Final: Ch 16 Tyrosine Kinases, Ras/MAPK Flashcards
3 things transcription of genes is influenced by
Chromatin structure
Epigenetic modifications of histones
What TFs and proteins the cell contains
4 basic types of signal transduction pathway
Receptor-associated kinase (RTK/cytokine receptor)
Cytosolic kinase (GPCR)
Protein subunit dissociation
Protein cleavage
kinases phosphorylate what two types of kinases
tyrosine kinase (cancer)
serine/thr kinase
tyrosine kinases phosphorylate __________ as a part of activation
eachother
What 2 categories of receptor activate tyrosine kinases?
Receptor tyrosine kinase
Cytokine receptors
the target of a kinase is often a…
kinase
specifically, tyrosine kinases phosphorylate what?
specific tyrosine residues on target proteins
Autophosphorylate the receptor
what do the phosphorylated (by a tyrosine kinase) target proteins do?
activate one or more signaling pathways for cell proliferation, differentiation, survival, and metabolism
2 types of receptors that activate tyrosine kinases
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
for cytokine receptors, the tightly bound ______ is known as a…
kinase, JAK kinase (just another kinase)
are tyrosine kinase receptors dimers?
most are monomeric, but ligand binding induces conformational change to form a dimer receptor
ex. insulin receptor is already a dimer
2 ways kinases are inactivated
by a phosphatase - remove phosphate from target protein
by endocytosis - internalize the receptor
what types of signaling molecule activate RTKs?
soluble or membrane bound peptides or hormones
ex. growth factors EGF, NGF
3 domains of a RTK
extracellular ligand-binding domain
1 transmembrane alpha helix
cytosolic domain with protein tyrosine kinase activity
activation lip
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
how does the conformational change in the phosphorylated activation lip activate kinase activity?
reduces the Km for ATP or the substrate
how does activation of epidermal growth factor receptor by EGF work?
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
_ RTSs participate in signaling by the members of the ___ family of signaling molecules
4, EGF
in humans, the _ members of the ___ family are…
4, HER (human epidermal growth factor receptor)
HER 1-4
HER1 - homodimerization
HER2- heterodimerization
Gene amplification of HER2
Error in DNA replication makes multiple copies of HER2
HER2 makes cells more sensitive to EGF
amplification of the HER2 gene
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
an effective therapy for HER2 overexpression
monoclonal antibodies for the HER2 protein
cytokines
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
prolactin
a cytokine that during pregnancy, causes the epithelial cells lining the mammary gland ducts to differentiate into acinar cells that produce milk
interferons
cytokines that are secreted after viral infection
act on nearby cells to induce enzymes that make the cells resistant to viral infection
many cytokines induce formation of important types of _____ cells
blood
ex. erythropoietin (Epo) - triggers production of RBC
binding of a cytokine to its receptor activates a tightly bound ___ protein tyrosine kinase
JAK
all cytokines evolved from a common ancestral protein and have a similar tertiary structure consisting of…
4 long alpha helices folded together
cytokine receptors do not posses _______ enzymatic activity
intrinsic - instead a JAK kinase is tightly bound to the cytosolic domain
JAK stands for
just another kinase
the 4 members of the JAK kinase family 3 domains…
N-terminal receptor binding domain
C-terminal kinase domain (poorly active)
middle domain that regulates kinase activity
how are JAK kinases activated?
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
what purpose do the phosphorylated tyrosine residues serve?
binding sites for proteins that have phosphotyrosine binding domains
ex. SH2 domain
what determines which phosphotyrosine an SH2 will bind?
variations in the hydrophobic pocket In the SH2 domain
phosphotyrosine binding domain (PTB)
found on multidocking proteins
Bind phosphorylated tyrosines on the receptor
multidocking protein
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
JAK kinases activate ____ transcription factors
STAT TFs
3 domains of STAT
N-terminal DNA binding domain
SH2 domain that binds phosphotyrosines in a cytokine’s cytosolic domain
C-terminal domain with a critical tyrosine residue
how does JAK-STAT work?
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
are the genes available to be activated by any STAT different?
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
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
reduces (desensitization)
desensitization example HER1/EGF
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
do HER1 mutants that lack kinase activity undergo accelerated endocytosis in the presence of ligand?
no
perhaps b/c activation of kinase activity in normal HER1 induces conformational change that exposes a sorting motif that signals endocytosis
can internalized receptors signal from within endosomes before their degradation?
