PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase
2004; Springer Nature; Volume: 23; Issue: 16 Linguagem: Inglês
10.1038/sj.emboj.7600345
ISSN1460-2075
AutoresFrauke Leenders, Kristin Möpert, Anett Schmiedeknecht, Ansgar Santel, Frank Czauderna, Manuela Aleku, Silke Penschuck, Sibylle Dames, Maria Sternberger, Thomas Röhl, Axel Wellmann, W. Arnold, Klaus Giese, Jörg Kaufmann, Anke Klippel,
Tópico(s)Peptidase Inhibition and Analysis
ResumoArticle29 July 2004free access PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase Frauke Leenders Frauke Leenders atugen AG, Berlin, Germany Search for more papers by this author Kristin Möpert Kristin Möpert atugen AG, Berlin, Germany Search for more papers by this author Anett Schmiedeknecht Anett Schmiedeknecht atugen AG, Berlin, Germany Search for more papers by this author Ansgar Santel Ansgar Santel atugen AG, Berlin, Germany Search for more papers by this author Frank Czauderna Frank Czauderna atugen AG, Berlin, Germany Search for more papers by this author Manuela Aleku Manuela Aleku atugen AG, Berlin, Germany Search for more papers by this author Silke Penschuck Silke Penschuck atugen AG, Berlin, GermanyPresent address: H Lundbeck A/S, Valby, Denmark Search for more papers by this author Sibylle Dames Sibylle Dames atugen AG, Berlin, Germany Search for more papers by this author Maria Sternberger Maria Sternberger atugen AG, Berlin, Germany Search for more papers by this author Thomas Röhl Thomas Röhl atugen AG, Berlin, Germany Search for more papers by this author Axel Wellmann Axel Wellmann Pathologisches Institut der Unikliniken, Bonn, Germany Search for more papers by this author Wolfgang Arnold Wolfgang Arnold atugen AG, Berlin, Germany Search for more papers by this author Klaus Giese Klaus Giese atugen AG, Berlin, Germany Search for more papers by this author Jörg Kaufmann Jörg Kaufmann atugen AG, Berlin, Germany Search for more papers by this author Anke Klippel Corresponding Author Anke Klippel atugen AG, Berlin, Germany Search for more papers by this author Frauke Leenders Frauke Leenders atugen AG, Berlin, Germany Search for more papers by this author Kristin Möpert Kristin Möpert atugen AG, Berlin, Germany Search for more papers by this author Anett Schmiedeknecht Anett Schmiedeknecht atugen AG, Berlin, Germany Search for more papers by this author Ansgar Santel Ansgar Santel atugen AG, Berlin, Germany Search for more papers by this author Frank Czauderna Frank Czauderna atugen AG, Berlin, Germany Search for more papers by this author Manuela Aleku Manuela Aleku atugen AG, Berlin, Germany Search for more papers by this author Silke Penschuck Silke Penschuck atugen AG, Berlin, GermanyPresent address: H Lundbeck A/S, Valby, Denmark Search for more papers by this author Sibylle Dames Sibylle Dames atugen AG, Berlin, Germany Search for more papers by this author Maria Sternberger Maria Sternberger atugen AG, Berlin, Germany Search for more papers by this author Thomas Röhl Thomas Röhl atugen AG, Berlin, Germany Search for more papers by this author Axel Wellmann Axel Wellmann Pathologisches Institut der Unikliniken, Bonn, Germany Search for more papers by this author Wolfgang Arnold Wolfgang Arnold atugen AG, Berlin, Germany Search for more papers by this author Klaus Giese Klaus Giese atugen AG, Berlin, Germany Search for more papers by this author Jörg Kaufmann Jörg Kaufmann atugen AG, Berlin, Germany Search for more papers by this author Anke Klippel Corresponding Author Anke Klippel atugen AG, Berlin, Germany Search for more papers by this author Author Information Frauke Leenders1, Kristin Möpert1, Anett Schmiedeknecht1, Ansgar Santel1, Frank Czauderna1, Manuela Aleku1, Silke Penschuck1, Sibylle Dames1, Maria Sternberger1, Thomas Röhl1, Axel Wellmann2, Wolfgang Arnold1, Klaus Giese1, Jörg Kaufmann1 and Anke Klippel 1 1atugen AG, Berlin, Germany 2Pathologisches Institut der Unikliniken, Bonn, Germany *Corresponding author. atugen AG, Robert-Rössle-Str. 10, 13125 Berlin, Germany. Tel.: +49 30 9489 2832; Fax: +49 30 9489 2827; E-mail: [email protected] The EMBO Journal (2004)23:3303-3313https://doi.org/10.1038/sj.emboj.7600345 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K. Introduction The phosphoinositide 3-kinase (PI3K)/PTEN signaling pathway regulates a diverse set of cellular responses including growth, development, survival, motility, adhesion, immune cell function and glucose transport (Katso et al, 2001; Roymans and Slegers, 