Dissertation

Brk tyrosine kinase signalling in the gastrointestinal tract

zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von

Dipl. Biol. Andrea Hägebarth
geb. am 11. Oktober 1977 in Mühlhausen, Thüringen

Dekan: Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, Ph.D.

Gutachter:
1. Prof. Dr. Harald Saumweber
2. Prof. Angela L. Tyner, Ph.D.
3. Prof. Dr. Walter Birchmeier

eingereicht: 27. April 2005

Datum der Promotion: 18. August 2005

Zusammenfassung

Für viele entwicklungsbiologische Prozesse ist die Tyrosin Kinase vermittelte Signaltransduktion von entscheidender Bedeutung. Die Tyrosin Kinase Brk (breast tumor kinase) stellt den Prototypen nicht N-terminal myristoylierter, Nicht-Rezeptor Tyrosin Kinasen dar. Die Expression dieser intrazellulären Kinase ist auf epitheliale Gewebe beschränkt und wird während der Entwicklung differentiell reguliert, wobei höchste RNA- und Proteinniveaus im Darmepithel detektiert wurden. In normalen Geweben ist die Brk Expression auf nichtproliferierende, terminal differenzierte Zellen beschränkt. Zusätzlich zu der entwicklungsregulierten und zelltypspezifische Expression in normalen Geweben, wurde eine Überexpression dieser Kinase in einigen epithelialen Tumoren festgestellt. Die genaue biologische Funktion dieser Tyrosin Kinase ist bis heute unbekannt. In der vorliegenden Arbeit wurde der Einfluss von Brk auf die Eigenschaften des murinen Darmepithels untersucht.

Um die regulatorische Funktion von Brk im murinen Darmepithel zu untersuchen, wurde das brk Gen in der Maus mittels homologer Rekombination unterbrochen. Brk knockout Mäuse zeigten keine offensichtlichen Defekte in ihrer Entwicklung jedoch eine erweiterte Proliferationszone in den Krypten des Darmepithels und verlängerte Villi im Bereich des Dünndarms. Eine genauere Untersuchung zeigte, dass die Inaktivierung von Brk zu einer erhöhten Akkummulation von nukleärem β-catenin in den Krypten der knockout Mäuse führte. Ausserdem konnte eine Hochregulierung des β-catenin Zielgens c-myc in den Krypten des Dünn- und Dickdarms festgestellt werden. Zusätzlich zeigten Brk knockout Mäuse eine Aktivierung des Akt-Signaltransduktionswegs und damit eine vermutliche Induktion von Überlebenssignalen in ihrem Darmepithel. Diese Ergebnisse zeigen, dass die Inaktivierung der Brk Tyrosine Kinase zu einer Störung des Fliessgleichgewichts zwischen Zellproliferation und –differenzierung mit aberranter Signaltransduktion im Darmepithel führt.

Weiterhin traten bei Brk knockout Mäuse chronische Entzündungen des Darmepithels sowie eine erhöhte Sensibilität gegenüber dem Reizmittel DSS auf. Im Gegensatz dazu, zeigten Wildtyp Mäuse eine mit der Literatur übereinstimmende Reaktion zu DSS. Interessanterweise wurde zusätzlich eine Induktion der Brk Expression im Darmepithel behandelter Wildtyp Mäuse vorgefunden. Diese Ergebnisse verdeutlichen das durch die Abwesenheit der Brk Tyrosin Kinase verursachte Ungleichgewicht in der Homöostase des Darmepithels. Ausserdem lassen sie eine schützende Funktion dieser Kinase in Entzündungsprozessen vermuten.

Apoptose - programmierter Zelltod - spielt eine grosse Rolle in der Entwicklung und Aufrechterhaltung der Integrität des Darmepithels. Die Deregulation dieses Prozesses hat häufig krankhafte Erscheinungen wie Krebs zur Folge. Trotz einer Aktivierung des Akt-Signaltransduktionswegs in Brk knockout Mäusen wurden keine Unterschiede im Apoptoseniveau unbehandelter Brk knockout und Wildtyp Mäuse gefunden. Die Behandlung dieser Mäuse mit γ-Strahlung resultierte jedoch in einer signifikanten Beeinträchtigung der Apoptosereaktion in knockout Mäusen. Im Gegensatz zu Wildtyp Mäusen waren Brk knockout Mäuse resistent gegenüber Bestrahlung, was die Anhäufung onkogener Mutationen und damit die Entwicklung von Krebs fördert. Ausserdem wurde erneut eine Induktion der Expression des Brk-Proteins im Darmepithel behandelter Wildtyp Mäuse festgestellt, was eine schützende Funtion dieser Kinase im Darmepithel weiter unterstreicht.

