Lecture

1
Lecture 9. July 13,2001 Cell signaling
Receptor tyrosine kinasesNO and NYC. Chapter 15.
Axiom 9 Multitasking is essential for success
2
The importance of G-proteins
3
The significance of signal transduction in the
nervous system"
4
Peptide signaling
5
How do cells regulate Ca2?Sequester in ER,
bind to proteins!

• Calmodulin binds 4 Ca2 ions
• In most cases, CM Ca2 binds to an effector
• Accordingly, CM Ca2 dissociates from target
• CM/kinase

6
Signaling by Tyrosine Kinases
7
Protein Tyrosine Kinases (PTK)

• 5 classes in the superfamily
• AGC group PKA, PKG, PKC, Rac, G-protein kinases
• CaMK group kinases regulated by Ca2/CaM
• CMGC group cyclin-dependent kinases ERK, MAP,
  Casein kinase
• PTK group conventional protein tyrosine
  kinases Src, Abl, Fak, PDGF, IR
• 5. OPK Other Protein Kinases

8
Tyrosine kinases can be cytosolic or integral
membrane receptors
.
Substrate
Single Membrane spanning Hydrophobic domain.
How many does the GPCR have?
No membrane-spanning domain
9
What are the substrates for TKs?Src, is a
non-receptor tyrosine kinase
Src, is the product of the first proto-oncogene
to be characterized.
Fariba Fana
10
Src and other proteins that have Src-homology
domains can bind to RTKs!
Even though Src is a cytoplasmic Tyrosine Kinase,
Src homology domains (SH)
11
Other proteins have homologies to Src domainsSH
Src-homology regions

• SH1 domain Catalytic domain of the protein
  kinase activity
• SH2 and SH3 domains mediate protein-protein
  interactions in cellular signaling cascades
  very common in proteins outside the Src family.
• SH2 binds peptides with consensus
  (PTyr-Met/Val-X-Met)
• SH3 b-barrel. Interacts with proline-rich
  peptide targets
• SH4 domain myristylation and membrane-localizatio
  n signal

12
The Src homology 2 (SH2) domain has been found in
a number of signal transduction pathways.

• Its primary function is to bind phosphotyrosines
  and in doing so localizing different proteins
  necessary to transmitt the proper function
  Pawson, 1997.
• Elegant experiments using a combinatorial
  approach showed that specificity for a particular
  protein is encoded in the amino acids following
  the phosphotyrosine Songyang, 1993. Monday, April
  20 1998 0826

SH2 Domains
13
SH3

• b-barrel of 5-6 anti-parallel b-strands.
• Binds a polyproline helix
• The loss of binding can lead to a constitutively
  active Src molecule and cancer

Williams, 1998
14
Receptor tyrosine kinasesAll are single
membrane-spanning proteins

• General Relevance
• Tyrosine phosphorylation is frequently an EARLY
  event in signaling.
• Amplification by downstream signaling elements
  greatly amplifies the effects of low levels of
  tyrosine phosphorylation that are most directly
  induced by extracellular triggers. Example PLC
  and PI3K
• Activation of multiple kinases (kinase cascades)
  including ser/thr as well as tyrosine kinases, is
  a frequent consequence of these early events.
  Example MAP Kinase
• There is often cross-talk between tyrosine
  kinase-induced pathways and other, e.g. G
  protein, signaling pathways.

15
You know that Im going to ask you why you should
care about receptor tyrosine kinases!
Activation of receptor tyrosine kinases
ultimately leads to cell division or
differentiation, for example, during embryonic
development. Other functions

• Growth control
• Cell-cell recognition
• Cell cycle control
• Immune responses
• Development
• Differentiation

Are these processes important? What happens if a
check point looses function?
16
Tyrosine Kinases and associated genes and
proteins are implicated in developmental defects
and cancer.

• Excessive activation of receptor tyrosine kinases
  can lead to uncontrolled growth and malignant
  transformation.
• Many defective or viral forms of tyrosine kinases
  and associated proteins are oncogenic
• v-src
• abl
• erbB

17
Classes of Receptor Tyrosine kinases

• EGFreceptor, NEU/HER2,HER3
• Insulin receptor
• PDGF
• FGF
• VEGF
• Eph

18
What makes a RTK active?

• a) Conformational change
• Insulin Receptor Kinase (IRK) Binds insulin
• Autophosphorylates
• Activates substrates including IRS-1 (insulin
  receptor substrate 1) by tyrosine phosphorylation
• b) Dimerization
• PDGF Receptor Binds platelet derived growth
  factor (PDGF)
• Monomeric integral membrane protein
• Autophosphorylates
• Activates enzymes including PI3 kinase,
  Phospholipase Cg and GAP (GTPase activating
  protein) by tyrosine phosphorylation
• 2. EGF Receptor
• Monomeric integral membrane protein
• 3. Eph Receptor family Erythropoietin producing
  hepatocellular carcinoma cell line
• Binds to Ephrins (ligand)
• Role in neurogenesis (neuronal pathfinding)

19
Response of the insulin receptor kinase (IRK) to
ligand binding Fig. 15.20

• Heterotetramer (2a, 2b)
• Insulin binding leads to change in structure
  (different from other RTKs)
• Conformation change activates b-subunit TK
  activity
• b subunit phosphorylates Tyr residues on
  cytoplasmic domains as well as downstream
  substrates (IRS)

