BIO 402/502 Advanced Cell & Developmental Biology I

1. BIO 402/502 Advanced Cell & Developmental BiologyI Section I: Dr. Berezney Lectures 10/11 Protein Kinase-Linked Receptors

2. Enzyme-linked Receptors • These receptors are commonly involved in growth, proliferation, differentiation, or survival. Because of this, their ligands are collectively called growth factors. • Growth factors usually act locally in very limited amounts (10-9 to 10-11M). • The effects of enzyme-linked receptors typically are slow requiring the expression of new genes • One exception is growth factor regulation of the cytoskeleton, which is rapid and can cause changes in cell shape or migration within seconds to minutes. • Defects in enzyme-linked receptors (because these enzymes are involved in proliferation, survival, and differentiation) frequently lead to cancer. In fact, most known cancer-causing mutations are in proteins involved in enzyme-linked receptor signaling pathways. • This lecture will focus on: • 1. Receptor tyrosine kinases • 2. Tyrosine kinase-associated receptors • 3. Receptor serine/threoninekinases

3. Structure of Receptor Tyrosine Kinases (RTKs) Extracellular domain Binds to the ligand (growth factor) Is highly divergent between RTKs Transmembrane domain All RTKs transverse the membrane only once Is highly divergent between RTKs Y Intracellular domain: All RTKs have a tyrosine kinase (TK) domain The TK domain is highly conserved among all RTKS RTKs all contain tyrosine residues outside of the kinase domain that are critical for receptor function Y Y TK Y Y Y

4. Receptor Tyrosine Kinases • Some kinase domains have “kinase insert regions” of unknown function. • The function of most of the conserved extracellular regions are not known (they do not bind to ligands). • They can be divided into 16 subclasses based on their structure. Seven families are shown here.

5. Activation of an RTK • When not activated, RTKs are monomeric (almost all). • Binding of ligand to the extracellular domain causes dimerization (also called cross-linking) of two RTK molecules. This can be accomplished by: • The ligand is a monomer and causes a conformational change in the receptor that exposes a binding domain in the RTK that promotes its dimerization (EGF). • The ligand is a homodimer that thus automatically dimerizes the receptor (PDGF, NGF). • The receptor already exists as a dimer (insulin). • The ligand is clustered together by binding to extracellular sulfate proteoglycans (FGFs). • The RTK undergoes intermolecular auto or trans or trans-autophosphorylationon tyrosines. This further stimulates the kinase activity of the RTK.

6. Ephrins and Eph Receptors • Ephrins are “repulsive” signals that stop neurons from growing into the incorrect areas of the brain. • 2. Ephrins are unusual ligands in that many of them are transmembrane proteins. • Even though ephrins are monomeric, they are clustered in the plasma membrane of the cell that • presents them. This allows for the cross-linking of their receptors, the Eph proteins. • Ephrins and Eph receptors carry out bidirectional signaling: when ephrin binds to the Eph receptor, the ephrin undergoes conformational changes that promotes a signal into the cell that presented the ephrin. • This bidirectional signaling is critical for development of the nervous system as a means of stopping the mixing of neurons from two different, neighboring regions.

7. Dominant-Negative Inhibitors of RTKs • Normally, upon ligand binding, the RTK dimerizes and trans-autophosphorylates. This phosphorylation further activates the kinase. • If a receptor mutant is expressed with an inactive kinase (kinase-dead), it will dimerize but not trans-autophosphorylate. • This will block activation of the receptor. • For this to work, the kinase-dead version of the receptor must be expressed at levels higher than the normal receptor. This type of inhibition is called dominant-negative inhibition. Fig 15-53, 5th Edition

8. RTKs Act as Platforms for Assembly of Signaling Complexes (Docking) • Several tyrosines are phosphorylated in RTKs. These phosphorylated tyrosine residues act as docking sites for signaling molecules. • These docking molecules can be enzymes, or they can be adaptor molecules that further recruit additional proteins to this newly-formed signaling complex. • Often, the molecules that bind to the phosphorylated RTK become substrates and are also tyrosine phosphorylated. • Phosphorylation of docking proteins generates additional binding sites for other proteins, allowing more signaling proteins to associate with the receptor complex.

9. The PDGF Receptor Signaling Complex • The PDGF receptor is phosphorylated on several tyrosines (5 are shown here) • 2. All three of these bound substrates are enzymes or regulators of enzymes. • 3. Phospholipase C-g, like PLC-b, produces DAG and IP3. It is activated when it is phosphorylated on a critical tyrosine residue by the PDGF receptor. • 4. Binding to phosphorylated tyrosine residues requires a specific recognition domain in the protein.

10. Fig 15-53, 5th Edition • Src Homology Region-2 (SH2) Domains • Binding to phosphotyrosine residues requires either a SH2 domain or, less frequently, a phosphotyrosine binding (PTB)domain. • 2. SH2 domains have two binding sites: one for the phosphotyrosine, and a second that binds to a specific amino acid residue (the specificity pocket). • 3. In the example above, this SH2 domain binds to isoleucine only when it is three amino acids “downstream” of the phosphotyrosine. Therefore, there is a great deal of specificity built into the SH2 domain binding. • 4. SH2 domains are modular, and can be “plugged into” proteins to allow them to associate with RTKs, or other tyrosine-phosphorylated proteins.

11. The monomeric GTPase: Ras • 1. Ras is a GTPase that acts as a monomer. • Ras belongs to a large family of monomeric GTPases that function in receptor signaling, regulation of the cytoskeleton, and in vesicular transport. • Ras is a molecular switch that is turned on and off by the exchange of GDP for GTP. • This switching is controlled by two classes of proteins. • GAPs enhance the GTPase activity of Ras, thereby shutting Ras off once it converts its GTP to GDP. • Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP to GTP, thereby activating Ras. • Ras promotes proliferation and differentiation pathways activated by RTKs. • Dominant-negative forms of Ras block these two pathways. Conversely, hyperactive forms of Ras promote proliferation without any extracellular signal. • Ras is a major mediator of cancer. ~30% of human cancers have hyperactive forms of Ras.