Zusammenfassend kann gesagt werden, dass die Brk Tyrosin Kinase eine entscheidende Rolle in der Aufrechterhaltung der Homöstase und Integrität des Darmepithels spielt. Insbesondere scheint Brk als entscheidender Faktor zur Bestimmung der Sensitivität epithelialer Zellen zu genotoxischem Stress zu fungieren. Entgegen der bisher vermuteten onkogenen Funktion in epithelialen Tumoren scheint Brk im normalen murinen Darmepithel “Tumor Suppressor” ähnliche Funtionen innezuhaben.

Eigene Schlagworte: Brk, Sik, PTK6, Tyrosine Kinase, Darmepithel

Abstract

The Breast tumor kinase Brk is a prototypical non-myristoylated, non-receptor tyrosine kinase. Brk expression is epithelial-specific with the highest level of expression in the gastrointestinal tract. In normal tissues, Brk expression is developmentally regulated, and is restricted to cells exiting the cell cycle and undergoing terminal differentiation. Interestingly, Brk expression is upregulated in some epithelial tumors. To date, the biological role of this intracellular tyrosine kinase in differentiation and oncogenesis remains poorly understood. The present study is focused on gaining a better understanding of the physiological role of Brk in the gastrointestinal tract.

To determine the role of Brk in the gastrointestinal tract, we disrupted mouse brk by homologous recombination. Loss of Brk in the mouse resulted in increased intestinal epithelial cell turnover and the appearance of longer small intestinal villi suggesting a role for Brk in the maintenance of intestinal tissue homeostasis. Brk deficient mice displayed enhanced accumulation of nuclear β-catenin and upregulation of the β-catenin target gene c-myc in the crypt compartment of small and large intestine. In addition, Brk deficient mice exhibited increased Akt kinase activity, suggesting an increase in pro-survival signaling. Furthermore, chronic inflammation was observed in Brk deficient mice, and they showed increased susceptibility to a colon injury model utilizing DSS. Interestingly, wild-type mice exhibited a significant upregulation of nuclear Brk protein throughout the intestinal epithelium in response to DSS.

Even though Brk deficient mice showed increased Akt pro-survival signaling, there was no corresponding difference in base-line apoptosis in untreated wild-type and knockout animals. However, subjected to γ-irradiation, Brk deficient animals were significantly impaired in the apoptotic response, a crucial mechanism protecting the stem cells in the small intestinal crypt against the accumulation of oncogenic mutations and the development of cancer. Wild-type mice, however, exhibited normal levels of apoptosis following γ-irradiation accompanied by a rapid induction of Brk expression in crypt cells. The ability of Brk to sensitize cells to apoptotic stimuli was also observed in vitro in Rat1A fibroblasts overexpressing the kinase.

These recent findings suggest that Brk plays a crucial role in the maintenance of intestinal tissue homeostasis and integrity. In addition, Brk may function to protect the intestinal epithelium against DNA-replication-induced errors and hence the development of cancer. Contrary to reported oncogenic properties of Brk in other epithelial tissues, Brk appears to have tumor suppressor-like functions in the mouse gastrointestinal epithelium.

