Funktionelle Charakterisierung von Interaktionspartnern der Polo-like Kinase 1 (Plk1)

Die Regulation des Zellzyklus wird durch Phosphorylierung und Dephosphorylierung gesteuert. Schlüsselenzyme sind dabei die Cdk/Cyclin-Komplexe. Für den Eintritt und das Durchlaufen der Mitose von besonderer Bedeutung ist die Polo-like Kinase 1 (Plk1). Zur Aufklärung Mitose-spezifischer Signalwege wurden in dieser Arbeit neue Interaktionspartner der Plk1 gesucht. Die Bedeutung dieser Interaktionen für Zellzyklus und Mitose sollten näher charakterisiert werden. In dieser Arbeit konnte gezeigt werden, daß Plk1 direkt mit humanem Cyclin B1, der regulatorischen Untereinheit des M-phase-promoting-factors (MPF), interagiert. Dabei konnte festgestellt werden, daß Plk1 in der Lage ist, Cyclin B1 innerhalb dessen cytoplasmic retention signals (CRS) am Ser-133 zu phosphorylieren. Ferner konnte gezeigt werden, daß die Phosphorylierung der CRS in einer synergistischen Weise durch Plk1 und MAPK (Erk2) stattfindet. Vorphosphorylierung der CRS durch MAPK an Ser-126 und Ser-128 führt nicht nur zu einer 3-4 fach stärkeren Phosphorylierung durch Plk1, sondern auch zur Entstehung neuer Phosphorylierungs-Stellen für Plk1. Die hier aufgeführten Daten zeigen, daß durch synergistische Phosphorylierung der CRS, an der Plk1 beteiligt ist, der rapide nukleäre Import des Cyclin B1/Cdk1-Komplexes in der Prophase gesteuert wird. Dieser rapide Import in den Kern ist Voraussetzung für die Auflösung der Kernmembran in der Prometaphase. Damit konnte gezeigt werden, daß Plk1 dazu beiträgt, den MPF in den Kern (zu seinen Substraten) zu bringen, um früh-mitotische Ereignisse zu induzieren. Mitotische Kinasen (wie MPF, aber auch MAPK) phosphorylieren ihre Substrate oft an Ser/Thr-Pro-Motiven. Sind diese Motive phosphoryliert (pSer/Thr-Pro), so können sie von Pin1, einer Peptidyl-Prolyl-cis/trans-Isomerase isomerisiert werden. In dieser Arbeit wurde gezeigt, daß Plk1 mit Pin1 interagiert. Dabei bindet Plk1 an die Protein-Interaktionsdomäne (WW-Domäne) von Pin1, ist aber gleichzeitig in der Lage, Pin1 innerhalb seiner katalytischen Domäne (PPIase Domäne) zu phosphorylieren. Es konnte gezeigt werden, daß das Ser-65 in Pin1 eine Phosphorylierungs-Stelle für Plk1 darstellt. Da bekannt war, daß Vortäuschung einer Phosphorylierung an Ser-16 oder Ser-67 die Pin1-Aktivität hemmt, wurde ein ähnlicher Effekt für Ser-65 erwartet. Jedoch führte die Vortäuschung der Phosphorylierung an Ser-65 nicht zu einer Reduktion der PPIase-Aktivität von Pin1. In dieser Arbeit konnte erstmals gezeigt werden, daß Pin1 über das Proteasom abgebaut wird. Die Vortäuschung der Ser-65-Phosphorylierung führt zur Stabilisierung des Pin1-Proteins. Damit ist Plk1 das erste bekannte Enzym, welches Pin1 durch Phosphorylierung stabilisiert

Targeting BET bromodomain proteins in solid tumors

There is increasing interest in inhibitors targeting BET (bromodomain and extra-terminal) proteins because of the association between this family of proteins and cancer progression. BET inhibitors were initially shown to have efficacy in hematologic malignancies; however, a number of studies have now shown that BET inhibitors can also block progression of non-hematologic malignancies. In this Review, we summarize the efficacy of BET inhibitors in select solid tumors; evaluate the role of BET proteins in mediating resistance to current targeted therapies; and consider potential toxicities of BET inhibitors. We also evaluate recently characterized mechanisms of resistance to BET inhibitors; summarize ongoing clinical trials with these inhibitors; and discuss potential future roles of BET inhibitors in patients with solid tumors

Pituitary tumor-transforming gene expression is a prognostic marker for tumor recurrence in squamous cell carcinoma of the head and neck