2001). Class IA PI3K molecules are heterodimers consisting of a regulatory subunit, p85, and a catalytic subunit, p110, whereby several different isoforms exist for each subunit (Wymann and Pirola, 1998). PI3K is only transiently activated after growth factor stimulation of normal cells, and is rapidly turned off through tumor suppressor PTEN function. PTEN negatively regulates PI3K signaling by dephosphorylating its second messenger phospholipid products, thereby ensuring that activation of the pathway occurs in a transient and controlled fashion (Vazquez and Sellers, 2000). PTEN is one of the most frequently inactivated tumor suppressor genes in human cancer (Trotman and Pandolfi, 2003), and its loss results in chronic activation of the PI3K pathway and correlates with increased metastatic behavior (Wang et al, 2003). Sustained PI3K activation can also be achieved by activated forms of PI3K itself and correlates with increased invasiveness or growth in semi-solid matrices (Jimenez et al, 1998; Klippel et al, 1998; Kobayashi et al, 1999). Conventional gene expression analysis using microarrays to identify cancer therapy targets is hampered by the chromosomal instability of tumor cells and typically results in a large number of differentially expressed genes with uncertain disease relevance. Instead of comparing disease end points, we investigated the molecular changes induced after simulating PI3K hyperactivation experimentally, which allowed to compare pairs of otherwise isogenic cells under normal growth conditions in subsequent gene profiling studies (Kaufmann et al, 2004). This approach was combined with three-dimensional (3D) culture conditions using basement membrane-containing extracellular matrix, which provides for a more relevant system to recapitulate tumor cell growth and metastasis than standard 2D culture conditions using plastic culture surfaces (Bissell et al, 2003). In fact, controlled modulation of PI3K signaling has little phenotypic consequences on 2D cell growth (Stolarov et al, 2001), but dramatically affects the growth phenotype under 3D culture conditions (Kaufmann et al, 2004). A number of kinases and other signaling molecules, which mediate PI3K-regulated events, represent candidate cancer therapy targets (Luo et al, 2003). However, most of these proteins act rather upstream in the PI3K signaling cascade and also regulate multiple functions in normal cells (Wymann et al, 2003). Therefore, their inhibition is likely to cause side effects. Here, we describe the identification, validation and biochemical characterization of a novel kinase molecule, which mediates invasive prostate cancer cell growth further downstream of a hyperactive PI3K pathway in cell-based assays and a mouse tumor model. Results PKN3 mediates growth on basement membrane downstream of PI3K Sustained activation of PI3K signaling is required for growth of invasive PTEN−/− PC-3 prostate cancer cells on basement membrane matrix, whereas modulation of MEK-MAP kinase signaling has little effect in this cell type (Kaufmann et al, 2004; Supplementary Figure 1). By expression profiling, we identified genes that are PI3K-dependently expressed in cells grown on basement membrane-containing matrigel (Kaufmann et al, 2004). One of these genes encoded a kinase with homology to the protein kinase C superfamily: PKN3, also known as PKNβ (Manning et al, 2002; Mukai, 2003), has not yet been linked to the PI3K/PTEN pathway or to invasive growth. After verifying its PI3K-dependent expression (Supplementary Figure 2A), we tested whether PKN3 expression is required for PI3K-induced cell growth under 3D culture conditions. PC-3 cells were treated with three different PKN3-specific antisense molecules, the so-called GeneBlocs (GBs) (Sternberger et al, 2002), and subsequently seeded on matrigel (Figure 1A). GBs specific for inhibiting expression of PTEN or p110β, an isoform of the catalytic subunit of PI3K, served as negative or positive controls. In PC-3 cells, p110β appears to represent the predominant isoform for mediating PI3K signaling as compared to p110α (Supplementary Figure 3). Parallel samples were analyzed to confirm specific reduction of PKN3 mRNA expression (not shown; see reduction of protein expression in Figure 1B). Inhibition of PKN3 expression interfered with growth on matrigel to the same extent as inhibition of p110β (Figure 1A), which was previously shown to be required for growth of PC-3 cells on basement membrane matrix (Czauderna et al, 2003b; Supplementary Figure 3). PTEN GB treatment had no effect on the PTEN−/− PC-3 cells in the matrigel assay, similar to a mismatch control antisense molecule (mm) of PKN3 GB3. Three GB molecules targeting distinct sites in the PKN3 mRNA all inhibited PC-3 cell growth on matrigel, whereas their corresponding four-nucleotide mismatch controls did not (Supplementary Figure 4A). To verify the GB-mediated PKN3 protein knockdown, we raised antibodies against the PKN3 kinase domain. PKN3-specific GBs that interfered with PC-3 cell growth on matrigel also efficiently reduced PKN3 protein levels, whereas none of the control antisense molecules had any effect (Figure 1B). Importantly, the PKN3 GBs did not interfere with expression of the kinase molecules PKN1 or PKN2 (also known as PRK1 and PRK2), which are close homologs of PKN3 (Mukai, 2003). Both were shown to be regulated by PI3K (Flynn et al, 2000) and implicated in prostate cancer (Metzger et al, 2003). Furthermore, PKN1 mRNA expression was also PI3K-dependent in PC-3 cells grown on matrigel (Supplementary Figure 2B). In contrast to PKN3, however, neither PKN1 nor PKN2 protein knockdown interfered with the ability of PC-3 to grow on matrigel (Supplementary Figures 4B and C). These experiments suggest that PKN3, but not PKN1 or PKN2, is required for PC-3 growth on matrigel. Figure 1.PKN3 mediates growth on basement membrane matrix in PTEN−/− prostate cancer cells. (A) PC-3 cells transfected with 30 nM of the indicated GB antisense or mismatch molecules (mm) were grown for 48 h on matrigel; size bars: 200 μm. The experiment was reproduced by independent transfections. (B) PC-3 cells were grown for 48 h in the presence of the indicated sets of PKN3 GB or mm control. Protein extracts were analyzed by immunoblotting using PKN3-specific antiserum. The filter was reprobed using antibodies against PKN1 and PKN2. The PI3K subunits p110α and p85 served as loading controls. Download figure Download PowerPoint PKN3 is expressed in a PI3K-dependent manner and is upregulated in patient prostate tumor samples To investigate whether sustained PI3K signaling is not only required but may even be sufficient for increased expression of PKN3, we generated stably transfected pools of a normal human breast epithelial cell line (MCF-10A) expressing an inducible version of a constitutively active PI3K, M-p110*-ER, from promoters with different strengths. The activity of M-p110*-ER can be stimulated by the addition of 4-hydroxytamoxifen (4-OHT), which results in chronic activation of the PI3K pathway (Klippel et al, 1998). Both stable M-p110*-ER pools exhibited increased basal PI3K signaling compared to the control cell population, as indicated by the elevated phosphorylation of the downstream effector p70S6 kinase (p70S6K) (Figure 2A, lanes 1, 5 and 9). In cells with lower M-p110*-ER levels PI3K signaling was further enhanced by 4-OHT treatment (lanes 5 and 6), whereas it was almost maximal without 4-OHT in cells with high M-p110*-ER levels (lanes 9 and 10). Endogenous PKN3 protein expression was strongly induced in M-p110*-ER cells compared to control cells, and the induction level correlated with the respective level of PI3K signaling (compare lanes 1 and 2, 5 and 6, and 9 and 10); the identity of the PKN3 signal was confirmed by the disappearance of PKN3 in GB-treated samples (lanes 13 and 14). In MCF-10A cells, increased PKN3 expression was also dependent on chronic activation of the PI3K pathway, as indicated in samples treated with LY294002 (LY), a small-molecule inhibitor of PI3K (lanes 7 and 11). Rapamycin, which inhibits mTOR-p70S6K signaling, also abrogated PKN3 expression in MCF-10A cells (lanes 8 and 12), indicating that this PI3K effector pathway contributes to PKN3 induction in these cells. PKN1/2 expression was regulated by PI3K to some extent, but was not as readily induced by M-p110*-ER as PKN3. Taken together, this indicates that hyperactivation of the PI3K pathway is not only required but can also be sufficient for enhanced PKN3 expression. Figure 2.PKN3 is PI3K-dependently expressed and upregulated in prostate tumor samples. (A) Stable MCF-10A cells that direct expression of a 4-OHT-inducible version of an activated PI3K, M-p110*-ER, from a weaker (open arrow) or a stronger promoter (filled arrow, bold) were analyzed compared to vector-transfected cells. The cells were incubated in serum-free medium with or without DMSO (D), 200 nM 4-OHT, 10 μM LY or 