (C-20) (Santa Cruz Biotechnology); PKN1 and 2 antibodies (Becton Dickinson); Penta-His antibody (Qiagen); TetR antibody (MoBiTec); β-tubulin (AB-1) antibody (Calbiochem).
Techniques: Activity Assay, Modification, Functional Assay, Labeling, Binding Assay, Activation Assay, Phospho-proteomics, In Vitro, Autoradiography, Stable Transfection, Expressing, Mutagenesis
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![Characterization of PKN3 catalytic activity. (A) Full-length or truncated PKN3 versions were modified with the Myc epitope at the C-terminus and transiently expressed in HeLa cells. Schematic structures of the molecules are shown (top): Presumed functional regions (Mukai, 2003) are represented by white (ACC-finger domains), hatched (C2-like domain) and black (kinase domain) boxes. The fragment lacking the first 287 amino acids from the N-terminus is labeled ΔN. A protein fragment spanning the kinase domain at the C-terminus is labeled KD. KR588 (KR) and TA718 (TA) denote point mutations in the ATP-binding site (lysine to arginine or glutamic acid (KE588) at position 588 behaved identically) and in the activation loop (T-loop) phosphorylation site (threonine to alanine, position 718) of the catalytic domain. The first and last amino acids of each fragment are indicated. Cell extracts were analyzed using anti-PKN3 antiserum or anti-phospho(T-loop)-PKN1/2 antibody (P*-PKN1/2) as indicated. The position of wt and truncated PKN3 molecules is indicated by black arrows at the left of each filter; the position of endogenous phosphorylated PKN1/2 molecules is shown by a white arrowhead. Anti-Myc precipitates were tested for kinase activity in vitro using MBP as a substrate; radiolabeled MBP (32[P]MBP) was detected by autoradiography (bottom). (B) HeLa cells stably expressing a 4-OHT-regulatable version of PKN3, PKN3-ER, and its inactive version, PKN3Δkin-ER (carrying mutation KE588), were stimulated for 30 min with 200 nM 4-OHT in DMSO (D). Cell extracts were immunoblotted and tested for in vitro kinase activity after precipitation with anti-ER antibody. (C) Serum-starved cells were stimulated in a time course with or without insulin (10 μg/ml)±4-OHT. Activation of the PI3K pathway was confirmed with anti-phospho(S473)-Akt antibody (P*-Akt). (D) Quiescent cells were stimulated for 6 h as in (C) ±10 μM LY, 10 μM U0126 (U0) or 20 nM rapamycin (Rap). Inhibitor treatment was monitored by P*-Akt, phospho(T202/Y204)-MAP kinase (P*-MAPK) and phospho(T389)-p70S6K (P*-p70S6K) levels.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4518/pmc00514518/pmc00514518__7600345f4.jpg)