yes
evidence: binding signaling proteins like Grb-2 and Sos
2 things that can happen to internalized receptors
returned to cell membrane
degraded in lysosomes
ubiquitination
addition of the small protein ubiquitin to a lysine in a protein signals it for degradation
enzyme c-Cbl does this
SHP1
phosphatase that negatively regulates signaling from cytokine receptors
inactivates JAK protein by binding to active receptor
has 2 SH2 domains
SHP1 in inactive state
one SH2 domain of SHP binds the catalytic site so it is inactive
SHP1 in active state
The blocking SH2 domain binds a phosphotyrosine in the active receptor
Catalytic site placed near phosphate in JAK activation lip and removes
SOCS proteins
terminate signaling from cytokine receptors by negative regulation
SH2 domain binds activated receptor to block binding of other SH2 containing proteins (competitive inhibition)
SOCS-1 mechanism
binds critical phosphotyrosine in activation lip of activated JAK2 kinase
SOCS box
domain that recruits ubiquitin ligases
Ubiquitin is a signal for degradation in proteasomes
almost all RTK and cytokine receptors activate which pathway
Ras/MAP
Ras protein
small monomeric G protein
member of the GTPase superfamily of switch proteins
what does activated Ras promote at the membrane
formation of signal transduction complexes containing 3 sequentially acting protein kinases (kinase cascade)
The kinase cascade activates MAP kinases
what do members of the MAP kinase family do
translocate into the nucleus and phosphorylate many proteins
ex. TFs that regulate expression of proteins for the cell cycle
Ras activation is accelerated by
guanine nucleotide factor (GEF)
binds the Ras*GDP complex to cause dissociation of GDP
what deactivates Ras
GTPase activating protein (GAP)
catalyzes hydrolysis of GTP to GDP to inactivate Ras (Ras has a low intrinsic hydrolysis rate)
does Ras operate downstream of most RTKs and cytokine receptors?
yes
the compound eye of the fly is composed of 800 individual eyes called _______
ommatidia
each ommatidium consists of 22 cells, 8 of which are photosensitive neurons called retinula or R cells (R1-R8)
Sevenless (Sev)
an RTK that regulates development of R7 cell in an ommatidium
flies with mutant Sev gene
R7 cell does not form, flies can’t see UV light
Boss (bride of Sevenless)
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
fruit fly experiment with Sev + Ras
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)
always active Ras in Sev mutant fly
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
RTKs and JAK kinases are linked to Ras by _______ proteins
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
SoS (Son of Sevenless)
a guanine nucleotide exchange protein (GEF) that catalyzes conversion of inactive GDP-Ras to active GTP-Ras
binding of SoS to inactive Ras causes a conformational change that triggers..
dissociation of GDP so GTP can bind and activate Ras
signals pass from activated Ras to a cascade of protein _______, ending with ____ _______
kinases, MAP kinase
mitogen
a ligand that stimulates growth and division (cell cycle)
MAP kinase (MAPK mitogen activated protein kinase)
cytosolic kinase
gets phosphorylated by MEK and activated, then amplifies the signal by phosphorylating nuclear TFs
Melanoma
Always active B-Raf stimulates MEK/MAP to make skin cancer
Ras/MAPK pathway
active Ras*GTP binds N-terminal domain of Raf kinase
hydrolysis of RasGTP to RasGDP releases active Raf
Raf phosphorylates MEK which phosphorylates MAP
MAP phosphorylates nuclear TFs
mutant Raf kinase without N-terminal regulatory domain
always active and induce cultured cells to proliferate in absence of stimulation by growth factors
also tumor cells
does MAPK dimerize?
yes, after phosphorylation a conformational change occurs and it dimerizes
the dimer is translocated into the nucleus where it phosphorylates TFs
what does quiescent mean
non-growing
early response genes
induced well before cells enter S phase
ex. one of these genes encodes the TF c-Fos
c-Fos
TF that induces expression of many genes encoding proteins required for cells to move through the cell cycle
most RTKs that find growth factors utilize the MAPK pathway to activate genes encoding proteins like _-__, which propel the cell through the cell cycle
c-Fos (a TF)
the enhancer that regulates the c-Fos gene contains a _______ _______ ______
serum response element - an enhanced that binds many TFs
MAPK induces transcription of the c-Fos gene by direct activation of which TF, and indirect activation of another TF
direct: ternary complex factor
indirect: serum response factor
association of phosphorylated ternary complex factor with two molecules of phosphorylated serum response factor forms… I
an active trimeric factor that activates gene transcription
_____ transmit signals to MAPK in yeast mating pathways
GPCR
G-betagamma triggers a kinase cascade like Ras/MAPK cascade
_____ proteins separate multiple MAPK pathways
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
basic idea of scaffold proteins
coordinates responses by organizing free floating proteins
ex. MAPK on scaffold activated in Ras/MAPK pathway
MAPK-P can enter nucleus and phosphorylate TFs
mitogens up…
cell proliferation
cell cycle
active in cancer
weird note about nutrients for animal cells
90% defined medium, 10% fetal calf serum (growth factors)
eliminate 10% = serum starved cells
look at signaling pathways activated when it is added back
Dominant-negative mutation
A loss of function in cells with wild-type copies
Mutant prevents normal protein from functioning
