Signal Transduction I

1
Sinyal Transdüksiyonun I Hücreler Arasi Iletisim
1 Sinyal Iletimi 1 Yrd. Doç. Dr. Izzet
YELKOVAN SIVAS- 2005
2
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
3
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
4
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
5
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
6
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
7
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8
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
9
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
10
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
11
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12
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
13
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
14
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
15
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
16
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
17
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
18
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller.
19
External sinyal iletimi Hedef hücrelere sinyal
iletimi (1) Endokrine sinyal iletimi sinyal
molekülleri (hormonlar) endokrin organ
hücrelerince sentezlenirler sentezlendikleri
yerin uzaginda baska bir mikroçevredeki hedef
hücrelere etki ederler. (2) Parakrin sinyal
iletimi sinyal molekülleri ayni mikroçevreyi
paylasan bir hücre veya hücre grubu tarafinda
salinir ve yakin çevredeki diger hücreleri
etkiler (Neurotransmitterler ve
neurohormonlar). (3) Autokrin sinyal iletimi
sinyal molekülleri bir hücre veya hücre grubundan
salinir ve sadece kendileri yanit olustururlar
(bir çok büyüme faktörü). (4) Hücre hücre ve
hücre matriks arasi etkilesimlerde ise karisan
moleküller..
20
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
21
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
22
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
23
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
24
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
25
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
26
Özet
(1) Sinyal üreten hücre tarafindan sinyal
molekülünün sentezlenmesi (2) Sinyal üreten hücre
tarafindan sinyal molekülünün salinmasi. (3)
Sinyal molekülünün hedef hücreye tasinmasi (4)
Sinyalin hedef hücrede özgül reseptör protein
tarafindan tutulmasi (5) Hücre içi sinyal
transdüksiyon yolunu tetiklemesi (6) Hücre
metabolizmasinda veya gen ekspresyonunda
degisiklikler (hücresel yanit). (7) Sinyalin
sönümlenmesi, çogunlukla hücresel yanitin
sonlandirilmasi.
27

• Sinyal transdüksiyonu bilginin, hücre disindan
  sitoplazmaya veya çekirdege tasinmasi için
  gerçeklesen moleküler olaylarin tamamidir.
• Amplifikasyon (zaman ve nitelik) ikinci
  haberciler araciligi ile sinyallerin
  amplifikasyonu (genellikle küçük moleküllerin
  turnoverleri oldukça hizlidir)
• Çoklu kontrol

sinyal
Algilama - Kabul etme
Amplifikasyon
Transduksiyon
Hücresel yanitlar
28

• Hücresel yanitlar özellikle sinyal iletim
  yollarinin (pathway) açilmasina ve sonuçta
  asagidaki hücresel degisikliklere neden olabilir
• hücre döngüsü ilerleyisi
• gen ekspresyonu
• protein trafik
• hücre göçü
• hücre iskeleti mimarisi
• hücresel konumlanma
• metabol,zma
• hücresel kalimlilik

29
Farkli hücreler ayni sinyal molekülüne farkli
yanitlar üretebilirler

• neurotransmitter
• Kalp kas hücresi kontraksiyon gücünde ve
  oraninda azalma
• iskelet kas hücresinde kontraksiyon
• düz kas hücresinde relaksiyon
• tükrük bezi hücresinde salgi salgilama