20 nM rapamycin (R) overnight. Parallel samples were treated with 30 nM PKN3 GB3 or mismatch (mm) control to confirm the identity of the PKN3 signal. Cell extracts were analyzed using the indicated antibodies. Inhibition of PI3K signaling was confirmed by dephosphorylation of p70S6K at T389 (P*-p70S6K); MAP kinase phosphorylation at T202/Y204 (P*-MAPK) served as control. (B) Immunohistochemical analysis of two adjacent human prostate tumor tissue sections was performed using anti-PKN3 antiserum or pre-immune serum (left panels). The samples were hematoxylin counterstained to monitor the glandular tissue structure; size bars: 500 μm. Normal and tumor prostate tissue samples were compared by in situ hybridization using a PKN3-specific antisense probe; the specificity of the signal was confirmed by hybridizing adjacent tissue sections with the sense probe (right panels); size bars: 200 μm. Download figure Download PowerPoint Since loss of PTEN function and the concomitant activation of PI3K signaling have been correlated with the development of metastatic prostate cancer, we wanted to test whether PKN3 could be detected in tissue samples of patient prostate tumors. Immunohistochemical analysis of adjacent tissue sections was carried out using PKN3 preimmune or immune serum. Only the immune serum-treated samples exhibited regions with positive staining (Figure 2B, left). Close inspection revealed that most of the tumor cells in the sample stained positive for PKN3 while the surrounding nontumorigenic tissue was negative. These results were corroborated by in situ hybridization studies, where prostate tumor tissues showed elevated staining only with a PKN3-antisense probe compared to normal prostate tissue (Figure 2B, right). Induced inhibition of PKN3 expression interferes with formation of lymph node metastasis in an orthotopic mouse prostate tumor model For long-term loss of function studies to validate candidate targets in mouse tumor model systems, we recently established a vector-derived expression system for inducible shRNA molecules (Czauderna et al, 2003b). Doxycyline (Dox) treatment induces U6tetO promoter-controlled shRNA expression by inactivating the tetracycline repressor (TetR). We isolated stable PC-3 cell populations expressing two different shRNAs targeting PKN3. Dox-induced shRNA expression resulted in efficient knockdown of PKN3 protein levels after 48 h (Figure 3A). Pools of stable cells directing induced inhibition of p110α or expressing an unrelated control shRNA were analyzed in parallel. Respective cell populations were seeded on matrigel and photographed at different time points. As expected from experiments shown above (Figure 1), cells with shRNA-mediated knockdown of PKN3 expression exhibited impaired growth on extracellular matrix (Figure 3B). As described earlier, inhibition of p110β, but not of p110α, interferes with matrigel growth of PC-3 cells (Supplementary Figure 3; Czauderna et al, 2003b), whereas p110α represents the predominant PI3K subunit in other cell types (Figure 5A). Accordingly, induced inhibition of p110α had no effect in PC-3 cells in the experiment shown here and served as a control for Dox treatment and shRNA induction. This result confirmed that vector-derived inducible expression of PKN3 shRNA in these stable cell populations was indeed functional. Figure 3.Inducible knockdown of PKN3 expression inhibits metastasis in an orthotopic mouse prostate tumor model. (A) PC-3 cells stably transfected with vectors that direct Dox-dependent expression of two different PKN3 shRNAs were analyzed in parallel to p110α or an unrelated control shRNA. Cells were grown±100 ng/ml Dox, and extracts were analyzed by immunoblotting. Expression of the TetR was confirmed using an anti-TetR antibody; α-tubulin served as loading control. (B) PKN3(2) shRNA and p110α shRNA cells analyzed in (A) were seeded on matrigel and photographed after 2 and 4 days; size bars: 200 μm. (C) PC-3 cells with inducible PKN3(2) shRNA were transplanted intraprostatically into nude mice. One group of animals received Dox (black bars), the second group was mock-treated (white bars); each group consisted of eight animals. After 56 days, the mice were killed and evaluated for primary tumor and lymph node metastases development (top). Each bar represents the mean tumor volume±s.e. The reduction in lymph node metastases formation in the Dox-treated group is statistically significant according to the Mann–Whitney