Lecture 20 (exam 3)

Question 1

Enzyme coupled receptors

Answer 1

are either enzymes or are very closely associated with enzymes

• Many receptors are transmembrane proteins with single membrane-spanning domain.
  This is particularly true for those that regulate growth.
• Substrate binding causes dimerization
• Receptor dimers are enzymatically active (often with protein kinase activity)

Question 2

Enzyme coupled growth factor receptors

Answer 2

are structurally and functionally different from G protein linked receptors

most in this class are protein kinases, some are protein phosphatases or guanylyl cyclase activity

Question 3

Enzyme coupled receptors examples

Answer 3

• Receptor tyrosine kinases (RTKs)
• Tyrosine kinase associated receptors
• Receptor serine/threonine kinases
• receptor guanylyl cyclases
• receptor tyrosine phosphatases

Question 4

Receptor tyrosine kinases (RTKs)

Answer 4

phosphorylate on specific tyrosines (growth factor receptors)

Question 5

Tyrosine kinase-associated receptors

Answer 5

non-covalent association with intracellular tyrosine kinases (cytokine receptors)

Question 6

Receptor serine/threonine kinases

Answer 6

phosphorylate specific serines or threonines ((TGFβ receptors)

Question 7

Receptor guanylyl cyclases

Answer 7

synthesize cGMP

Question 8

Receptor tyrosine phosphatases

Answer 8

remove phosphate from tyrosines

Question 9

RTKs are…

Answer 9

the most abundant type of enzyme-coupled receptor, with about 60 members classified into 20 structural families

Question 10

RTKs regulate

Answer 10

cell proliferation, cell growth, cell differentiation, cell migration, and during during development, cell fate

Question 11

Growth factors

Answer 11

a class of signaling molecules

RTKs were discovered by studying GFs

Question 12

Mechanisms of Dimerization for RTKs

Answer 12

1. The ligand can be a dimer itself and thus bind to the two receptors simultaneously (as above).
2. A monomeric ligand can bind to 2 receptors simultaneously to bring them together.
3. Two ligands can bind independently to 2 receptors to bring them together.
4. Some receptors like the insulin receptor are already dimers, and the ligand just induces a conformational change that activates the kinase activity.

Question 13

RTKs Contain an Intracellular Tyrosine Kinase Domain

Answer 13

• In the inactive or basal state, RTKs are monomers anchored in
  the plasma membrane by a single transmembrane domain
• The intracellular domain of RTKs contains a ligand-activated
  tyrosine kinase domain
• The receptors themselves are the initial substrates of that
  tyrosine kinase activity
• Binding of ligand causes receptor monomers to dimerize in the
  the plasma membrane, which activates the tyrosine kinase activity
  of one or both monomers

Question 14

Dimerization of RTKs

Answer 14

1. Trans phosphorylation = autophosphorylation:
   the activated tyr kinase domains phosphorylate
   each other (‘standard’ mechanism)
2. Conformational changes in
   the domains in response to
   ligand binding ends up having
   one tyr kinase domain
   (activator) activating the other
   the Tyr kinase domain
   (receiver), and the receiver
   then phosphorylates both
   subunits (EGF-R mechanism)

Question 15

Once the Internal Domains of the RTK Are Phosphorylated, They Serve as

Answer 15

Docking sites for signaling proteins

Question 16

Binding of phosphotyrosine

Answer 16

SH2 Domain and PTB domain

Question 17

Binding of Phosphotyrosine: SH2 Domains

Answer 17

The first modular interaction domain was shown to depend on the PTM of the substrate

• ~100 amino acids in length
• Central β-sheet separates two α-helices
• One binding site on each side of βsheet:
  
  • Positive ‘phosphotyrosine pocket’
  • Variable ‘specificity pocket’
• Each binding pocket contributes roughly half of the binding energy
• Phosphorylation increases affinity
  1000x
• Bidentate ionic interaction with ArgβB5

Question 18

Binding of Phosphotyrosine: PTB Domains

Answer 18

Two orthogonal β-sheets form a
sandwich, capped by a single Cterminal α-helix

• Binds preferentially to “NPxpY”
  motif, where x can be any amino
  acid and pY is the phosphotyrosine

Question 19

‘Adaptors’ link-activated RTKs or other

Answer 19

signaling pathways

• adaptors have binding domains that scaffold multiple proteins, bringing them in close proximity
• adaptors have no enzymatic activity

Question 20

Grb2

Answer 20

is an adaptor that binds RTKs

links the MAP kinase cascade to RTKs in some cases

is composed of little more than a central SH2 domain

Question 21

SH3 domains bind

Answer 21

proline-rich regions

contain two binding grooves, each binds one proline and one other (hydrophobic) amino acid

polyproline type II (PPII) helices

Question 22

Polyproline type II (PPII) helices

Answer 22

are common recognition motifs (PxxP sequences)
- rigid, left-handed helix
- three residues per turn
- pseudo symmetry allows N-C or C-N binding

Question 23

RTKs are proto-oncogenes and often become…

Answer 23

oncogenic

The RTK can become mutated so that it dimerizes and becomes
constitutively active without the ligand, or the external domain can be truncated, which also activates the receptor without a ligand.

