Cellular signal transduction II. TGF-ß, the growth factor which inhibits growth?

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1 Cellular signal transduction II TGF-ß, the growth factor which inhibits growth?

2 Transforming growth factor ß (TGF-ß) Growth factor Stimulation of adhesion independent cell proliferation by tumor cells in serum-free medium/softagar Transforming Transformation of epithelial cells to cells with mesenchymal properties. Epithelial to Mesenchymal Transdifferentiation

3 Transforming growth factor ß (TGF-ß) Growth factor Stimulation of adhesion independent cell proliferation by tumor cells in serum-free medium/softagar Transforming Transformation of epithelial cells to cells with mesenchymal properties. Epithelial to Mesenchymal Transdifferentiation Epithel: Layer of close connected polarized cells Mesenchym: Layer of loose connected, non polarized cells

4 Transforming Growth Factor-ß-Induced Epithelial to Mesenchymal Transdifferentiation Control TGF-ß Bhowmick et al., Mol Cell Biol 12, (2001)

5 Transforming Growth Factor-ß-Induced Epithelial to Mesenchymal Transdifferentiation Link to Cadherins Bhowmick et al., Mol Cell Biol 12, (2001)

6 TGFß a pleiotropic cytokine - 3 isoforms are expressed in mammalian cells (TGFß 1-3) - sequence is highly conserved - TGFß1: expressed in endothelial, hämapoetic and connective tissue cells - TGFß2: expressed in epithelial und neuronal cells - TGFß3: expressed in mesenchymal cells

7 TGF-ß und related proteins TGF-ß-family TGF-ß Activin Nodal BM-family BM2 (bone morphogenic rotein) BM4 BM7

8 Secretion of TGF-ß1 as an inactive complex ß1-LA (80 kda) TGF-ß1 (25 kda) S S S S S S LTB ( kda) LA = Latency-associated protein LTB = Latent TGF-ß-binding protein

9 Secretion of TGF-ß1 as an inactive complex ß1-LA (80 kda) TGF-ß1 (25 kda) S S S S S S LTB ( kda) LA = Latency-associated protein LTB = Latent TGF-ß-binding protein

10 Secretion of TGF-ß1 as an inactive complex ß1-LA (80 kda) TGF-ß1 (25 kda) S S S S S S LTB ( kda) LA = Latency-associated protein LTB = Latent TGF-ß-binding protein

11 Secretion of TGF-ß1 as an inactive complexko ß1-LA (80 kda) TGF-ß1 (25 kda) S S S S S S LTB ( kda) LA = Latency-associated protein LTB = Latent TGF-ß-binding protein Activation of TGFß by proteolytical processes : - lasmin - Metalloproteinases (MMS)

12 TGFß a pleiotropic cytokine - 3 isoforms are expressed in mammalian cells (TGFß 1-3) - sequence is highly conserved - TGFß1: expressed in endothelial, hämapoetic and connective tissue cells - TGFß2: expressed in epithelial und neuronal cells - TGFß3: expressed in mesenchymal cells Signals for the proteolytic activation: - Stimulation of integrin receptors by bindin to the extracellular matrix - damage of vessels and activation of plasmin/thrombospondin

13 TGFß functions - Inhibition of cell proliferation - Induction of cell differentiation Tummorsupressor

14 TGFß funktions - Inhibition of cell proliferation Zellproliferation - Induction of cell differentiation Zelldifferenzierung Tummorsupressor - Supression of immune response

15 TGFß funktions - Inhibition of cell proliferation Zellproliferation - Induction of cell differentiation Zelldifferenzierung Tummorsupressor - Supression of immune response - Stimulation of extracellular matrix synthesis

16 TGFß funktions - Inhibition of cell proliferation Zellproliferation - Induction of cell differentiation Zelldifferenzierung Tummorsupressor - Supression of immune response - Stimulation of extracellualar matrix synthesis - Stimulation of angiogenesis