20
Three-dimensional structures of the insulin
receptor tyrosine kinase (IRK)

• IRK conformational change upon activation loop
  phosphorylation. The N-terminal lobe of IRK is
  colored white and the C-terminal lobe is colored
  dark grey. The activation loop (green) contains
  autophosphorylation sites Y1158, Y1162 and Y1163,
  and the catalytic loop (orange) contains the
  putative catalytic base, D1132. Also shown are
  the unbound/bound ATP analog and
  tyrosine-containing substrate peptide (pink).
  Hubbard, EMBO J. 16, 5572 (1997)

21
Once Tyr-Phosphorylated, the IRK activity
trigerrs a number of signaling pathways.

• Phosphatidylinositol 3-hydroxy kinase, makes
  PIP2,PIP3
• Grb2, Sos, activates Ras
• Activation of PI-PLC

22
Unlike IRK, most RTKs are present as a monomer in
the resting cell membrane
23
Receptor Tyrosine Kinases
Receptor protein-tyrosine kinases transmit
signals across the plasma membrane, from the cell
exterior to the cytoplasm.

24
Receptor tyrosine kinases
The interaction of the external domain of a
receptor tyrosine kinase with the ligand, often a
growth factor, up-regulates the enzymatic
activity of the intracellular catalytic domain,
which causes tyrosine phosphorylation of
cytoplasmic signaling molecules.
Fig. 15.23
25
Fibroblast Growth Factor Receptor Tyrosine Kinase

• During the metastasis of tumor cells, the
  adhesion between normal cells and cancerous cells
  must be broken. Many molecules involved in this
  process have been identified, and most are
  implicated in either heritable human diseases or
  cancer. For instance, members of the fibroblast
  growth factor receptor (FGFR) family have been
  linked widely to the development of cancer and
  disease.

• Dimeric assembly of 2 FGF2FGFR1 complexes. FGF2
  is colored orange, Ig-like domain 2 of FGFR1 is
  colored green, and Ig-like domain 3 of FGFR1 is
  colored cyan. Plotnikov et al., Cell 98, 641
  (1999)

26

• N-CAM modulates tumor-cell adhesion to matrix by
  inducing FGF-receptor signalingUGO CAVALLARO,
  JOACHIM NIEDERMEYER, MARTIN FUXA, Nature Cell
  Biology 3, 650-657 (July 2001)

27
Eph receptor kinase
Erythropoietin producing hepatocellular carcinoma
cell line

• Putative signaling effectors are in light
  shading.
• Proteins with known SH2 domains are depicted as
  oval shapes

28
RTKs can activate the Ras pathway of cellular
signaling

• Ras is a small G-protein (monomeric 21-kD)
• Mutant Ras proteins are unable to dissociate GTP,
  so they are stuck in the ON or proliferative
  state ras (gene) mutations found in 30 of human
  cancers.
• Do you think mutations in Ras-GAPs can lead to
  disease? Oui!

29
Steps in the activation of Ras by RTKs. Fig.
15.24
Raf is a PK that triggers MAP-K pathway
Raf
SH2 binds RTK, SH3 binds SOS
c-fos, c-jun Cell proliferation
Ras-GEF
30
NO signaling
31
Nitric oxide is a free radical

• It contains an unpaired electron
• .NO
• Role in macrophage killing of pathogens
• NO also acts as a second messenger that causes
  relaxation of smooth muscle

32
Signal transduction pathway mediated by NO and
cGMP.
PDE5
136252
33
Nitric oxide (NO) is a small membrane-permeating
free radical. It is synthesized as needed, since
it cannot be stored in vesicles. Consequently,
regulation of its synthesis is crucial.

• Nitric oxide synthase (NOS) converts arginine to
  NO and citrulline
• NOS is activated by Ca2/calmodulin
• NOS is inactivated by phosphorylation
• NOS is located only in neurons in CNS (2 of all
  cells)

34
Nitric Oxide Synthase Complexed with Dystrophin
and Absent from Skeletal Muscle Sarcolemma In
Duchenne Muscular Dystrophy

• J. E. Brenman , D. S. Chao , H. Xia , K. Aldape ,
  and D. S. Bredt 1
• 1 Department of Physiology, University of
  California, San Francisco School of Medicine
  94143-0444, USA

NOS is a dystrophin-binding protein
35
Pfizer web site

• VIAGRA enables many men with erectile dysfunction
  to respond to sexual stimulation. When a man is
  sexually excited, VIAGRA helps the penis fill
  with enough blood to cause an erection. After sex
  is over, the erection goes away.

36
How does Viagra work?

• Enlivens the male wunder horn with fresh sound?
• Sildenafil inhibits PDE5 (phosphodiesterase 5)
• cGMP build up in the cell
• Enhances the effects of NO
• Not for patients using nitrates (nitroglycerine)

37
Summary

• Be able to outline pathways for
• GPCR (Fig. 15.13)
• IRK activation and downstream effectors
• RTK and activation of Ras
• Compare and contrast activation events mediated
  by Ca2, cAMP and NO.

38
Finito

• For Monday
• Read Chapter 16, the Biology of Cancer