The Retinoblastoma Protein

1
The Retinoblastoma Protein

• J. William Harbour, MD
• Washington University, St. Louis

2
Clinical background

• 1 per 20,000 live births
• 200 cases per year in the U.S.
• Most cases present by 2 years old
• Survival gt95 in developed countries

3
Clinical presentation

• Leukocoria (80)
• Strabismus (20)

4
Growth patterns
5
Treatment
6
Clinical genetics

• Hereditary RB
• Non-hereditary RB

7
Non-hereditary RB

• 60 of patients
• Avg. age at diagnosis 12-24 months
• Unilateral tumor
• No second tumor risk

8
Hereditary RB

• 40 of patients
• Avg. age at diagnosis 6-12 months

9
Hereditary RB

• Familial cancer syndrome
• Bilateral eye tumors
• Autosomal dominant
• Second primary tumors
• Midline intracranial neuroectodermal tumors
• Osteosarcoma
• Soft tissue sarcomas
• Melanomas
• Epithelial cancers

10
Historical background
11
1951State of RB genetics
12
1971Knudson hypothesis
13
1971Knudson hypothesis
No germline mutation
10-x
10-x

• Unifocal, unilateral
• Nonhereditary
• No second cancers

10x
Germline mutation
10-x

• Multifocal, bilateral
• Autosomal dominant
• Frequent second cancers

10x
14
1980-83RB linked to 13q14
15
1983-86Recessive nature of RB confirmed
16
1985Loss of heterozyosity analysis

• Cavenee et al., Science 1985

17
1986Cloning of esterase D
18
1986-87Discovery of RB gene
19
Fung et al. 1987
HindIII genomic fragments...
Southern blot...
20
Germline RB gene mutations

• Mutations present in all(?) RBs
• No hotspots
• Most mutations inactivate protein

21
1988RB mutations in other cancers
...but limited spectrum of cancers
22
Early insights into Rb function
23
1987Rb is a nuclear phosphoprotein
24
1988-89 Rb binds to viral oncoproteins
25
1991-92Rb interacts with E2F/DP1 heterodimers
26
1989-92 Role of Rb in cell cycle

• Rb phosphorylation is cell cycle dependent

27
1990-92Role of Rb in cell cycle

• Rb is hyperphosphorylated in cycling cells and
  hypophosphorylated in quiescent cells

28
1989-96 Role of Rb in differentiation

• Rb is hypophosphorylated in differentiated cells

29
1995-96Role of Rb in apoptosis

• Rb inhibits E2F-induced apoptosis

30
1993Rb inhibits E2F transactivation
31
Model of Rb function early 1990's

• Rb masks E2F transactivation domain

cell cycle
differentiation
apoposis
32
Model of Rb function early 1990's

• Rb masks E2F transactivation domain

cell cycle
differentiation
apoposis
cell cycle
differentiation
apoposis
33
1992-95Intrinsic or "active" Rb repressor
activity
...fusion of Rb to DNA binding domains
34
E2F recruits Rb to "actively" repress
transcription
35
1999-2000Active repression regulates G1 arrest
36
How does Rb "actively" repress transcription?
37
How does Rb "actively" repress transcription?

• Recruitment of chromatin remodeling enzymes to
  E2F-responsive promoters
• Histone deacetylases
• SWI/SNF complexes
• DNA methyltransferases
• Polycomb complexes
• Histone methyltransferases

38
1998Rb recruits HDAC
39
2000Rb recruits SWI/SNF complexes
40
2000Rb recruits DNA methyltransferases
41
2001-02Rb recruits Polycomb complexes
42
2001-03Rb recruits histone methylases
...irreversible cell cycle arrest?
43
Rb is involved in a complex array of
multiprotein repressor modules
44
Multiple mechanisms of Rb transcriptional
repression

• Simple masking of E2F transactivation domain
• e.g. apoptosis?

45
Multiple mechanisms of Rb transcriptional
repression

• Reversible active repression
• e.g. cell cycle

46
Multiple mechanisms of Rb transcriptional
repression

• Irreversible gene silencing
• e.g. senescence, differentiation

47
Additional complexity

• Other pocket proteins
• p107 p130
• Multiple E2Fs
• E2F1-3 bind Rb
• E2F4 5 bind p107, p130

Stevaux Dyson, 2002
48
Rb protein structure function
49
Rb protein structure

• 928 amino acids
• 16 potential CDK phosphorylation sites

Repression
E2F binding
MDM2
LXCXE proteins
c-Abl
50
Rb-E7 crystal structure

• LXCXE binding site
• located in B box, which requires interaction with
  A box for active conformation

51
Rb-E2F crystal structure

• E2F binding site
• Distinct from LXCXE site
• Located in cleft at A-B interface
• Contains residues from both A and B boxes

52
How does phosphorylation regulate Rb?
53
Phosphorylation of Rb

• 16 potential cdk phosphoacceptor sites

54
Phosphorylation of Rb

• Rb is phosphorylated by cdk4/6-cyclin D and
  cdk2-cyclin E in G1/S

55
Early model for Rb phosphoregulation
ACTIVE
INACTIVE
56
Early model for Rb phosphoregulation
cdk-cyclins
ACTIVE
INACTIVE
57
Early model for Rb phosphoregulation
cdk-cyclins
ACTIVE
INACTIVE
phosphatases
58
Early model for Rb phosphoregulation
cdk-cyclins
G1
S
ACTIVE
INACTIVE
G1/0
M
phosphatases
59
Problems with simple "on-off" model

• Why multiple cdk-cyclins?
• Why don't cells undergo apoptosis during normal
  cell division if E2F is released?
• Rb-E2F complexes are present beyond G1
• Difference in phenotype between Rb-/- and p16-/-
  cells