17 Signal generation by TGF-ß-receptors 3 types of TGFß-receptors were identified in mammalian cells: TGFß-R I: 55 kda TGFß-R II: 75 kda TGFß-R III: kda roteoglycan without signal transduction properties

18 Signal generation by TGF-ß-receptors Ligand bindingdomain TßR-II TßR-I

19 Signal generation by TGF-ß-receptors TßR-II TßR-I Ligand bindingdomain Transmembranedomain

20 Signal generation by TGF-ß-receptors TßR-II TßR-I Ligand bindingdommain Transmembranedomain Kinasedomain

21 Signal generation by TGF-ß-receptors TßR-II TßR-I Ligandbindingdomain Transmembranedomain Kinasedomain TßR I and TßR-II are serine/threonine kinases

22 Signal generation by TGF-ß-receptors TßR-II TßR-I Ligandbindingdomain Transmembranedomain Kinasedomain Constitutive active kinase

23 Signal generation by TGF-ß-receptors TßR-II TGF-ß TßR-I Ligandbindingdomain Transmembranedomain Kinasedomain Kinase activated by phophorylation Constitutive active kinase

24 Signal generation by TGF-ß-receptors TßR-II TGF-ß TßR-I Ligandbindingdomain Transmembranedomain Kinasedomain Kinase activated by phophorylation Constitutive active kinase SGSGSG GS Domäne

25 Signal generation by TGF-ß-receptors TßR-II TGF-ß TßR-I Ligandbindingdomain Transmembranedomain Kinasedomain Kinase activated by phophorylation Constitutive active kinase formation of a receptor/ligand complex

26 Signal generation by TGF-ß-receptors TßR-II TGF-ß TßR-I Ligandbindingdomain Transmembranedomain Kinasedomain Constitutive active kinase Kinase activated by phophorylation hosphorylation of the GS-domain

27 Signal generation by TGF-ß-receptors TßR-II Ligandbindingdomain Transmembranedomain Kinasedomain Constitutive active kinase TGF-ß TßR-I Kinase activated by phophorylation Signal cascade

28 Determination of ligand specificity by combination of different members of the TßR-II- und TßR-I-family TGF-ß Activin/Nodal TßR-II TßR-I ActR-II ActR-IIB ActR-IB

29 Bestimmung der Ligandspezifität durch Kombination unterschiedlicher Mitglieder der TßR-II- und TßR-I-Familien TGF-ß Activin/Nodal TßR-II TßR-I ActR-II ActR-IIB ActR-IB BM2 BM4 BM7 GDF5 BM4 BM7 AMH/MIS BMR-II BMR-IA BMR-IB ActR-II ActR-IIB BMR-IA BMR-IB ActR-II ActR-IIB ALK2 AMHR-II BMR-IB

30 Signal generation by TGF-ß-receptors TßR-II Ligandbindingdomain Transmembranedomain Kinasedomain Constitutive active kinase TGF-ß TßR-I Kinase activated by phophorylation Signal cascade Smad-proteins

31 Structure of Smad-proteins Fusion of Sma: Analogon from C. elegans Mad: Analogon from Drosophila (Mad= Mothers against decapentaplegig ) Decapentaplegic = TGFß-Analogon from Drosophila

32 Structure of Smad-roteins DNA-Binding R-Smads (ß-hairpin ) NLS SXS -COO -

33 Structure of Smad-roteins MH1-domain Linker MH2-Domain DNA-Binding R-Smads (ß-hairpin ) NLS SXS -COO -

34 Structure of Smad-roteins hosphorylation site MH1-domain Linker MH2-Domain DNA-Binding R-Smads (ß-hairpin ) NLS SXS -COO -

35 Structure of Smad-roteins hosphorylation site MH1-domain Linker MH2-Domain R-Smads DNA-Binding (ß-hairpin ) NLS SXS -COO - Co-Smad -COO -

36 Structure of Smad-roteins hosphorylation site MH1-domain Linker MH2-Domain R-Smads DNA-Binding (ß-hairpin ) NLS SXS -COO - Co-Smad -COO - I-Smads H 2 N -COO -