BACKGROUND: The proto-oncogene pituitary tumor-transforming gene (PTTG) has been shown to be abundantly overexpressed in a large variety of neoplasms likely promoting neo-vascularization and tumor invasiveness. In this study, we investigated a potential role for PTTG mRNA expression as a marker to evaluate the future clinical outcome of patients diagnosed with primary cancer of the head and neck. METHODS: Tumor samples derived from primary tumors of 89 patients suffering from a squamous cell carcinoma were analyzed for PTTG mRNA-expression and compared to corresponding unaffected tissue. Expression levels were correlated to standard clinico-pathological parameters based on a five year observation period. RESULTS: In almost all 89 tumor samples PTTG was found to be overexpressed (median fold increase: 2.1) when compared to the unaffected tissue specimens derived from the same patient. The nodal stage correlated with PTTG transcript levels with significant differences between pN0 (median expression: 1.32) and pN+ (median expression: 2.12; P = 0.016). In patients who developed a tumor recurrence we detected a significantly higher PTTG expression in primary tumors (median expression: 2.63) when compared to patients who did not develop a tumor recurrence (median expression: 1.29; P = 0.009). Since the median expression of PTTG in patients with tumor stage T1/2N0M0 that received surgery alone without tumor recurrence was 0.94 versus 3.82 in patients suffering from a tumor recurrence (P = 0.006), PTTG expression might provide a feasible mean of predicting tumor recurrence. CONCLUSION: Elevated PTTG transcript levels might be used as a prognostic biomarker for future clinical outcome (i.e. recurrence) in primary squamous cell carcinomas of the head and neck, especially in early stages of tumor development

LIN-9 Phosphorylation on Threonine-96 Is Required for Transcriptional Activation of LIN-9 Target Genes and Promotes Cell Cycle Progression

<div><p>Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.</p></div

Phosphorylation of Thr-96 in LIN-9 is essential for transcriptional activation of cell cycle genes.

<p>(A) Indicated GFP-LIN-9 constructs were cotransfected into 293T cells, together with luciferase under the control of the promoters of cdc6, cyclin B1 or cyclin A2. Cell lysates were subjected to reporter gene assays using Promega Glo (Promega). Experiment was performed in triplicates. For the cdc6 promoter: n = 4, for the cyclin A and cyclin B promoters: n = 2; *p<0.05, **p<0.005, ***p<0.0005 (B) One of the three samples used in A was subjected to Western blot, using GFP antibody to detect expression of GFP-LIN-9 constructs. GFP-LIN-9^T96D migrates faster and is always expressed at lower levels than GST-LIN-9 wild type and T96A.</p

cyclin E is essential for phosphorylation of Thr-96 in LIN-9 in vivo in primary cells.

<p>(A) p-LIN-9^Thr-96 antibody is specific. 293T cells were cotransfected with plasmids encoding GFP-LIN-9, FLAG-Cdk3 and cyclin E, and cell extracts were subjected to immunoprecipitation using a monoclonal LIN-9 antibody. IP material was split into two, and incubated for 1 hour at 30°C with or without lambda phosphatase. Samples were subjected to Western Blot using our p-LIN-9^Thr-96 antibody (upper panel). Equal loading of LIN-9 was confirmed by incubating the same membrane with a rabbit antibody against LIN-9 (lower panel). (B) HUVECs were transfected with siRNA control or siRNA directed against Cyclin E1 (Santa Cruz) using Dharmafect 4 (Dharmacon) and harvested after 48 hours. Western Blots was performed with the indicated antibodies. (C) T98G cells were starved in serum-free medium for 36 hours followed by the addition of growth medium (supplemented with 10% serum). Cells were harvested by tripsynization at the indicated time after the addition of serum and use for Western blot or FACS analysis. Western blots were performed as described in Methods.</p

Phosphorylation of Thr-96 in LIN-9 accelerates entry into G2/M.

<p>293T cells were transfected with the indicated LIN-9 constructs or GFP control and 24 hours later synchronized by serum starvation. Cells were released into the cell cycle by the addition of complete growth medium and harvested at 0, 6 and 12 hours for FACS analysis.</p

LIN-9 is phosphorylated by and is found in a trimeric complex with Cdk3/cyclin E1.

<p>(A) GST-LIN-9 expressed in bacteria was subjected to in vitro kinase assays using indicated kinase complexes purchased from ProQinase. Asterisk indicates GST-cyclin autophosphorylation. The lower panel shows Coomassie blue staining of the same gel. (B) GST-LIN-9 was subjected to in vitro kinase assays using Flag-Cdk3 kinase complexes (Flag-IP) from human 293Tcells. Autoradiography visualizes incorporation of phospholabel and asterisks (white, placed to the left of the corresponding band) indicate autophosphorylation of cyclin E1 and cyclin A2, respectively (upper panel). Coomassie Brillant Blue (CBB) staining depicts loading of GST-LIN-9 and Flag-Cdk3 (second panel). Ten percent of IP material was subjected to Western blot to visualize amounts of co-precipitated cyclin A2 (third panel), cyclin E1 (fourth panel) and Flag-Cdk3 (lower panel). (C) 293T cells were cotransfected with Flag-Cdk3 and GFP-LIN-9, followed by Flag-IP. IP material was subjected to Western blots using LIN-9 antibody (upper panel), cyclin E1 antibody (middle panel) and Flag-antibody (lower panel).</p