30
Reseptörler
Hücre reseptorleri (almaçlari) dört farkli
grupta toplamislardir 1- Enzim bagli
reseptörler 2- Enzimli reseptörler 3-
Iyon-kanali bagli reseptörler 4- G-protein bagli
reseptörler
31
Reseptör tipleri
1- Enzim bagli reseptörler e.g. Protein kinase
reseptorleri PKR
Ligand
Membran
Inaktif enzim
32
Reseptör tipleri
1- Enzim bagli reseptörler e.g. Protein kinase
reseptörleri PKR
Ligand
Aktif enzim
33
Reseptör tipleri
2- Enzimli reseptörler e.g. cytokine reseptorler,
büyüme hormonu reseptorleri ve interferonlar
Inaktif enzim
34
Reseptör tipleri
2- Enzimli reseptörler e.g. cytokine reseptorler,
büyüme hormonu reseptorleri ve interferonlar
Aktif enzim
35
Reseptör tipleri
3- Iyon-kanalina bagli reseptörler e.g.
neurotransmitter-geçitli iyon kanallari
Ions
36
Reseptör tipleri
3- Iyon-kanalina bagli reseptörler e.g.
neurotransmitter-geçitli iyon kanallari
37
Reseptör tipleri
3- Iyon-kanalina bagli reseptörler e.g.
neurotransmitter-geçitli iyon kanallari
38
Reseptör tipleri
4- G-protein- bagli reseptörler e.g. epinefrine,
serotonin ve glukagon reseptörleri
enzyme
G protein
39
Reseptör tipleri
4- G-protein- bagli reseptörler e.g. epinefrine,
serotonin ve glukagon reseptörleri
enzyme
40
Reseptör tipleri
4- G-protein- bagli reseptörler e.g. epinefrine,
serotonin ve glukagon reseptörleri
enzyme
Activated G protein
41
Reseptör tipleri
4- G-protein- bagli reseptörler e.g. epinefrine,
serotonin ve glukagon reseptörleri
enzyme
42
Reseptör tipleri
4- G-protein- bagli reseptörler e.g. epinefrine,
serotonin ve glukagon reseptörleri
Activated enzyme
43
Principle
Reversible phosphorylation plays a major role in
signal transduction
Intracellular signaling pathways typically
involve phosphorylation cascades that are
reversibly and tightly controlled by protein
kinases and protein phosphatases.
These are switches in the cell.
44
Kinases Phosphatases
Protein kinases phosphorylate proteins.
Protein
ATP
45
Kinases Phosphatases
Protein kinases phosphorylate proteins. Protein
phosphatases dephosphorylate proteins.
Protein
ATP
46

• There are gt 500 protein kinases and gt 100
  protein tyrosine phosphatases in human.
• Defined (and conserved) in both protein
  sequence and in function.
• Can be predicted by examining the amino acid
  sequences.