60
p16-Rb pathway

• p16 requires Rb to inhibit cell growth

61
p16-Rb pathway

• p16 inhibits cdk4, preventing Rb
  hyperphosphorylation

62
p16-Rb pathway

• p16 mutated or methylated in many cancers
• p16 Rb mutations mutually exclusive
• Suggests linear pathway

63
But the tumor phenotypes are very different

• Rb-/- tumors
• High rate of apoptosis
• Rapid rate of proliferation
• Sensitivity to chemotherapy
• p16-/- tumors
• Low rate of apoptosis
• Slow rate of proliferation
• Resistance to chemotherapy

64
Rb phosphorylation is more complex
65
Sites in C-terminus regulate LXCXE binding
c-Abl
TAg
66
Sites in C-terminus regulate LXCXE binding
E2F
67
Progressive phosphorylation of Rb
68
Progressive phosphorylation of Rb

• Initially, the C-terminus is phosphorylated by
  cdk4-cyclin D
• This triggers an intramolecular interaction
  between phosphorylated C-terminus and lysine
  patch in the B box
• This intramolecular alteration displaces HDAC or
  other LXCXE binding proteins

69
Progressive phosphorylation of Rb

• The C-terminus recruits cdk2-cyclin E to the A-B
  interface, where it can now gain access to
  phosphorylate Ser567
• This occurs only when cdk2 activity is high
• Ser567 phosphorylation disrupts A-B interface
  and, consequently, the Rb-E2F interaction

70
Progressive phosphorylation model supported by
crystal structure
71
2003Stepwise Rb phosphorylation in living cells
Cell cycle Differentiation
Apoptosis
72
Model for stepwise phosphorylation and
inactivation of Rb
P
HDAC
P
P
P
Rb
Rb
cdk4
cyclin D
E2F
73
Model for stepwise phosphorylation and
inactivation of Rb
p16
P
HDAC
P
P
P
Rb
Rb
cdk4
cyclin D
E2F
Cell cycle Differentiation
74
Model for stepwise phosphorylation and
inactivation of Rb
p16
P
HDAC
P
P
P
HIGH cdk2
Rb
Rb
cdk4
cyclin D
(or Rb mutation)
E2F
Cell cycle Differentiation
75
Model for stepwise phosphorylation and
inactivation of Rb
Rb Degradation
p16
P
HDAC
P
P
P
P
P
P
HIGH cdk2
P
Rb
Rb
Rb
cdk4
cyclin D
(or Rb mutation)
E2F
567
P
Cell cycle Differentiation
Increased apoptosis
E2F
76
Model for stepwise phosphorylation and
inactivation of Rb
Rb Degradation
p16
P
HDAC
P
P
P
P
P
P
HIGH cdk2
P
Rb
Rb
Rb
cdk4
cyclin D
(or Rb mutation)
E2F
567
P
Cell cycle Differentiation
Increased apoptosis
E2F
Deregulated by p16 inactivation
77
Model for stepwise phosphorylation and
inactivation of Rb
Rb Degradation
p16
P
HDAC
P
P
P
P
P
P
HIGH cdk2
P
Rb
Rb
Rb
cdk4
cyclin D
(or Rb mutation)
E2F
567
P
Cell cycle Differentiation
Increased apoptosis
E2F
Deregulated by p16 inactivation
Deregulated by (complete) Rb inactivation
78
Rb in mouse models
79
Rb/-

• Pituitary adenomas
• Medullary thyroid carcinomas
• No retinoblastomas

80
Rb -/-

• Die at E12-15
• Defects in neurogenesis and erythropoeisis
• Massive apoptosis
• Lack of differentiation
• Excessive proliferation
• No retinal (or other) tumors

81
Partial rescue of Rb-/- phenotype

• E2F-/-
• p53-/-
• Id2-/-

82
Chimeric mice

• Rb-/- phenotype is largely rescued in chimeric
  mice containing Rb/ cells
• Pituitary tumors
• No retinal tumors
• Many effects of Rb-/- are not cell autonomous
• Maandag et al., EMBO J 1994 134260

83
Conditional Rb knockout

• Cre-LoxP system
• Conditional Rb knockout in CNS PNS
• Rescue of CNS apoptosis (but not PNS)
• CNS but not PNS was hypoxic
• Extra-embryonic effects on phenotype?
• MacPherson et al., Mol Cell Biol 2003 231044

84
2003Extra-embryonic effects of Rb

• Do placental changes due to Rb loss account for
  developmental phenotype in Rb-/- mice?

85
2003Extra-embryonic effects of Rb

• Rb-/- mice supplied normal placenta
• Tetraploid aggregation
• Conditional knockout

86
2003Extra-embryonic effects of Rb

• Normal placenta largely rescued Rb-/- phenotype

87
2003Extra-embryonic effects of Rb

• Authors' conclusion...

88
Conclusions

• Rb
• transcriptional repressor
• E2F is its major binding partner for access to
  promoters
• inhibits gene expression temporarily or
  permanently depending on co-repressors
• HDACs, SWI/SNF, DNA methyltransferases, histone
  methylases, and Polycomb complexes

89
Conclusions

• Rb
• part of complex network containing 3 pocket
  proteins and probably at least 8 E2Fs
• inhibits cell cycle and apoptosis
• promotes differentiation and senescence

90
Conclusions

• Rb
• probably does not play intrinsic role in cell
  cycle regulation
• temporarily or permanently arrest cells in
  response to internal or external cues as part of
  programs regulating cellular differentiation and
  senescence