Keywords: Brk, Sik, PTK6, Protein tyrosine kinase, intestine

Table of contents

• 1 Introduction
  • 1.1  Protein tyrosine kinases in signal transduction
  • 1.2 Brk family tyrosine kinases
  • 1.3 The non-receptor tyrosine kinase Brk
  • 1.4 Brk signaling substrates
  • 1.5 Morphology and homeostasis of the intestinal epithelium
  • 1.6  Intestinal neoplasia
  • 1.7 Wnt signaling in intestinal development and cancer
  • 1.8 Tyrosine kinase signaling in the gastrointestinal tract
  • 1.9 Apoptosis and survival signaling in the intestinal epithelium
  • 1.10 Inflammation and intestinal cancer
  • 1.11 Mouse models for colorectal cancer
  • 1.12 Aim of the study
• 2 Materials and Methods
  • 2.1  Mouse strains
  • 2.2 Mouse experiments
    • 2.2.1 Induction of colitis using dextran sodium sulfate
    • 2.2.2 Treatment of mice with AOM and DSS in a colon carcinogenesis model
    • 2.2.3 γ-irradiation of wild-type and Brk knockout mice
  • 2.3 Tissue preparation and histology
  • 2.4 Immunohistochemical techniques
  • 2.5 Expression constructs, cell culture and stable cell lines
  • 2.6 Cell treatments
  • 2.7 Flow cytometry
  • 2.8 Ribonuclease protection assays
  • 2.9 Protein lysates and immunoblotting
  • 2.10 Immunoprecipitations
  • 2.11  In vitro Akt kinase assays
• 3 Results
  • 3.1  Brk is required for intestinal homeostasis
    • 3.1.1  Brk protein expression is restricted to differentiated cells
    • 3.1.2 Increased epithelial cell turnover in Brk knockout mice
    • 3.1.3  Increased accumulation of nuclear β-catenin in the absence of Brk
    • 3.1.4  Regulation of β-catenin activity by Brk may be mediated by Akt
  • 3.2 Brk signaling protects from intestinal inflammation
    • 3.2.1  Chronic inflammation in Brk deficient mice
    • 3.2.2 Brk protects the intestinal epithelium from cytokine-mediated injury
    • 3.2.3  Increased susceptibility of Brk deficient mice to DSS inhibits tumor development in the AOM/DSS tumorigenesis model
  • 3.3 Brk sensitizes cells to apoptosis in vivo and in vitro
    • 3.3.1  Brk is required for DNA-damage induced intestinal apoptosis
    • 3.3.2 Enhanced pro-survival Akt and MAPK signaling in Brk knockout mice
    • 3.3.3 Induction of Brk in the intestine following ionizing radiation
    • 3.3.4  Expression of Brk sensitizes cells to apoptosis in vitro
• 4 Discussion
  • 4.1  Regulation of intestinal homeostasis by Brk
  • 4.2 Brk signaling and inflammation
  • 4.3 Regulation of apoptosis by Brk signaling
  • 4.4 Brk signaling in tumor suppression and cancer development
• Abbreviations
• Cited Literature
• Acknowledgments
• Eidestattliche Erklärung