47
Assaying protein kinases
Protein kinase Substrate ATP (may be
radioactive ATP, usually 32P-g-ATP) Mg2
Buffer
Methods
48
Phosphorylation
Kinases and phosphatases can be divided
into (a) transmembrane proteins or
intracellular proteins. (b) serine/threonine-speci
fic or tyrosine-specific (but also a class of
dual-specific)
49
Phosphorylation
Tyrosine Phosphorylation
Tyrosine phosphorylation is rare in the cell
(only lt0.1 of total protein phosphorylation),
but these are important in cellular
regulation. The importance of protein tyrosine
kinases (PTK) in normal cellular regulation is
evident from the fact that many of these are
encoded by proto-oncogenes (see Cancers lectures
for how proto-oncogenes are turned into
oncogenes).
50
Phosphorylation
Receptor protein tyrosine kinase
Found in all multicellular organisms (but not
unicellular organisms like yeast). Contain a
protein tyrosine kinase domain in the cytoplasmic
side. Protein kinase activity is stimulated by
binding of ligands to the extracellular side.
Most known ligands for receptor PTKs are
secreted, soluble proteins but membrane-bound
and extracellular matrix-bound ligands can also
activate receptor PTKs.
51
Receptors
(A) Extracellular region
N
- Binds ligand. - Typically several hundred
amino acids in length and is composed of
different patterns of cysteine residues and
various structural sequence motifs, e.g. Ig
(immunoglobulin)-like domains leucine-rich
domains. - Most receptor PTKs are modified by N-
and O-linked glycosylation.
C
52
Receptors
(B) Transmembrane region
N
- All receptor PTKs have a single transmembrane
region (contains hydrophobic amino acids). -
After the transmembrane region, there are a few
basic amino acids.
C
53
Receptors
(C) Cytoplasmic region
N
- Composed of a juxtamembrane region,
C
54
Receptors
(C) Cytoplasmic region
N
- Composed of a juxtamembrane region, followed by
the protein kinase catalytic domain (PTK),
C
55
Receptors
(C) Cytoplasmic region
N
- Composed of a juxtamembrane region, followed by
the protein kinase catalytic domain (PTK), then
followed by a C-terminal region.
C
56
Receptors
(C) Cytoplasmic region
N
- The protein kinase catalytic domain is
conserved in sequence of 250 aa in length
(conservation from 32-95).
C
57
Receptors
(C) Cytoplasmic region
N
- The protein kinase catalytic domain is
conserved in sequence of 250 aa in length
(conservation from 32-95). - The C-terminal
region varies from a few up to 200 aa in length -
most of the tyrosine phosphorylation occurs here.
However, a major tyrosine phosphorylation site
is located in the catalytic domain - its
phosphorylation is required for kinase activation
in many cases.
C
58
Receptors
Receptor protein tyrosine kinase-initiated signal
transduction
Principle (1) Ligand binds to the extracellular
domain (2) Receptor oligomerization (3) tyrosine
autophosphorylation of the receptor subunits
59
Receptors
Receptor protein tyrosine kinase-initiated signal
transduction
Principle (1) Ligand binds to the extracellular
domain (2) Receptor oligomerization (3) tyrosine
autophosphorylation of the receptor subunits
60
Receptors
Receptor protein tyrosine kinase-initiated signal
transduction
Principle (1) Ligand binds to the extracellular
domain (2) Receptor oligomerization (3) tyrosine
autophosphorylation of the receptor subunits
61
Receptors
Autophosphorylation of receptors serves two
purposes (1) activates catalytic activity of
the PTK. (2) changes the conformation of the
receptor that allows it to bind to next
cytoplasmic singaling molecules in the cascade
62
Receptors
(I) Ligand binding induces receptor
oligomerization
Ligands (polypeptides) reach the receptors by
bloodstream via diffusion, or by basement
membrane or extracellular matrix deposition, or
as cell-bound forms on neighboring cells.
63
Receptors
(I) Ligand binding induces receptor
oligomerization
Ligands (polypeptides) reach the receptors by
bloodstream via diffusion, or by basement
membrane or extracellular matrix deposition, or
as cell-bound forms on neighboring cells.
Ligand binding is reversible, specific, and with
high affinity.
64
Receptors
(I) Ligand binding induces receptor
oligomerization
Ligands (polypeptides) reach the receptors by
bloodstream via diffusion, or by basement
membrane or extracellular matrix deposition, or
as cell-bound forms on neighboring cells.
Ligand binding is reversible, specific, and with
high affinity.
65
Receptors
Examples PDGF (platelet-derived growth factor)
PDGF are dimeric - homodimers or heterodimers of
A and B chains.
66
Receptors
Examples PDGF (platelet-derived growth factor)
PDGF are dimeric - homodimers or heterodimers of
A and B chains. PDGF A chain binds only a PDGF
receptor, but B chain binds both a and b
receptors. Hence PDGF-AA induces aa receptor
homodimers, and PDGF-AB induces aa- and ab-
receptor formation.
67
Receptors
Examples PDGF (platelet-derived growth factor)
Different composition of the PDGF appears to have
different cellular responses. Apart from the
ligands, the extracellular domains of the
