Final: Ch 16 Tyrosine Kinases, Ras/MAPK

Question 1

3 things transcription of genes is influenced by

Answer 1

Chromatin structure

Epigenetic modifications of histones

What TFs and proteins the cell contains

Question 2

4 basic types of signal transduction pathway

Answer 2

Receptor-associated kinase (RTK/cytokine receptor)

Cytosolic kinase (GPCR)

Protein subunit dissociation

Protein cleavage

Question 3

kinases phosphorylate what two types of kinases

Answer 3

tyrosine kinase (cancer)

serine/thr kinase

Question 4

tyrosine kinases phosphorylate __________ as a part of activation

Answer 4

eachother

Question 5

What 2 categories of receptor activate tyrosine kinases?

Answer 5

Receptor tyrosine kinase

Cytokine receptors

Question 6

the target of a kinase is often a…

Answer 6

kinase

Question 7

specifically, tyrosine kinases phosphorylate what?

Answer 7

specific tyrosine residues on target proteins

Autophosphorylate the receptor

Question 8

what do the phosphorylated (by a tyrosine kinase) target proteins do?

Answer 8

activate one or more signaling pathways for cell proliferation, differentiation, survival, and metabolism

Question 9

2 types of receptors that activate tyrosine kinases

Answer 9

receptor tyrosine kinases (RTK) - the tyrosine kinase is an intrinsic part of the receptor’s pp chain (encoded by same gene)

cytokine receptors - receptor and kinase bound tightly together but encoded by different genes

Question 10

for cytokine receptors, the tightly bound ______ is known as a…

Answer 10

kinase, JAK kinase (just another kinase)

Question 11

are tyrosine kinase receptors dimers?

Answer 11

most are monomeric, but ligand binding induces conformational change to form a dimer receptor

ex. insulin receptor is already a dimer

Question 12

2 ways kinases are inactivated

Answer 12

by a phosphatase - remove phosphate from target protein

by endocytosis - internalize the receptor

Question 13

what types of signaling molecule activate RTKs?

Answer 13

soluble or membrane bound peptides or hormones

ex. growth factors EGF, NGF

Question 14

3 domains of a RTK

Answer 14

extracellular ligand-binding domain

1 transmembrane alpha helix

cytosolic domain with protein tyrosine kinase activity

Question 15

activation lip

Answer 15

flexible domain in cytosolic side of RTK

unphosphorylated without ligand binding - blocks kinase activity (or binding of ATP like in the insulin receptor)

after ligand binding, the receptor sometimes and one kinase phosphorylates a tyrosine residue in the activation lip of the other to activate its kinase activity

Question 16

how does the conformational change in the phosphorylated activation lip activate kinase activity?

Answer 16

reduces the Km for ATP or the substrate

Question 17

how does activation of epidermal growth factor receptor by EGF work?

Answer 17

EGF ligand binds and forms an asymmetric kinase dimer

the activator kinase binds the juxtamembrane segment of the receiver kinase

this causes a conformational change that removes the activation lip from the kinase active site of the receiver

the active kinase phosphorylates tyrosine residues in the receptor

Question 18

_ RTSs participate in signaling by the members of the ___ family of signaling molecules

Answer 18

4, EGF

Question 19

in humans, the _ members of the ___ family are…

Answer 19

4, HER (human epidermal growth factor receptor)

HER 1-4

HER1 - homodimerization

HER2- heterodimerization

Question 20

Gene amplification of HER2

Answer 20

Error in DNA replication makes multiple copies of HER2

HER2 makes cells more sensitive to EGF

Question 21

amplification of the HER2 gene

Answer 21

25% of breast cancer - overexpression of HER2 protein in tumor cells

makes the tumor cells sensitive to growth my low levels of any member of the EGF family

Question 22

an effective therapy for HER2 overexpression

Answer 22

monoclonal antibodies for the HER2 protein

Question 23

cytokines

Answer 23

small, secreting signaling molecules that control growth and differentiation of specific cells

ex. interleukins (proliferation and functioning of T cells and Ab producing B cells) –> TNF-alpha

Question 24

prolactin

Answer 24

a cytokine that during pregnancy, causes the epithelial cells lining the mammary gland ducts to differentiate into acinar cells that produce milk

Question 25

interferons

Answer 25

cytokines that are secreted after viral infection act on nearby cells to induce enzymes that make the cells resistant to viral infection

Question 26

many cytokines induce formation of important types of _____ cells

Answer 26

blood ex. erythropoietin (Epo) - triggers production of RBC

Question 27

binding of a cytokine to its receptor activates a tightly bound ___ protein tyrosine kinase

Answer 27

JAK

Question 28

all cytokines evolved from a common ancestral protein and have a similar tertiary structure consisting of...

Answer 28

4 long alpha helices folded together

Question 29

cytokine receptors do not posses _______ enzymatic activity

Answer 29

intrinsic - instead a JAK kinase is tightly bound to the cytosolic domain

Question 30

JAK stands for

Answer 30

just another kinase

Question 31

the 4 members of the JAK kinase family 3 domains...