37 Interaction domains of Smad-roteins Co-factor interaction

38 Members of the Smad-family

39 Members of the Smad-family

40 Members of the Smad-family

41 Members of the Smad-family

42 Activation of R-Smads by TßR-I-dependent phosphorylation of the C-terminal domain -NH 2 SXS -COO - TßR-I AT AD SXS -COO -

43 Smad-Activation by the TGF-ß signal cascade TßR-II TGF-ß TßR-I -NH 2 SXS -COO - R-Smad

44 Smad-Activation by the TGF-ß signal cascade TßR-II TGF-ß TßR-I SARA -NH 2 SXS -COO - R-Smad SARA = Smad anchor for receptor activation

45 Smad-Activation by the TGF-ß signal cascade TßR-II TGF-ß TßR-I SARA -NH 2 SXS -COO - R-Smad SARA = Smad anchor for receptor activation arrests R-Smads in the cytosol

46 Smad-Activation by the TGF-ß signal cascade TßR-II TßR-I TGF-ß SARA SXS -COO - -NH 2 R-Smad SARA = Smad anchor for receptor activation facilitates TßR-Smad interaction

47 Smad-Activation by the TGF-ß signal cascade TßR-II TßR-I TGF-ß SARA SXS -COO - -NH 2 R-Smad SXS -COO -

48 Smad-Activation by the TGF-ß signal cascade TßR-II TßR-I TGF-ß SARA SXS -COO - -NH 2 R-Smad SXS -COO - SXS -COO - -COO - SXS -COO - -NH 2 SMAD-4 Co-Smad -COO -

49 Smad-Activation by the TGF-ß signal cascade TßR-II TßR-I TGF-ß SARA SXS -COO - -NH 2 R-Smad SXS -COO - SXS -COO - -COO - SXS -COO - -NH 2 Co-Smad Translocation into the nucleus -COO -

50 Smad-Activation by the TGF-ß signal cascade H 2 N TßR-II TßR-I TGF-ß SARA -COO - X I-Smad SARA -NH 2 SXS -COO - R-Smad SXS -COO - -COO - SXS -COO - X SXS -COO - X -NH 2 Co-Smad Translokation into the nucleus -COO -

51 Smad-dependent activation of transcription SXS-COO - -COO - SXS -COO - 5' 3' C A G A C Transactivation? smad-binding element

52 Smad-dependent activation of transcription SXS-COO - -COO - SXS -COO - 5' 3' C A G A C Transactivation? smad-binding element frequency of the consensus sequence: 1/1024 bp (4 Basen, entanucleotide: 4 5 bp) Sequence is located in nearly every promoter Gene specificity is generated by combonation with other factors

53 Smad-dependent activation of transcription SXS-COO - -COO - SXS -COO - 5' 3' C A G A C Transactivation? smad-binding element frequency of the consensus sequence: 1/1024 bp (4 Basen, entanucleotide: 4 5 bp) Sequence is located in nearly every promoter Gene specificity is generated by combonation with other factors

54 Smad-dependent activation of transcription SXS-COO - -COO - SXS -COO - 5' 3' C A G A C Transactivation? smad-binding element frequency of the consensus sequence: 1/1024 bp (4 Basen, entanucleotide: 4 5 bp) Sequence is located in nearly every promoter Gene specificity is generated by combination with other factors

55 Smad-dependent activation of transcription SXS-COO - -COO - SXS -COO - 5' 3' C A G A C Transactivation? smad-binding element Affinity of smads for the consensus sequence is low

56 Smad-dependent activation of transcription SXS-COO - -COO - SXS -COO - 5' 3' C A G A C Transactivation? smad-binding element Affinity of smads for the consensus sequence is low multiple SBE increase binding affinity, other factors increase stability

57 Smad-dependent activation of transcription DNA-binding cofactors SXS-COO - -COO - SXS -COO - Transactivation 5' C A G A C Smad Binding Element