receptors are also involved in the dimer
formation.
68
Receptors
Examples EGF (epidermal growth factor)
Ligands are monomeric. Ligands induce both
homo- and heterodimers of their receptors. (see
later)
69
Receptors
(II) Tyrosine phosphorylation of receptors
Ligand binding
70
Receptors
(II) Tyrosine phosphorylation of receptors
Ligand binding Receptor oligomerization
71
Receptors
(II) Tyrosine phosphorylation of receptors
Ligand binding Receptor oligomerization
Juxtapositioning of the cytoplasmic domains of
the receptors Conformational changes
Conformation change because of juxtapositioning
72
Receptors
(II) Tyrosine phosphorylation of receptors
The initial phosphorylation (occurs on Tyr857 in
PDGF receptor) represent the major
autophosphorylation site in PTK, is normally
buried in the active site, preventing access of
Mg2-ATP.
Protein kinases are inactive
73
Receptors
(II) Tyrosine phosphorylation of receptors
Receptor dimerization induces conformational
changes that allows Mg2-ATP to bind, and
trans-phosphorylation occurs on the tyrosine
residue in the other receptor of the complex.
Protein kinases activated
Autophosphorylation (trans-phosphorylation)
74
Receptors
(II) Tyrosine phosphorylation of receptors
Other tyrosine residues in the receptors can then
be phosphorylated by the activated receptor PTK.
These phosphorylations serve as molecular
switches to specifically bind cytoplasmic
signaling molecules (see later).
75
Receptors
Summary
76
Turning off or quenching of the receptor
PTK-initiated signaling - dephosphorylation -
receptor internalization (endocytosis, may be
autophosphorylation-mediated) - negative feedback
loop by phosphorylation
77
Turning off or quenching of the receptor
PTK-initiated signaling - dephosphorylation -
receptor internalization (endocytosis, may be
autophosphorylation-mediated) - negative feedback
loop by phosphorylation
Phosphatase
78
Turning off or quenching of the receptor
PTK-initiated signaling - dephosphorylation -
receptor internalization (endocytosis, may be
autophosphorylation-mediated) - negative feedback
loop by phosphorylation
79
Turning off or quenching of the receptor
PTK-initiated signaling - dephosphorylation -
receptor internalization (endocytosis, may be
autophosphorylation-mediated) - negative feedback
loop by phosphorylation
80
Interaction of receptors with cytoplasmic proteins
The next step in PTK-mediated signaling involves
interaction with cytoplasmic proteins that
contain protein-protein interaction modules
CONCEPT Protein modules direct specific
interactions in signal transduction pathways.
Various modules are frequently found in the same
proteins.
81
(I) Src Homology 2 (SH2) domain
SH2 domain recognizes phosphotyrosine-containing
motifs. SH2 conserved regions of 100 aa.
First identified as homology regions between
members of the Src family (see later).
82
SH2
SH2 binds to phosphotyrosine and the immediate
C-terminal residues (3-5) in a sequence-specific
fashion. e.g. the autophosphorylated tyrosine
residue in a receptor PTK binds specifically to
one or more SH2-containing proteins, but may not
bind to other SH2-containing proteins.
83
SH2
Experiment Incubation of different SH2 domains
with degenerate phosphotyrosine-containing
peptide library ? wash out unbound peptides ?
protein sequencing of the bound peptides to
determine binding specificity.
These experiments showed that the consensus
binding sequences for different SH2 domains are
pY-X-Z-X or pY-Z-X-Z (pY phosphotyrosine, Z
specific aa, X any aa).
84
SH2
3-D crystal and NMR structural analysis reveal
that SH2 domain is a folded, globular structure
protubing from the rest of the protein, with N-
and C-terminals close together. The
phosphopeptide binding site is a pocket on the
surface of the structure. Like a plug
(the phosphotyrosine-containing peptide) inserted
into a socket (the SH2 domain) (Cell 72 779
(1993)).
85
(No Transcript)
86
One side of the pocket is lined with conserved
basic aa and binds the phosphotyrosine
87
The other side of the pocket is more variable and
allows specific recognition of the residues at
the C-terminal of the phosphotyrosine.
88
Variations in the nature of the hydrophobic
socket in different SH2 domains allow them to
bind to phosphotyrosine adjacent to different
sequences.
89
SH2
90
SH2
The ability of SH2 to distinguish between
phosphotyrosine and phosphoserine/threonine is
mainly due to a conserved Arg residue in SH2
(Arg175 in Src) (this is actually the only
invariant residue of the SH2). This residue is
buried in the bottom of the binding pocket, and
only the long phosphotyrosine side chain can
achieve the optimal binding.
Experiment Substitution of the Arg175-equivalent
in Abls SH2 domain with lysine (using
site-directed mutagenesis) ? complete loss of
phosphotyrosine binding.
91
Site-directed mutagenesis
To change specific base(s) of a piece of DNA.
Methods
92
Site-directed mutagenesis
Many methods, here are some (I)
Methods
93
Site-directed mutagenesis
(I) Cut with restriction enzymes
Methods
94
Site-directed mutagenesis