test. Representative in situ pictures of three animals of each group are shown (bottom). The primary prostate tumor is labeled with 'T' and the position of lumbar and renal lymph node metastases is indicated by white arrows. (D) RNA samples extracted from PC-3 prostate tumors of seven animals of each group were analyzed by Northern blotting for induction of PKN3 shRNA; tRNAVal served as loading control (top). PKN3 mRNA levels after shRNA induction were quantified by Taqman analysis relative to p110α mRNA (bottom); the results are mean of triplicates±s.e. Black bars indicate the results for Dox-treated animals, white bars for the control group. (E) PC-3 cell populations stably expressing shRNA specific for p110α (negative control), p110β (positive control) and PKN3 were analyzed by time-lapse-video microscopy on matrigel. Pictures taken at the indicated times after seeding are shown at × 2.5 magnification. Download figure Download PowerPoint Next, stable PKN3 shRNA PC-3 cells were transplanted intraprostatically into nude mice (Stephenson et al, 1992). The animals were split into two groups, one group was treated with Dox to induce shRNA expression and the second group was mock-treated. After 56 days the mice were killed and analyzed for primary tumor formation and lymph node metastasis. The Dox-treated group of mice showed a small effect on primary tumor growth; however, metastases formation was strongly inhibited (Figure 3C). Similar results were obtained in a separate experiment with a cell population expressing the second PKN3 shRNA; also, Dox treatment of mice transplanted with untransfected PC-3 cells did neither affect tumor nor metastases formation (not shown). Northern-blot analysis of RNA extracted from the primary tumors showed that samples obtained from Dox-treated mice exhibited on average increased expression of PKN3 shRNA compared to samples from the control group (Figure 3D, top panel). In fact, overall there was an inverse correlation between the level of shRNA induction and the amount of PKN3 mRNA detected in these samples: high shRNA levels in tumors resulted in reduced levels of PKN3 mRNA (Figure 3D, lower panel). Leakiness in shRNA expression as well as limited knockdown in PKN3 mRNA in certain samples may have been caused by loss of TetR expression after 56-day implantation or by contaminating PKN3 signals from mouse cells in the respective human tumor cell material. Nevertheless, the data strongly suggest that the decreased PKN3 levels in the primary tumors were indeed the cause for the reduced metastases formation. This result correlates with the results obtained from the matrigel growth assay and indicates that PKN3 may be an important mediator of invasive signaling. The finding that PKN3 supports metastasis formation rather than tumor growth suggests that PKN3 might modulate cell adhesion and/or migration. shRNA-mediated reduction in PKN3 expression appeared to affect PC-3 cell morphology and the structure of the tubulin network, indicating cytoskeletal rearrangements (Supplementary Figure 5). To analyze whether these alterations affect migration and motility, PC-3 cell populations expressing shRNA specific for p110α, p110β and PKN3 were analyzed by time-lapse-video microscopy on matrigel, since changes caused by reduced PKN3 levels are more evident under 3D growth conditions (Figure 3E). Equal plating of the cells was observed 1 h after seeding. Subsequent time points indicated that cells expressing p110β- or PKN3-specific shRNAs were impaired in forming network-like structures on matrigel, which correlated with reduced cell numbers compared to the p110α shRNA control. The entire video clips for the p110α shRNA control and the PKN3 shRNA cells can be viewed in the Supplementary section. The data indicate that motility and cell–cell contacts might influence PC-3 cell growth on matrigel. PKN3 is an AGC-type kinase and its catalytic activity is regulated by PI3K After validating PKN3 as a downstream effector of activated PI3K by independent gene silencing approaches, we wanted to characterize its catalytic activity to investigate whether it could serve as a target for therapeutic intervention in cancer. We transiently expressed various recombinant PKN3 derivatives in HeLa cells (Figure 4A, top), and immunoblotting of cell extracts with PKN3-specific antiserum revealed approximately equal expression of the different proteins (upper blot). The cell extracts were analyzed in parallel using an