58 Smad-dependent activation of transcription DNA-binding cofactors SXS-COO - -COO - SXS -COO - Transactivation 5' C A G A C A T C A C A Smad Binding Element Activin Response Element

59 Smad-dependent activation of transcription DNA-binding cofactors SXS-COO - -COO - SXS -COO - Transactivation FAST 5' C A G A C A T C A C A Smad Binding Element Activin Response Element

60 Smad-dependent activation of transcription DNA-binding cofactors SXS-COO - -COO - SXS -COO - Transactivation FAST 5' C A G A C A T C A C A Smad Binding Element Activin Response Element FAST inrease binding of the smad-complexes to the DNA but does not act as a transacivator

61 Smad-dependent activation of transcription DNA-binding cofactors DNA-binding partners of smads are - FAST for Smad2 and Smad3 - OAZ for Smad1, Smad5 and Smad8

62 Smad-dependent activation of transcription DNA-binding cofactors DNA-binding partners of smads are - FAST for Smad2 and Smad3 - OAZ for Smad1, Smad5 and Smad8 DNA-binding partners increase signal specificity - only genes are activated which contains SREs and recognition sites for the cofactors - cofactors are specific for Smads - cofactors are cell specific expressed

63 Smad-dependent regulation of transcription Co-activators or -repressors

64 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - 5' 3' C A G A C Smad Binding Element

65 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - Histon-Acetyl-Transferase 5' 3' C A G A C Smad Binding Element

66 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - Histone Histon-Acetyl-Transferase Histon-Ac 5' 3' C A G A C Smad Binding Element

67 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - Histone Histon-Acetyl-Transferase Histon-Ac 5' 3' C A G A C Smad Binding Element Chromatin loosening Increased transcription

68 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - Histone Histon-Acetyl-Transferase Histon-Ac 5' 3' C A G A C Chromatin loosening Smad Binding Element SXS -COO - -COO - Repressor Increased transcription SXS -COO - 5' 3' C A G A C Smad Binding Element

69 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - Histone Histon-Acetyl-Transferase Histon-Ac 5' 3' C A G A C Chromatin loosening Smad Binding Element SXS -COO - -COO - Repressor Increased transcription SXS -COO - Histon-Deacetylase 5' 3' C A G A C Smad Binding Element

70 Smad-dependent regulation of transcription Co-activators or -repressors SXS -COO - -COO - Coactivator SXS -COO - Histone Histon-Acetyl-Transferase Histon-Ac 5' 3' C A G A C Chromatin loosening Smad Binding Element SXS -COO - -COO - Repressor Increased transcription SXS -COO - Histon-Ac Histon-Deacetylase Histone 5' 3' C A G A C Smad Binding Element Chromatin condensing Decreased transcription

71 Activation of the TGFß-R/SMAD signal cascade leads to a cell cycle arrest in epithelial cells Wy?

72 Cyclin-dependent kinases: Motors and switches of the cell cycle Dr. F. Neuschäfer-Rube

73 CDKs: Motors of the cellcycle How act CDKs as motors of te cell cycle? Which substrates are phophorylated by CDKs during the cell cycle?

74 CDK Substrates: Initiation of the S-hase Fuction of CDKs in initiation of the S-hase

75 CDK Substrates: Initiation of the S-hase Retinoblastom-rotein (Rb) - Key - substrate of the S-hase - nuclear protein, 110 kda NH2- A B -COOH

76 CDK Substrates: Initiation of the S-hase Retinoblastom-rotein (Rb) - Key - substrate of the S-hase - nuclear protein, 110 kda NH2- A B -COOH Binding of transcription factor E2F

77 CDK Substrates: Initiation of the S-hase Retinoblastom-rotein (Rb) - Key - substrate of the S-hase - nuclear protein, 110 kda NH2- A B -COOH Binding of transcription factor E2F E2F: central transcription factor for the induction of S-hase genes