(I) Replace with fragment containing the
mutation
Methods
95
Site-directed mutagenesis
(II) PCR with oligonucleotide containing the
mutation
Methods
96
Site-directed mutagenesis
(II) PCR with oligonucleotide containing the
mutation
Methods
97
Site-directed mutagenesis
(II) A second PCR using the first PCR product as
one of the primers
Methods
98
Site-directed mutagenesis
(II) A second PCR using the first PCR product as
one of the primers
Methods
99
SH2
The ability of SH2 to distinguish between
phosphotyrosine and phosphoserine/threonine is
mainly due to a conserved Arg residue in SH2
(Arg175 in Src) (this is actually the only
invariant residue of the SH2). This residue is
buried in the bottom of the binding pocket, and
only the long phosphotyrosine side chain can
achieve the optimal binding.
Experiment Substitution of the Arg175-equivalent
in Abls SH2 domain with lysine (using
site-directed mutagenesis) ? complete loss of
phosphotyrosine binding.
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(II) Src Homology 3 (SH3) domain
SH3 modules bind proline-rich sequences. SH3
domains 60 aa residues long. SH3 binds to
proline-rich peptides of 10 aa long (containing
the sequence X-P-p-X-P X tend to be aliphatic,
p tend to be proline).
101
SH3
Crystal structure and NMR structure of SH3
domains show that they contain elongated binding
clefts, where hydrophobic pockets contact the
polyproline peptide helix - these peptides are
pseudosymmetrical and can potentially bind in
either orientation (Cell 76 933 (1994)).
102
ligand and SH3 domain binding
103
Signal Transduction by the SRC Family
Peyton Rous (1911) discovered that fibrosarcoma
could be transmitted between chickens in
cell-free extracts of the tumor. Subsequently
(1970), the viral oncogene v-src was discovered
that could cause cellular transformation (see
Cancer lectures) . This gave way to the
discovery of proto-oncogenes and much of signal
transduction pathways. As one of the first
proto-oncogene and tyrosine kinase described, SRC
has provided a prototype for understanding signal
transduction involving tyrosine phosphorylation.
But even now, the exact functions of SRC in
normal cells are still unclear.
104
SRC
SRC family of Protein tyrosine kinases 8 known
members of the family SRC, LCK, BLK, HCK,
FGR,YES, LYN, FYN - with 60-75 amino acid
identity between them (outside the unique region
- see below).
105
SRC
Sequences (a) myristylation sequence (b)
unique region (c) SH3 domain (d) SH2
domain (e) catalytic domain (f) regulatory
region
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
106
SRC
Myristylation sequence All SRC family members
are membrane associated - although they do not
have any hydrophobic transmembrane nor membrane
anchor sequences. The membrane association is
due to co-translational addition of the 14-carbon
fatty acid myristic acid to the glycine residue
at position 2 (conserved in the SRC family),
follows the proteolytic removal of the initiator
methionine.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
107
SRC
Myristylation sequence The N-terminal sequences
of SRC are sufficient for myristylation.
Addition of residue 1-7 of SRC to the N-terminal
of pyruvate kinase is sufficient to make the
fusion protein being myristylated. Mutation of
SRC from Gly2 to Ala2 ? no myristylation.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
108
SRC
Myristylation sequence Myristylation is
necessary, but not sufficient, for stable
association of SRC with cell membranes. e.g.
fusion protein of pyruvate kinase with residue
1-7 of SRC can be myristylated, but it is not
associated with membranes.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
109
SRC
Unique region This region is thought to be
involved in the interaction of SRC family with
specific cellular proteins, some of which may be
substrates of SRC protein kinases. This is
the region that differs the most between
different members of the SRC family.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
110
SRC
SH3 and SH2 as discussed above
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
111
SRC
Catalytic domain Catalyze transfer of
g-phosphate from ATP to tyrosine residues on
protein.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
112
SRC
Catalytic domain Catalyze transfer of
g-phosphate from ATP to tyrosine residues on
protein. Autophosphorylation site (Tyr416 in
SRC) enhances kinase activity.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
113
SRC
Regulatory domain The C-terminal 16-19
residues of SRC family contain site of tyrosine
phosphorylation (Tyr527 in SRC) that plays a key
role in the regulation of the activity of SRC.
Phosphorylation of Tyr527 ? inhibition of
kinase activity.
Myristylation
Y
Y
SH3
SH2
Protein kinase
Unique
Membrane
114
SRC
Evidence
The viral oncogenes products of v-src, v-yes,
and v-fgr all are truncated versions of their
normal proto-oncogene that lack the C-terminal
Try527. These viral oncogene products have
higher catalytic activity than the normal
protein.
Kinase activity

