Androgen-stimulated PAK6 activation promotes cell motility and invasion in LNCap cells stably expressing PAK6-WT, PAK6-5A and PAK6-5E.

<p>(A) Androgen stimulation promotes transwell cell migration of LNCap-PAK6-WT and LNCap-PAK6-5A cells. LNCap-PAK6-5E cells, albeit with elevated baseline motility, do not respond to androgen stimulation with increased migration. Data represent the mean ± SEM (n=3). **<i>p</i><0.01, ***<i>p</i><0.001 compared to their respective –DHT controls; Student’s <i>t</i>-test. (B) Androgen stimulation promotes matrigel invasion of LNCap PAK6-WT and LNCap-PAK6-5 cells. Similarly, LNCap-PAK6-5E cells, albeit with elevated baseline invasion, do not respond to androgen stimulation with increased invasion. Data represent the mean ± SEM (n=3). *<i>p</i><0.05, **<i>p</i><0.01 compared to their respective –DHT controls; Student’s <i>t</i>-test. (C) Representative micrographs of invaded LNCap and PAK6-derivertives cells depicted in (B). Scale bar: 50 μm. </p

Schematic diagram of PAK6 AR-binding domain phosphorylation site mutations.

<p>The AR-binding domain constitutive non-phosphorylation mutant PAK6-5A is generated by substituting three serine and two threonine residues with alanine (A). The AR-binding domain constitutive phosphorylation mimic mutant PAK6-5E is generated by substituting Serine/Threonine residues with glutamic acid (E).</p

Mapping PAK6-AR interacting domain by mammalian two-hybrid assay.

<p>(A) Schematic depiction of full-length PAK6 and boundaries of truncated PAK6mutants encompassing various domains in the left panel. The right panel shows the results of mammalian two-hybrid assays. The interaction was determined by measuring the luciferase activities of HEK 293 cells transiently transfected with PAK6 full-length and deletion mutants fused in-frame downstream of VP16 transactivation domain in a pACT vector and GAL4 DBD fused AR in a pBIND vector. Data represent the mean ± SEM (n = 3). *<i>p</i><0.05, compared to full-length PAK6, one-way ANOVA. (B) The left panel shows the schematic depiction of full-length AR and boundaries of AR truncated mutants encompassing various domains. A mammalian two-hybrid assay was performed by co-transfecting HEK-293cells with pACT-AR or truncated AR mutants, as indicated, with pBIND-PAK6 and pG5-Luc reporter construct. In both sets of experiment, a renilla luciferase reporter activity was used as a transfection internal control. The luciferase activity was normalized against the internal control and the vector basal control groups. Data represent the mean ± SEM (n = 3). *<i>p</i><0.05, compared to full-length AR, one-way ANOVA.</p

Androgen-stimulated PAK6 activation is mediated by androgen receptor. LAPC4 cells were plated overnight in IMDM supplemented with 10% charcoal-stripped fetal bovine serum.

<p>(A) Time course (0 h: non-treated) study of PAK6 activation in response to 10 nM dihydrotestosterone (DHT) stimulation in LAPC4 cells. (B) A DHT ten-fold serial dilution (0 nM: non-treated; and DHT-treated 0.1 nM to 1,000 nM) dose response of PAK6 activation. (C) 10 nM DHT does not activate PAK6 kinase activity in androgen receptor negative PC3-PAK6 cell. (D) PAK6 <i>in </i><i>vitro</i> kinase assay demonstrates knocking down of AR by siRNA diminishes androgen-stimulated PAK6 activation in LAPC4 cells. Middle panels depict the expression levels of AR and PAK6 in LNCap-PAK6 cells in response to AR specific siRNA. (E) Whole lysate of LNCap, LAPC4 and PC3 blot against anti-PAK6,anti-AR and anti-actin.</p

Androgen-stimulated PAK6 activation promotes cell motility and invasion in LNCap cells stably transfected with tetracycline-inducible PAK6 vector (LNCap-tetON-PAK6).

<p>(A) Anti-PAK6 immuno-blot of tetracycline inducible PAK6 expression in two different clones of LNCap cells depicts the induction of PAK6 expression by doxycycline (1 μg/ml). (B) In vitro kinase activities of doxycycline-induced PAK6 kinase from individual clones using histone H4 as an exogenous substrate. (C) Androgen stimulation promotes transwell cell migration of doxycycline-induced LNCap-tetOn-PAK6 cells. Data represent the mean ± SEM. (n=6). **<i>p</i><0.01, ***<i>p</i><0.001 compared to their respective –DHT control; Student’s <i>t</i>-test. (D) Androgen stimulation promotes matrigel invasion of doxycycline-induced LNCap-tetON-PAK6 cells. All experiments were repeated at least three times, with consistent results. Data represent the mean ± SEM. (n=6). ***<i>p</i><0.001 compared to their respective –DHT control; Student’s <i>t</i>-test .</p

Phosphorylation site mutants of PAK6-5A and PAK6-5E exhibit altered ability to interact with AR and androgen-stimulated kinase activation.

<p>(A) Interactions between AR and PAK6-5A, PAK6-5E are assessed by a mammalian two-hybrid assay. Full-length AR is cloned in-frame downstream of VP16 transactivation domain with a HA-tag using a pACT vector. PAK6-wt, -5A and 5E were cloned in-frame downstream of GAL4 DBD with a HA tag using a pBIND vector. A mammalian two-hybrid assay was performed by co-transfecting HEK-293cells with pACT-AR or AR truncated mutants, as indicated, with pBIND-PAK6 and pG5-Luc vector. Cells were treated with vehicle control or DHT (10 nM) for 20 h followed by dual luciferase assay. A renilla luciferase reporter activity was used as a transfection internal control. The luciferase activity was normalized by the internal control and the vector basal control groups. Data represent the mean ± SEM (n=3, *<i>p</i><0.01 compared to their respective vehicle controls; Student’s <i>t</i>-test). (B) A reciprocal immunoprecipitation assay was performed using AR and PAK6-WT, -5A and -5E mutants. HEK-293 cells were transfected with equal amount of AR and PAK6 expression vectors in the presence or absence of 10 nM DHT. Twenty hours post-transfection either AR or PAK6 was immunoprecipitated using antibodies specifically for either molecule then reciprocally probed by western blot for presence of co-precipitated interacting proteins as depicted. (C) A representative experiment of androgen-stimulated activation of PAK6-WT, PAK6-5A and PAK6-5E in an <i>in </i><i>vitro</i> kinase assay. The upper phosphorylation band is PAK6 autophosphorylation. The lower bands are the phosphorylation of the exogenous substrate histone H4. (D) Quantification of relative kinase activities of in vitro kinase assays from (C). Data represent the mean ± SEM (n=4, *<i>p</i><0.01 compared to their respective vehicle controls; Student’s <i>t</i>-test). </p