Lecture #9. July 13,2001 Cell signaling: Receptor tyrosine kinases

NO and NYC. Chapter 15.

Axiom #9: Multitasking is essential for success

The importance of G-proteins

      

    

                 

The Nobel Prize in Physiology or Medicine 1994

"for their discovery of G-proteins and the role of these proteins in signal transduction in cells"

                                                               

Alfred G. Gilman Martin Rodbell

       

USA USA

University of Texas, Southwestern Medical Center Dallas, TX, USA

National Institute of Environmental Health Sciences Research Triangle Park, NC, USA

1941 - 1925 - 1998

The significance of “signal transduction in the nervous system"

      

    

                 

The Nobel Prize in Physiology or Medicine 2000

"signal transduction in the nervous system"

                

               

                

               

                

               Arvid Carlsson Paul

Greengard Eric R Kandel

           

Sweden USA USA

Göteborg University

Göteborg, Sweden

Rockefeller University New York, NY, USA

Columbia University

New York, NY, USA

1923 - 1925 - 1929 -

Peptide signaling   

    

                 

The Nobel Prize in Physiology or Medicine 1999

"for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell"

                               

Günter Blobel

   

USA

Rockefeller University New York, NY, USA and National Institute of Environmental Health Sciences Research Triangle Park, NC, USA

1936 -

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

Signaling by Tyrosine Kinases

Protein Tyrosine Kinases (PTK)5 classes in the superfamily

1. AGC group: PKA, PKG, PKC, Rac, G-protein kinases

2. CaMK group: kinases regulated by Ca2+/CaM

3. CMGC group: cyclin-dependent kinases ERK, MAP, Casein kinase

4. PTK group: conventional protein tyrosine kinases Src, Abl, Fak, PDGF, IR

5. OPK: Other Protein Kinases

Tyrosine kinases can be cytosolic or integral membrane receptors

.

Single Membrane spanning Hydrophobic domain. How many does the GPCR have?

No membrane-spanning domain

Substrate

What are the substrates for TKs?

Src, is a non-receptor tyrosine kinase

Fariba Fana

Src, is the product of the first proto-oncogene to be characterized.

Src and other proteins that have Src-homology domains

can bind to RTKs!

Src homology domains: (SH)

Even though Src is a cytoplasmic Tyrosine Kinase,

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: -barrel. Interacts with proline-rich peptide targets

• SH4 domain: myristylation and membrane-localization signal

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 08:26

SH2 Domains

SH3

-barrel of 5-6 anti-parallel -strands.

Binds a polyproline helix

The loss of binding can lead to a constitutively active Src molecule and cancer

Williams, 1998

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.

You know that I’m 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?

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

Classes of Receptor Tyrosine kinases

1. EGFreceptor, NEU/HER2,HER3

2. Insulin receptor

3. PDGF

4. FGF

5. VEGF

6. Eph

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: 1. PDGF Receptor: Binds platelet derived growth factor (PDGF)

Monomeric integral membrane proteinAutophosphorylates Activates enzymes including PI3 kinase, Phospholipase C and GAP (GTPase activating

protein) by tyrosine phosphorylation

2. EGF ReceptorMonomeric integral membrane protein

3. Eph Receptor family: Erythropoietin producing hepatocellular carcinoma cell lineBinds to Ephrins (ligand)Role in neurogenesis (neuronal pathfinding)

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

• Heterotetramer (2, 2)

• Insulin binding leads to change in structure (different from other RTKs)

• Conformation change activates -subunit TK activity

subunit phosphorylates Tyr residues on cytoplasmic domains as well as downstream substrates (IRS)

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)]

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

Unlike IRK, most RTKs are present as a monomer in the

resting cell membrane

Receptor Tyrosine KinasesReceptor protein-tyrosine kinases transmit signals across the plasma membrane, from the cell exterior to the cytoplasm.

Receptor tyrosine kinasesThe 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

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 FGF2:FGFR1 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)]

• 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)

Eph receptor kinase

• Putative signaling effectors are in light shading.

• Proteins with known SH2 domains are depicted as oval shapes

Erythropoietin producing hepatocellular carcinoma cell line

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!

Steps in the activation of Ras by RTKs. Fig. 15.24

Ras-GEF

Raf

Raf is a PK that triggers MAP-K pathway

SH2 binds RTK, SH3 binds SOS

c-fos, c-junCell proliferation

NO signaling

      

   

                  

The Nobel Prize in Physiology or Medicine 1998

"for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system"

            

                   

            

                   

                 

              Robert F. Furchgott

Louis J. Ignarro

Ferid Murad

           

USA USA USA

SUNY Health Science Center Brooklyn, NY, USA

UCLA School of Medicine Los Angeles, CA, USA

University of Texas, Health Science Center Dallas, TX, USA

1916 - 1941 - 1936 -

      

 

 

    

Nitric oxide is a free radical

• It contains an unpaired electron

• .N=O

• Role in macrophage killing of pathogens

• NO also acts as a second messenger that causes relaxation of smooth muscle

Signal transduction pathway mediated by NO and cGMP.

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PDE5

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)

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

Muscular Dystrophy

NOS is a dystrophin-binding protein

• 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

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.

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)

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.

Finito

• For Monday

• Read Chapter 16, the Biology of Cancer