Y

SH3
SH2
Protein kinase
Unique
115
SRC
Evidence
Mutation of Try527 in SRC family ? increase
kinase activity
Kinase activity


116
SRC
Evidence
Dephosphorylation of Tyr527 by phosphatase ?
increase kinase activity
Kinase activity

Phosphatase

117
SRC
Tyr527 is not phosphorylated by SRC itself.
- Mutation of SRC in the ATP binding site, render
the enzyme inactive, is still fully
phosphorylated on Tyr527 when expressed in cells.

ATP binding site
K
R
Note No autophosphorylation, but Tyr527 still
phosphorylated
118
SRC
i.e. other kinase(s) is responsible for
phosphorylation of SRC Tyr527. One may be CSK
(for c-SRC kinase or C-terminal SRC kinase).
Evidence - CSK can phosphorylate SRC and related
proteins Tyr527 in vitro and in vivo. - In cell
lines from CSK KO mice (targeted deletion of CSK
genes which die in utero), SRC has increased
kinase activity.
119
SRC
Crystal structure of SRC
120
SRC
Intramolecular interaction
Interacton of SH3 with the proline-rich sequence
present between SH2 and catalytic domain (the
linker region) is not shown here (see previous
slide)
SH3
SH2
Unique
INACTIVE
121
SRC
Y
Y
SH3
SH2
Protein kinase
Unique
ACTIVE
122
SRC
Adapted from Schwartzberg (1998) Oncogene 171463
123
SRC
?
If the above model is correct, how do you expect
the kinase activity will change if the SH2 domain
of SRC is mutated?
124
SRC
Functions of SRC family
Different members of the SRC family have
different patterns of expression. Patterns of
expression provides some indications of the
function.
125
SRC
Functions of SRC family
Potential substrates of SRC
Transformation by activated forms of SRC is
accompanied by increased tyrosine phosphorylation
of a number of cellular proteins, some of which
must be substrates of SRC and are critical for
the oncogenic capability of SRC.
126
SRC
Functions of SRC family
Potential substrates of SRC
(a) Signal transducing proteins - many potential
substrates are identified discuss typical
transfection experiments in signal transduction
research - what are the problems?
(b) Cytoskeletal proteins - on activation, a
portion of SRC become associated with
cytoskeleton. - nonactivated SRC and
nontransforming mutants of v-SRC are not
associated with cytoskeleton. - transformation is
associated with large changes in cytoskeleton
organization (see Transformation lectures).

127
SRC
Functions of SRC family
Src in PDGF signaling
Quiescent cells PDGF ? PDGF binds to PDGF
receptor ? receptor oligomerization and
phosphorylation (see above) ? SRC, YES, and FYN
binds to Tyr579 and Tyr581 in the juxtamembrane
domain of PDGF receptor.
Src
128
SRC
Functions of SRC family
SRC family in heamopoietic signal transduction
First observed that T cell surface
glycoproteins CD4 and CD8 were physically
associated with LCK later on it was shown that
SRC family of PTK are involved in a number of
signal transduction pathways that are initiated
through a diverse group of cell surface
proteins. Cross-linking of CD4 on T cells with
anti-CD4 antibodies ? stimulation of LCK
activity. The role of LCKK-CD4 in T cells
appears to be the augmentation of signals
initiated by T cell receptors.
129
SRC
Functions of SRC family
SRC family in heamopoietic signal transduction
FYN is important in T cell signaling. FYN
co-immunoprecipitated with T cell receptors.
Transgenic mice overexpressing FYN in thymocytes
? T cell receptors became hyper-responsive and
overexpression of an kinase-inactive FYN ?
suppressed T cell receptors response.
Thymocytes from FYN-/- knockout mice did not
response to T cell receptors stimulation.
130
Randy Y.C. Poon Department of Biochemistry Hong
Kong University of Science and Technology Clear
Water Bay Hong Kong bcrandy_at_ust.hk