Images

• Fig. 1: Structure of Src and Brk tyrosine kinases. Src and Brk tyrosine kinases share 44% amino acid identity. Both Src and Brk proteins contain SH3 and SH2 domains that regulate protein-protein interactions as well as a conserved catalytic SH1 domain. The tyrosine at 527 in Src and at 447 in Brk regulates kinase activity. Phosphorylation on these tyrosine residues results in the intramolecular formation of an inactive conformation involving both SH2 and SH3 domains. The lysine at 295 in Src and at 219 in Brk correlates with the ATP binding site and its mutation results in a dominant-negative protein. Tyrosines 416 in Src and 341 in Brk reside in the activation loop and are autophosphorylated resulting in increased kinase activity. In contrast to Src, Brk lacks an aminoterminal consensus myristoylation sequence.
• Fig. 2: Schematic presentation of the mammalian digestive tract. (A) Mammalian digestive tract. Digestion starts in the mouth, where food is being macerated and partially digested. The esophagus serves as an alimentary organ, transporting food into the stomach, where it is digested by various enzymes. Most of the nutrient absorption occurs in the small intestine whereas water is absorbed mainly in the large intestine. The cecum serves for the storage of food. (B) Structure of the small intestine. Putative stem cells reside immediately above the Paneth cells. Progenitors stop proliferating at the crypt-villus junction and express differentiation markers. Enteroendocrine, absorptive, and mucus-producing cells migrate upward, whereas Paneth cells migrate downward and localize at the bottom of the crypt. (C) Structure of the large intestine. Stem cells reside at the crypt bottom. Progenitors are amplified by constant division along the bottom two thirds of the crypts, whereas cell cycle arrest and differentiation occur when progenitors reach the top third of the crypts. Paneth cells are absent in the colon.
• Fig. 3: CRC development. Correlation between CRC progression and the accumulation of genetic alterations according to Fearon & Vogelstein (1990) (Fearon and Vogelstein, 1990).
• Fig. 4: Expression of Brk protein in the gastrointestinal tract. Immunohistochemistry for Brk in adult wild-type jejunum and colon using anti-Brk antibodies or anti-rabbit IgG antibodies as controls and DAB as a substrate (brown). Nuclei were counterstained with hematoxylin (blue). Size bar represents 50 µm. Brk expression is restricted to differentiated epithelial cells.
• Fig. 5: Brk mRNA and protein expression in the gastrointestinal tract. (A) RNase protection analysis for Brk mRNA synthesis in homozygous mutant and wild-type mice. The loading control was performed with a probe for cyclophilin. (B) Western blot analysis for Brk synthesis in neonatal (14 days) and adult (8 weeks) wild-type (WT) and mutant (Brk -/-) ileum and colon. The loading control was performed with anti-β-actin antibodies.
• Fig. 6: Defects in intestinal epithelial homeostasis in the absence of Brk signaling. Photomicrographs of immunohistochemical staining for PCNA (A) and BrdU (B) (brown) from sections of jejunum and colon of wild-type (WT) and knockout (Brk -/-) mice. Mice were injected with 50 µg BrdU/ g bodyweight and sacrificed 2 hours later. Sections were counterstained with hematoxylin (blue). Size bars represent 50 µm. The amount of proliferation indicated by PCNA and BrdU positive cells is increased in the mutant epithelium.
• Fig. 7: Loss of Brk expression affects the crypt-villus morphology. (A, C) Representative sections of the distal ileum and distal jejunum from wild-type (WT) and knockout (Brk -/-) mice stained with hematoxylin/eosin. Size bars represent 100 µm. (B, D) Histograms of average villus length in wild-type and knockout distal ileum (B) and jejunum (D). Error bars represent ± SD. P-values were determined using the Student’s test. The average villus length is drastically increased in the mutant mucosa.
• Fig. 8: Increased nuclear β-catenin expression in mutant intestinal epithelium. (A, B) Immunohistochemical analysis of wild-type (WT) and knockout (Brk -/-) distal jejunum and colon sections with antibodies against β-catenin and IgG as controls. Staining of β-catenin shows accumulation in the nucleus of cells at the crypt base (arrowheads), but is absent from the nucleus of cells along the crypt-villus axis, where it is only membrane-localized. Size bars represent 50 µm. Nuclear β-catenin is detected in more crypt cells of mutant intestinal epithelium compared to wild-type mice.
• Fig. 9: Expression of nuclear β-catenin and its target gene c-myc in mutant intestinal epithelium. Representative sections of the distal jejunum (A) and colon (B) from wild-type (WT) and knockout (Brk -/-) adult animals were stained with anti-β-catenin and anti-c-myc antibodies. Immunostaining with anti-IgG antibodies served as controls. Size bars represent 50 µm. Brk mutant mice exhibit increased nuclear β-catenin accumulation and upregulated expression of the β-catenin target gene c-myc in the intestinal crypts.