Answer 31

N-terminal receptor binding domain C-terminal kinase domain (poorly active) middle domain that regulates kinase activity

Question 32

how are JAK kinases activated?

Answer 32

just like in RTKs - ligand binding induces conformational change to form a dimer where the JAKs are close together so they can phosphorylate each other this phosphorylation enhances ATP/substrate affinity to increase kinase activity

Question 33

what purpose do the phosphorylated tyrosine residues serve?

Answer 33

binding sites for proteins that have phosphotyrosine binding domains ex. SH2 domain

Question 34

what determines which phosphotyrosine an SH2 will bind?

Answer 34

variations in the hydrophobic pocket In the SH2 domain

Question 35

phosphotyrosine binding domain (PTB)

Answer 35

found on multidocking proteins Bind phosphorylated tyrosines on the receptor

Question 36

multidocking protein

Answer 36

serve as docking sites for multiple signal transduction proteins ex. IRS-1 (insulin receptor substrate) is phosphorylated by the receptor and the tyrosines (on IRS-1) serve as docking sites for SH2-containing signaling proteins

Question 37

JAK kinases activate ____ transcription factors

Answer 37

STAT TFs

Question 38

3 domains of STAT

Answer 38

N-terminal DNA binding domain SH2 domain that binds phosphotyrosines in a cytokine's cytosolic domain C-terminal domain with a critical tyrosine residue

Question 39

how does JAK-STAT work?

Answer 39

SH2 domain on STAT binds the activated cytokine receptor at a phosphotyrosine JAK kinase phosphorylates STAT's C-terminal tyrosine STAT dissociates from the receptor and dimerizea, exposing a nuclear-localization signal the STAT dimer moves into the nucleus and binds specific enhancers to alter gene expression

Question 40

are the genes available to be activated by any STAT different?

Answer 40

yes in mammary glands, STAT5 induces transcription of genes encoding milk proteins when STAT5 is activated in erythroid progenitor cells, it induces transcription of the bcl-xl gene that prevents apoptosis

Question 41

prolonged treatment of cells with ligand often _____ the number of available cell-surface receptors so there is a less robust response to ligand than at the beginning of treatment

Answer 41

reduces (desensitization)

Question 42

desensitization example HER1/EGF

Answer 42

in absence of EGF, HER1 receptors are long lived (receptor-mediated endocytosis occurs at a slow rate) after EGF binding, endocytosis of HER1 is increased and only a few receptors return to the membrane, most are degraded in lysosomes

Question 43

do HER1 mutants that lack kinase activity undergo accelerated endocytosis in the presence of ligand?

Answer 43

no perhaps b/c activation of kinase activity in normal HER1 induces conformational change that exposes a sorting motif that signals endocytosis

Question 44

can internalized receptors signal from within endosomes before their degradation?

Answer 44

yes evidence: binding signaling proteins like Grb-2 and Sos

Question 45

2 things that can happen to internalized receptors

Answer 45

returned to cell membrane degraded in lysosomes

Question 46

ubiquitination

Answer 46

addition of the small protein ubiquitin to a lysine in a protein signals it for degradation enzyme c-Cbl does this

Question 47

SHP1

Answer 47

phosphatase that negatively regulates signaling from cytokine receptors inactivates JAK protein by binding to active receptor has 2 SH2 domains

Question 48

SHP1 in inactive state

Answer 48

one SH2 domain of SHP binds the catalytic site so it is inactive

Question 49

SHP1 in active state

Answer 49

The blocking SH2 domain binds a phosphotyrosine in the active receptor Catalytic site placed near phosphate in JAK activation lip and removes

Question 50

SOCS proteins

Answer 50

terminate signaling from cytokine receptors by negative regulation SH2 domain binds activated receptor to block binding of other SH2 containing proteins (competitive inhibition)

Question 51

SOCS-1 mechanism

Answer 51

binds critical phosphotyrosine in activation lip of activated JAK2 kinase

Question 52

SOCS box

Answer 52

domain that recruits ubiquitin ligases Ubiquitin is a signal for degradation in proteasomes

Question 53

almost all RTK and cytokine receptors activate which pathway

Answer 53

Ras/MAP

Question 54

Ras protein

Answer 54

small monomeric G protein member of the GTPase superfamily of switch proteins

Question 55

what does activated Ras promote at the membrane

Answer 55

formation of signal transduction complexes containing 3 sequentially acting protein kinases (kinase cascade) The kinase cascade activates MAP kinases

Question 56

what do members of the MAP kinase family do

Answer 56

translocate into the nucleus and phosphorylate many proteins ex. TFs that regulate expression of proteins for the cell cycle

Question 57

Ras activation is accelerated by

Answer 57

guanine nucleotide factor (GEF) binds the Ras*GDP complex to cause dissociation of GDP

Question 58

what deactivates Ras

Answer 58

GTPase activating protein (GAP) catalyzes hydrolysis of GTP to GDP to inactivate Ras (Ras has a low intrinsic hydrolysis rate)

Question 59

does Ras operate downstream of most RTKs and cytokine receptors?