78 CDK Substrates: Initiation of the S-hase Retinoblastom-rotein (Rb) - Key - substrate of the S-hase - nuclear protein, 110 kda NH2- A B -COOH Binding of transcription factor E2F Rb act as a tumor supressor gene

79 CDK Substrates: Initiation of the S-hase Retinoblastom-rotein (Rb) - Key - substrate of the S-hase - nuclear protein, 110 kda NH2- A B -COOH Binding of transcription factor E2F - Rb act as a tumor supressor gene - Rb can be hyperphosphorylated

80 CDK Substrates: Initiation of the S-hase Rb Rb E2F Repression of E2F-controlled genes

81 CDK Substrates: Initiation of the S-hase Cyclin E CDK 2 Rb Rb Rb E2F Repression of E2F-controlled genes E2F Induktion of E2F-controlled genes

82 E2F: Initiator of the S-hase Cyclin E E2F-controlled genes CDK 2 DNA-ol I Rb E2F dnt-synth.

83 E2F: Initiator of the S-hase Cyclin E E2F-controlled genes CDK 2 Cyclin E DNA-ol I Rb E2F dnt-synth. E2F positive autoregulatory feedback loop!

84 E2F: Initiator of the S-hase Cyclin E E2F-controlled genes CDK 2 Cyclin E Rb E2F DNA-ol I dnt-synth. forced" transition of the restriction point positive autoregulatory feedback loop! E2F S-hase

85 Regulation of CDK activity: Inhibitors Cyclin Cyclin inactive CDK CDK inactive Cyclinconcentration AT CDK-Inhibitors CKI Cyclin CDK T160 hosphorylation Dephosphorylation active AT CDK CKI inactive T160 i T14 Y15 Cyclin CDK inactive T160

86 Regulation of CDK activity: Inhibitors Example: CKI p21 Isosteric inhibition of CDK activity by binding in the active centre Cyclin E CDK 2 CKI p21 G1 S-hase

87 Inhibition of cell cycle progression by TGF-ß

88 Inhibition of cell cycle progression by TGF-ß TGF-ß

89 Inhibition of cell cycle progression by TGF-ß TGF-ß p15, p21

90 Inhibition of cell cycle progression by TGF-ß TGF-ß p15, p21 p27 Cyclin/CdK

91 Inhibition of cell cycle progression by TGF-ß TGF-ß p15, p21 p27 Rb/E2F Cyclin/CdK Rb-

92 Inhibition of cell cycle progression by TGF-ß TGF-ß p15, p21 p27 Cyclin/CdK Rb/E2F E2F S-hase Gene Rb-

93 Inhibition of cell cycle progression by TGF-ß TGF-ß myc p15, p21 p27 Cyclin/CdK Rb/E2F E2F S-hase Gene Rb-

94 Regulation of cell cycle by c-myc Myc Cell cycle progression G1 S

95 Target genes of the transctiption factor c-myc (+) Cyclin D increased formation of CyclinD/Cdk4 complexes (+) Cyclin E increased formation of CyclinE/Cdk2 complexes (+) cdc25a-hosphatase activation of CyclinD/Cdk4 complexes (+) p27-sequestration factor activation of CyclinE/Cdk2 complexes

96 Regulation of cell cycle by c-myc Myc Cell cycle progression G1 S

97 Inhibion of cell cycle progression by TGF-ß TGF-ß myc 27-sequestration factor p15, p21 p27 27/SF Cyclin/CdK Rb/E2F E2F S-hase Gene Rb-

98 Role of TGF-ß- signal chains in tumor formation Abolishment of TGF-ß-dependent inhibition of cell cycle progresion by mutations in TßR or Smad will lead to tumor formation

99 Role of TGF-ß- signal chains in tumor formation Abolishment of TGF-ß-dependent inhibition of cell cycle progresion by mutations in TßR or Smad will lead to tumor formation In late states tumor formation is enhanced by TGF-ß secretion via : Immun suppression Epithelial to mesenchymal transdifferenziation enhanced formation of metastasis increased neovascularisation