• Fig. 10: Increased Akt kinase activity in the intestinal epithelium of Brk mutant mice. Endogenous Akt was immunoprecipitated from total cell lysates of the distal ileum of inbred (2.8 G12) and outbred (2.8) wild-type (WT) and knockout (KO) mice. In vitro kinase assays were performed with purified recombinant GSK-3 and ATP. Reactions were stopped at 0 (control) and 30 minutes and subjected to Western blotting with phospho-GSK-3β and total GSK-3 β antibodies. Equal amounts of total GSK-3β protein were present in the reactions. Loss of Brk results in increased Akt kinase activity in the intestinal epithelium.
• Fig. 11: Chronic inflammation in the intestinal epithelium of adult outbred Brk deficient mice (2.8). (A) Gross microscopical examination of intestinal sections from outbred 2.8 knockout mice. Sections from small intestine and colon were stained with hematoxylin/eosin. Increased amount of lymphoid tissue, Peyer’s patches, was observed in mutant epithelium. (B) RNase protection assays were performed with total RNA from distal ileum of adult (8 weeks) wild-type (WT) and knockout (Brk-/-) oubred (2.8) animals and ³²P-labeled antisense multiprobes specific for cytokines. Rpl32 and GAPDH expression were examined as controls. Brk mutant mice exhibit increased expression of IL-18 and IL-6 in the intestinal epithelium. (C) Immunohistochemical analysis of the apical epithelial barrier. Immunostaining with antibodies specific for ZO-1 and β-catenin on colonic sections from wild-type (WT) and mutant (Brk -/-) animals. No differences in the expression and position of these tight (ZO-1) and adherens (β-catenin) junction markers between wild-type and mutant mice were observed. Size bars represent 50 µm.
• Fig. 12: Colonic epithelial damage in inbred Brk deficient mice following DSS administration. (A) Study design. Mice were subjected to 5 days of 3% DSS (5d) in drinking water followed by 3 days water (8d). Arrows indicate days 0, 5 and 8 of DSS treatment. (B, C) Representative photomicrographs of colons from inbred (2.8 G12) wild-type (WT) and knockout (Brk -/-) mice stained with hematoxylin/eosin at days 0, 5 and 8 of DSS treatment. Brk knockout mice exhibit increased epithelial injury with severe crypt loss after treatment. By day 8 after treatment, the inflammatory cell infiltration has become more extensive in mutant animals (indicated by arrow). Size bars represent 100 µm. (D) RNase protection assays were performed with total RNA from distal colon of inbred (2.8 G12) wild-type (WT) and knockout (Brk-/-) animals and ³²P-labeled antisense multiprobes specific for cytokines. Rpl32 and GAPDH expression was examined as controls. Brk mutant mice exhibit increased expression of pro-inflammatory cytokines, such as IL-6, but a decreased expression of the epithelial specific cytokines IL-18, in the colon after DSS treatment.
• Fig. 13: Induction of nuclear Brk tyrosine kinase expression upon DSS treatment in wild-type mice. (A, B) Western blot analyses were performed with total cell lysate from distal ileum (A) and distal colon (B) of wild-type (WT) and knockout (Brk-/-) animals using antibodies against Brk. Expression of β-actin was examined as a control for protein loading. Brk protein expression increased with treatment. (C, D) Immunohistochemical analysis of colons from wild-type (WT) animals at days 0, 5 and 8 of DSS treatment with anti-Brk antibodies or anti-IgG antibodies as a control. Size bars represent 50 µm. Induction of nuclear Brk protein expression throughout the colonic epithelium of wild-type mice after DSS treatment.
• Fig. 14: Inflammation-related mouse colon carcinogenesis model. (A) Study design. Arrows indicate initial AOM injection at 0 weeks and study end at week 20 respectively. The black box indicates the 7 day treatment with 2% DSS. (B) Increased mortality in wild-type mice upon treatment. Survival of wild-type (WT) and mutant (Brk -/-) mice was monitored over time of treatment. (C, D) Macroscopic view of colons of wild-type (WT) and mutant (Brk -/-) mice after AOM/DSS treatment. Size bars represent 1 cm. Due to their increased susceptibility to inflammation, Brk deficient mice appear less susceptible to AOM/DSS induced tumor formation in the colon.
• Fig. 15: Resistance to DNA-damage induced apoptosis in the absence of Brk. (A) Radiation –induced apoptosis of cells in the small intestine. Sections of distal jejunum from untreated and γ-irradiated (8 Gγ) wild-type (WT) and knockout (Brk -/-) mice at 6 and 72 hours post irradiation were prepared, analyzed by staining for cleaved caspase 3 and detected with Avidin-FITC (apoptotic cells stain green). Size bar represents 100 µm. (B, C) Histogram (B) and frequency histogram (C) of apoptotic cells per crypt-villus unit. Approximately 100 crypt-villus units per section were scored. Values shown are mean ± S.D. from two sections from at least three different mice per group. (D) Western blot analysis of irradiated ileum. Total cell lysates from distal ileum of wild-type (WT) and knockout (Brk -/-) mice 6 hours after irradiation (8 Gγ) were subjected to Western blotting with anti-cleaved caspase 3 antibodies. Immunoblotting with anti-Brk and anti-β-actin antibodies served as controls for the genotypes and protein loading respectively. Brk deficient mice show increased resistance to radiation-induced apoptosis in the small intestine.