Answer 59

yes

Question 60

the compound eye of the fly is composed of 800 individual eyes called _______

Answer 60

ommatidia each ommatidium consists of 22 cells, 8 of which are photosensitive neurons called retinula or R cells (R1-R8)

Question 61

Sevenless (Sev)

Answer 61

an RTK that regulates development of R7 cell in an ommatidium

Question 62

flies with mutant Sev gene

Answer 62

R7 cell does not form, flies can't see UV light

Question 63

Boss (bride of Sevenless)

Answer 63

protein expressed on the surface of R8 cells - is the ligand for the Sev RTK on the surface of the R7 precursor cell no Boss, no R7 cells

Question 64

fruit fly experiment with Sev + Ras

Answer 64

temperature sensitive Sev mutant formed R7 cells at an intermediate temp 2nd mutation in 1/3 other important proteins in the pathway produced no R7 cells (SH2 adapter, Sos-a GEF, Ras)

Question 65

always active Ras in Sev mutant fly

Answer 65

produces R7 cells even though no Sev proves that activation of Ras is a critical step in intracellular signaling by most if not all RTKs and cytokine receptors

Question 66

RTKs and JAK kinases are linked to Ras by _______ proteins

Answer 66

adapter - serve as a link, not catalytic GRB2 - links activated receptor (SH2) and SoS (SH3) SoS (Son of Sevenless) - a GEF that speeds up GTP binding to Ras

Question 67

SoS (Son of Sevenless)

Answer 67

a guanine nucleotide exchange protein (GEF) that catalyzes conversion of inactive GDP-Ras to active GTP-Ras

Question 68

binding of SoS to inactive Ras causes a conformational change that triggers..

Answer 68

dissociation of GDP so GTP can bind and activate Ras

Question 69

signals pass from activated Ras to a cascade of protein _______, ending with ____ _______

Answer 69

kinases, MAP kinase

Question 70

mitogen

Answer 70

a ligand that stimulates growth and division (cell cycle)

Question 71

MAP kinase (MAPK mitogen activated protein kinase)

Answer 71

cytosolic kinase gets phosphorylated by MEK and activated, then amplifies the signal by phosphorylating nuclear TFs

Question 72

Melanoma

Answer 72

Always active B-Raf stimulates MEK/MAP to make skin cancer

Question 73

Ras/MAPK pathway

Answer 73

active Ras*GTP binds N-terminal domain of Raf kinase hydrolysis of Ras*GTP to Ras*GDP releases active Raf Raf phosphorylates MEK which phosphorylates MAP MAP phosphorylates nuclear TFs

Question 74

mutant Raf kinase without N-terminal regulatory domain

Answer 74

always active and induce cultured cells to proliferate in absence of stimulation by growth factors also tumor cells

Question 75

does MAPK dimerize?

Answer 75

yes, after phosphorylation a conformational change occurs and it dimerizes the dimer is translocated into the nucleus where it phosphorylates TFs

Question 76

what does quiescent mean

Answer 76

non-growing

Question 77

early response genes

Answer 77

induced well before cells enter S phase ex. one of these genes encodes the TF c-Fos

Question 78

c-Fos

Answer 78

TF that induces expression of many genes encoding proteins required for cells to move through the cell cycle

Question 79

most RTKs that find growth factors utilize the MAPK pathway to activate genes encoding proteins like _-__, which propel the cell through the cell cycle

Answer 79

c-Fos (a TF)

Question 80

the enhancer that regulates the c-Fos gene contains a _______ _______ ______

Answer 80

serum response element - an enhanced that binds many TFs

Question 81

MAPK induces transcription of the c-Fos gene by direct activation of which TF, and indirect activation of another TF

Answer 81

direct: ternary complex factor indirect: serum response factor

Question 82

association of phosphorylated ternary complex factor with two molecules of phosphorylated serum response factor forms... I

Answer 82

an active trimeric factor that activates gene transcription

Question 83

_____ transmit signals to MAPK in yeast mating pathways

Answer 83

GPCR G-betagamma triggers a kinase cascade like Ras/MAPK cascade

Question 84

_____ proteins separate multiple MAPK pathways

Answer 84

scaffold allow the kinases of one pathway to interact with one another, but not with kinases from other pathways ex. Ksr (kinase suppressor of Ras) binds MEK and MAPK

Question 85

basic idea of scaffold proteins

Answer 85

coordinates responses by organizing free floating proteins ex. MAPK on scaffold activated in Ras/MAPK pathway MAPK-P can enter nucleus and phosphorylate TFs

Question 86

mitogens up...

Answer 86

cell proliferation cell cycle active in cancer

Question 87

weird note about nutrients for animal cells

Answer 87

90% defined medium, 10% fetal calf serum (growth factors) eliminate 10% = serum starved cells look at signaling pathways activated when it is added back

Question 88

Dominant-negative mutation

Answer 88

A loss of function in cells with wild-type copies Mutant prevents normal protein from functioning