Anything that results in a constitutively active kinase domain is bad news

Question 24

Mutation of the Her2 and EGF Receptors Leads to

Answer 24

activation without ligand

both receptors bind EGF

Her2/Neu is often mutated or overexpressed in ‘hormone-independent’ breast cancer (BCa)

Question 25

Major pathways activated by RTKs

Answer 25

cell proliferation, cell growth, cell survival

Question 26

RTK Pathways (II): Phospholipase C-y

Answer 26

* Phospholipase C-γ functions the same as PLC-β: cleavage of PIP2 to make two secondary messengers (DAG and IP3). * This allows RTKs to increase cytoplasmic Ca2+ concentration and activate Protein Kinase C

Question 27

Compare/contrast PLC-β and PLC-γ

Answer 27

* Major differences are in binding domains in the c-terminus * PLC-γ has several Tyrosine residues that are phosphorylated by the activated RTK

Question 28

How do you know if something has been phosphorylated?

Answer 28

1. Western blot with phosphotyrosinespecific antibody (or anti-phosphoserine, etc. antibody) 2. Radio-labeled phosphate incorporation 3. Tandem mass spectroscopy (MS/MS) Electron Transfer Dissociation (ETD)

Question 29

3. Tandem mass spectroscopy (MS/MS) Electron Transfer Dissociation (ETD)

Answer 29

is a gentle fragmentation method that does not cleave off the phosphates, and allows the precise identification of phosphorylated residues.

Question 30

MAP kinase cascade

Answer 30

Mitogen activated protein kinase

Question 31

mitogens

Answer 31

signaling molecules that induce cell proliferation such as growth factors, estrogen and testosterone by triggering mitosis

Question 32

Signaling from RTK to Ras

Answer 32

Grb2 recognizes a specific phosphorylated tyrosine on the RTK by means of an SH2 domain and recruits Sos by means of two SH3 domains. Sos stimulates guanine nucleotide exchange, which activates Ras

Question 33

Grb2

Answer 33

is just an adaptor protein, composed of SH2 and SH3 domains - Central SH2 domain binds pTyr on receptor - Flanking SH3 domains bind proline-rich PPII helices on the protein SOS

Question 34

SOS

Answer 34

is a guanine nucleotide exchange factor * Sos is a cytoplasmic protein and recruitment by Grb2 affects its localization to the membrane, which is the only place its target is found * The binding domain of Sos can fold up and autoinhibit the GEF domain when not bound to Grb2 * Sos catalyzes the exchange of GTP for GDP in Ras

Question 35

Ras

Answer 35

is a key component of the MAP kinase cascade All of the Ras Family Members Function as Binary Switches

Question 36

GTPase Activating Proteins (GAPs)

Answer 36

reduce Ras activation by increasing its ability to hydrolyze GTP (~100-fold). Keep about 95% of Ras inactive in the absence of a signaling molecule. Usually called Ras-GAPs.

Question 37

Guanine Exchange Factors (GEFs)

Answer 37

accelerate Ras activation by promoting the exchange of GTP for the bound GDP ex. SOS

Question 38

The Importance of Being Turned Off (MAPK)

Answer 38

* The intrinsic GTPase activity of Ras is weak and only hydrolyzes GTP slowly * GTPase Activating Proteins (GAPs, Ras-GAPs) act to enhance the weak GTPase activity of monomeric G proteins and promote conversion to the inactive state * All oncogenic forms of Ras analyzed from human cancers have mutations that interfere with the ability of Ras to interact with Ras-GAP * These oncogenic forms of Ras are mostly in the GTP-bound (active) state and continually signal to cells to promote cell division

Question 39

The MAP Kinase Cascade Linked to an RTK

Answer 39

Phosphorylation of Myc, Jun, & Fos leads to the transcription of genes involved in cell proliferation

Question 40

GTP-bound Ras interacts directly with the

Answer 40

ser/thr-specific protein kinase Raf (a MAP kinase kinase kinase) to activate it

Question 41

Raf

Answer 41

was first discovered as an oncogene; oncogenic forms of Raf do not require signals from Ras, Ras-GEF or RTK to promote cell division. (~75% of melanomas are caused by Raf)

Question 42

Raf only has one type of catalytic substrate -

Answer 42

the protein kinase Mek (a MAP kinase kinase)

Question 43

Mek

Answer 43

MAP kinase kinase is unusual in that it contains both ser/thr protein kinase activity and tyrosine-protein kinase activity (on separate structural domains)

Question 44

Activated Mek

Answer 44

only has one catalytic substrate family, MAP kinases, most commonly the protein kinase Erk

Question 45

Activation of MAP kinase requires

Answer 45

phosphorylation of a threonine and a tyrosine that are separated by only a single amino acid MAP kinases have many target

Question 46

Activated MAP kinase can act on target proteins in both the cytoplasm and nucleus:

Answer 46

One action of MAP kinase is to activate gene transcription of genes involved in cell proliferation by phosphorylating and activating transcription activators