• Fig. 16: Pro-survival signaling in Brk deficient mice. Total cell lysates (TCL) from distal ileum of wild-type (WT) and knockout (Brk -/-) animals at 0, 6 and 72 hours after whole-body γ-irradiation (8 Gγ) were prepared. (A) Lysates from irradiated (6 hours) wild-type and knockout mice were analyzed by Western blotting and immunoprecipitation (IP). Endogenous Akt was immunoprecipitated and subjected to Western blotting with phospho-Akt specific antibodies. IP for IgG served as a control. Equal amounts of Akt were present in each immunoprecipitation as confirmed by immublotting for total Akt. Brk deficient mice exhibit increased phospho-Ser473 Akt. (B) Akt in vitro kinase assays. The lysates analyzed in (A) were subjected to Akt kinase assay. Endogenous Akt was immunoprecipitated with immobilized Akt antibody, and incubated with purified recombinant GSK-3 and ATP in kinase reaction buffer. Reactions were stopped at 0 (control or C) and 30 minutes and subjected to immunoblotting with Phospho-GSK-3β and total GSK-3β antibodies. Increased Akt kinase activity is detected in mice deficient for Brk. (C) Western blot analysis of TCL from untreated and irradiated (6 and 72 hours) wild-type and knockout mice with antibodies against phospho- and total Erk1/2. Expression of Brk and β-actin were examined as controls for genotype and protein loading respectively. Increased phosphorylation of Erk1/2 was observed in Brk deficient mice.
• Fig. 17: Brk protein expression is induced by γ-irradiation. (A) RNase protection assays were performed with total RNA from distal ileum of untreated and irradiated wild-type animals and ³²P-labeled antisense probes specific for Brk and cyclophilin as control. (B) Western blot analysis of untreated and irradiated wild-type mice with lysates from distal ileum and antibodies against Brk and β-actin as a loading control. Increased Brk protein levels were detected in irradiated mice. (C) Immunohistochemical analysis of Brk expression on distal jejunum sections of untreated and irradiated wild-type animals. Immunostaining for IgG served as a control. Brk protein expression is restricted to the villus of untreated mice, whereas it is also detected in the crypt of irradiated mice. Size bar represents 50 µm.
• Fig. 18: Growth properties of stable Rat1A cells. (A) Western blot analysis of stable Rat1A fibroblasts. Total cell lysates of Rat1A cells stably overexpressing the expression vector pLXSN (Vector), wild-type Brk (Brk WT) and constitutively activated Brk (Brk Y-F) were analyzed by immunoblotting with anti-Brk antibodies and anti-β-actin antibodies as a control for protein loading. Rat1A cells do not express endogenous Brk. (B) Cell cycle analysis. Stable Rat1A populations were synchronized and arrested in G₀ by serum starvation for 48 hours. Cells were released into G₁ with the addition of 10% FBS, harvested at 0 and 16 hours post stimulation, and analyzed for cellular DNA content by flow cytometry. (C) Exponential growth curves of the indicated stable Rat1A populations. The total amount of cells per 6 cm plate was counted for 5 consecutive days. Values are means ± S.D. of 2 experiments with at least 3 plates per cell line and time point. No differences in growth and cell cycle were observed in Rat1A fibroblasts stably overexpressing Brk WT or Brk Y-F.
• Fig. 19: Brk sensitizes Rat1A fibroblasts to apoptosis induced by serum starvation or UV-irradiation/serum starvation. Stable Rat1A populations overexpressing vector alone (Vector), wild-type (Brk WT) and activated Brk (Brk Y-F) were plated at equal density and apoptosis was induced by 24 hours serum starvation or by serum starvation and ultraviolet irradiation (50 J/m²). Control cells were grown in 10% FBS. The percentage of cells with condensed chromatin was determined by DAPI staining. (A) Representative photomicrographs of DAPI-stained populations after 24 hours serum starvation. The arrows indicate cells with condensed chromatin (B) Histograms of percentage of apoptotic cells in untreated and serum starved populations. Percentage of apoptotic cells was determined microscopically after staining cells with DAPI. The values are means ± S.D. of three independent experiments. (C) FACS analysis of apoptosis. Untreated and serum starved populations were fixed, stained with propidium iodide (PI) and subjected to FACS analysis. The percentages of cells in sub G1 are indicated. (D) Cells were subjected to UV irradiation and at the time points indicated, chromatin condensation and the percentage of apoptotic cells were determined by DAPI staining. Values represent the average of three independent experiments ± S.D. Cell lines expressing Brk show an earlier onset of apoptosis and undergo apoptosis to a greater extent when compared to control cell lines (vector).
• Fig. 20: Illustration of a putative regulatory role of Brk tyrosine kinase signaling. Brk represses Akt kinase activity, which is required to fully activate β-catenin. Thus, Brk inhibits nuclear-accumulation of β-catenin and transcription of β-catenin target genes in differentiated cells. Wnt signaling is also required but not sufficient to fully activate β-catenin.
• Fig. 21: Brk tumor suppressor qualities. Brk is required for intestinal homeostasis. It is positioned to inhibit cell proliferation in the intestinal epithelium. Loss of Brk results in increased epithelial cell turnover and chronic inflammation in the intestine. In addition, Brk protects the intestine against injury and oncogenic mutations. The Brk tyrosine kinase is required for the induction of intestinal apoptosis following genotoxic stress.