antibody that recognizes the activation loop phosphorylation (T-loop) site in the catalytic domain of PKN1 or PKN2 (middle blot). Phosphorylation of this conserved threonine residue, which is present in PKN3 at position 718, is required for activation of AGC-type kinases (Parekh et al, 2000). Endogenous PKN1/2 and all recombinant PKN3 molecules were detected by this phospho-specific antibody, except for the fragment carrying a threonine to alanine substitution at position 718 (TA718), indicating that wild-type (wt) PKN3 is phosphorylated at this residue. The PKN3 derivatives were precipitated from the lysate via their Myc tag and after a series of stringent washes subjected to an in vitro protein kinase assay using myelin basic protein (MBP) as a substrate in the presence of radiolabeled ATP. The fragment comprising just the kinase domain exhibited catalytic activity, which was specific, since fragments with mutations in the catalytic center (lysine to arginine, KR588) or the T-loop phosphorylation site (TA718) had no detectable activity (lower panel). The full-length molecule, however, had substantially increased kinase activity compared to the catalytic domain fragment. ΔN, the derivative lacking the N-terminal region, appeared to be inactive despite the fact that it overlaps the kinase domain fragment, which was active by itself, and was phosphorylated at T718. This suggests that the middle region of PKN3 imposes a negative regulatory function on the catalytic domain that is relieved in the presence of the N-terminus in the full-length protein. Our results were confirmed with HA-tagged versions of PKN3 (not shown). Figure 4.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. Download figure Download PowerPoint Having established a biochemical assay for PKN3 enzymatic activity we wanted to test whether its catalytic function is regulated by PI3K as shown for other AGC-type kinases (Parekh et al, 2000). In order to investigate the requirements for PKN3 activation without using overexpression, we established stable HeLa cell pools expressing an inducible version of PKN3, PKN3-ER, and its kinase-deficient version, PKN3Δkin-ER. The PKN3-ER fusion protein had no detectable kinase activity in the absence of 4-OHT, also increased phosphorylation at the T-loop site (T718) was 4-OHT dependent (Figure 4B). PKN3Δkin-ER had no activity confirming specificity of the 4-OHT-induced response. Consistent with transient transfection experiments (Supplementary Figure 6), phosphorylation at T718 in the inactive PKN3Δkin-ER was weaker in this inducible system than in the active kinase molecule. Next, HeLa PKN3-ER cells were starved in serum-free medium overnight and subsequently stimulated in the presence or absence of 4-OHT with or without insulin for the indicated times (Figure 4C). Insulin treatment strongly activated the PI3K pathway in HeLa cells, as indicated by increased phosphorylation of the downstream effectors Akt and p70S6K, but had little or no effect on the MEK-MAP kinase pathway (Figure 4D). PKN3-ER kinase activity as well as T-loop phosphorylation were substantially increased in the presence of 4-OHT plus insulin (Figure 4C, lanes 1–5) compared to 4-OHT treatment alone (lanes 6–10). In contrast to the immediate Akt activation, PKN3-ER activity increased steadily during 6 h insulin treatment, suggesting that PKN3 is not a direct PI3K effector and requires activation of additional components within the signaling cascade, which then mediate PKN3 stimulation further downstream. Similar data were obtained with serum stimulation (not shown). To investigate the contribution of various signaling pathways to PKN3 activation, 6 h insulin stimulation was carried out in the absence or presence of LY, U0126 or Rapamycin. Successful inhibitor treatment was monitored by reduced phosphorylation of Akt, MAP kinase or p70S6K (Figure 4D). Inhibition of PI3K strongly interfered with the insulin-mediated increase in PKN3 activity and also blocked PKN3 basal activity (lanes 3 and 8), whereas inhibition of MEK-MAP kinase signaling had no substantial effect (lanes 4 and 9). Rapamycin did not affect the insulin-mediated increase in PKN3 activity, but it reduced the basal activity (lanes 5 and 10), suggesting that mTOR-p70S6K signaling downstream of PI3K can contribute to PKN3 stimulation to a certain extent. This set of experiments suggests that activation of PI3K can regulate PKN3 activ
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