12. See Fig 15-58, 5th Edition Activation of Ras by RTKs 1. An adaptor protein such as Grb2, associates with activated RTK via its SH2 domain 2. Grb2 recruits a Ras GEF to the receptor complex. 3. The recruitment of the Ras GEF to the plasma membrane brings it within close proximity to Ras, where it exchanges GDP for GTP in Ras and activates Ras. 4. Ras is generally activated by GEFs, rather than being activated by inhibition of GAPs. 5. Ras is a critical mediator of RTK signaling, and Ras is responsible for the activation of multiple “downstream” signaling pathways.

13. PIP3 PIP2 Activity of Phosphoinositide 3-kinases • Phosphoinositide 3-kinases (PI 3-kinases) phosphorylate the 3 hydroxyl position in the inositol ring of phosphatidylinositol lipids. • 2. PI 3-kinase is the major signaling molecule involved in cell growth. If dividing cells did not grow as well, they would get smaller and smaller until they died. PI 3-kinase promotes the cell growth necessary to allow cells to divide and mature. • PI 3-kinases are activated by RTKs, but are also activated by GPCRs and other types of receptors. • 4. PI 3-kinases also promote the survival of multiple cell types by inhibiting apoptosis.

14. Generation of Inositol Phospholipid Docking Sites by PI 3 Kinase • Inositol lipids phosphorylated on the 3-OH position are second messengers. • PI(3,4)P2, and PI(3,4,5)P3 are the two products that are involved in the promotion of cell growth and survival. • PI 3-kinases have two subunits, a regulatory subunit an a catalytic subunit. • PI 3-kinases can phosphorylate the 3-OH position in PI, PI(4)P, and PI(4,5)P2. This yields PI(3)P1, PI(3,4)P2, and PI(3,4,5)P3.

15. PI 3-kinase Promotes Cell Survival via Activation of PKB/Akt • The activation of an RTK leads to the activation of PI 3-kinase. In this example, PI 3-kinase binds directly to the RTK, is phosphoryated and activated by it. • The production of phosphoinositol lipids in the PM leads to the recruitment (to the PM) of proteins that have Pleckstrin homology (PH)domains. Some PH domains bind specifically to PI(3,4)P2 and PI(3,4,5)P3, which translocates them to the membrane. • Two of these proteins are protein kinase B (PKB/Akt) and PDK1 (phosphoinositide – dependent protein kinase I).When PKB(Akt) binds to PI(3,4,5)P3it undergoes a conformational change that allows it to be phosphorylated by PDK1 and mTOR • 4. Phosphorylation of PKB (Akt) by PDK1 and mTor leads to activation and release from the plasma membrane where it can phosphorylate other intracellular signaling proteins. • 5. One substrate of PKB(Akt) is the pro-apoptotic protein BAD. When PKB phosphorylates BAD it inactivates it, thus blocking apoptosis and allowing the cell to survive.

16. c.t. c.t. • Signaling Networks • Signal diversity: most receptors activate multiple intracellular signaling pathways. This is one mechanism that allows a single receptor to have multiple effects on a cell. • Cross-talk: most signaling pathways contain points where they can be regulated by other signaling pathways. This enables one signaling pathway to “branch out” and affect other target proteins. • Redundancy: many pathways are activated by more than one receptor. Thus, different signal molecules can have similar effects on a cell. • Signal amplification: multiple steps in an intracellular signaling pathway allows for the signal to be amplified along the way. Thus, very small amounts of a ligand can have dramatic effects.

17. Non-Receptor Protein Tyrosine Kinases (nrPTKs) • 1. These exist in the cytoplasm as soluble proteins, or they are membrane associated (typically via a lipid modification). • The nrPTKs can be divided into at least 10 main families based on amino acid similarities • These kinases are often used by receptors with no intrinsic tyrosine kinase activity. • Receptors that recruit and activate nrPTKs initiate signaling pathways that are similar to the RTKs. • nrPTKs induce the assembly of substantial signaling complexes, similar to the activity of RTKs. • Receptors Without Intrinsic Tyrosine Kinase Activity • That Utilize nrPTKs • Receptors that rely on cytoplasmic TKs consists of a large and diverse group of receptors. • Some rely on the Srcfamily of kinases, the largest subfamily of cytoplasmic TKs. Example of receptors that require Srckinases are the T cell receptors. • 3. A number of receptors rely on the Janus family (JAK) of tyrosine kinases. Examples of receptors that use JAKs are cytokine receptors and some hormone receptors, such as growth hormone.

18. JAK-STAT Pathway Activated by Cytokines • Receptor oligomerization leads to activation of the associated JAK kinases. • The activated JAKs phosphorylate the receptor, which provides a docking site for STAT proteins. • The STATs are phosphorylated by JAKs, which causes their dissociation from the receptor and their dimerization. • The dimerized STATs translocate into the nucleus and promote gene transcription.

19. Example of Ser-Thr Kinase Receptors [STKR] TGFb Family • A large family of secreted factors. • 2. Have very diverse functions, often in development. • 3. All are characterized by a “cysteine knot” structure that accounts for their stable dimeric form. • 4 Most members activate Ser-Thr kinase receptors [STKR].

20. tetrameric receptor complex Smad-dependent Signaling Pathway Activated by TGFβ TGFβ receptors  SMADs  Nucleus  TGFβ Response Element in Target Gene