Final: Ch 16 PI, SerK, Hedgehog Flashcards
do phosphoinositide pathways have long-term effects on gene expression?
yes
phosphoinositide pathways end with a variety of kinases, especially….
protein kinase C (PKC)
protein kinase B (PKB)
PKC & PKB play key roles in what 2 things
cellular growth and metabolism
hormonal stimulation of some GPCR leads to activation of ________ C
phospholipase C (PLC)
what does PLC do after activation
cleaves PIP2 to generate the second messengers DAG and IP3
signaling via the IP3/DAG pathway leads to…
an increase in cytosolic Ca and activation of PKC
which isoform of PLC is activated by GPCRs
beta
which isoform of PLC is activated by RTKs and cytokine receptors?
gamma - contains SH2 domains
what do the SH2 domains of PLC-gamma do
bind specific phosphotyrosines on the activated receptors
positions the PLC close to PIP2, and PLC gets phosphorylated
recruitment of PI-3 kinase to activated receptors leads to synthesis of _ phosphorylated phosphatidylinositols
2
how does the PI-3 kinase RTK/cytokine receptor pathway work
PI-3 kinase recruited to membrane by binding SH2 domains to phosphotyrosine on the cytosolic domain of activated RTK/cytokine receptors
catalytic domain of PI-3 kinase close to phosphoinositide substrates
phosphorylates 3’ carbon
what do the products of the PI-3 kinase pathway do
act as docking sites for signal transducing proteins so the signal is passed downstream
ex. cell division, prevent apoptosis
polyoma virus
DNA virus that transforms mammalian cells to uncontrolled growth
uses oncoprotein middle T
researchers discovered PI-3 kinase when studying middle T
protein kinase B (PKB)
serine/threonine kinase also called Akt
Also has a PH domain that binds to the 3-phosphates from PI-3
since the inositol phosphates are present on the cytosolic face of the membrane, binding recruits the ______ protein to the cell membrane
entire protein
following hormone stimulation (rise in PI 3-phosphates), what does PKB do
binds the membrane bound 3-phosphates via the PH domain (releases active catalytic site) and becomes localized at the plasma membrane
need two other kinases (PDK1 and PDK2 to be fully active)
PDK1 and PDK2
PDK1 is recruited to the membrane via binding of PH domain - eventually encounters PKB and phosphorylates a thr residue in the activation lip
PDK2 phosphorylates a serine not in the activation lip
once fully activated, PKB can…
dissociate from the membrane and phosphorylate target proteins within the cell
activated PKB can phosphorylate and inactivate ___-apoptotic proteins like ___
pro-apoptotic, Bad
short-term effect that prevents activation of an apoptosis pathway that leads to death
PKB and the Forkhead TF FOXO3a
PKB promotes survival of cultured cells by phosphorylating FOXO3a
this reduces the TF’s ability to induce expression of many pro-apoptotic genes
the PI-3 pathway is negatively regulated by ____ _________
PTEN phosphatase
overexpression in cultured mammalian cells promotes apoptosis by reducing the level of the PI 3-phosphates and hence activation and anti-apoptotic effect of PKB
is the PTEN gene deleted in many types of advanced human cancers?
yes, contributes to uncontrolled growth b/c of elevated levels of PI 3-phosphates
inositol phosphate (IP) releases stores of ____
Ca2+
transforming growth factor B (TGF-B) superfamily and the transforming growth factor B receptor superfamily
receptor serine kinases and signaling molecules
phosphorylate and activate the Smads TFs that regulate growth and differentiation pathways
in unstimulated cells, the Smads are located in the ______, but when activated they move into the ________ to regulate transcription
cytosol, nucleus (via NLS)
the primary function of the 3 human TGF-B isoforms is to….
strongly prevent cellular proliferation
induce synthesis of proteins that inhibit the cell cycle
TGF-B prevents growth in both…
the secreting cell (autocrine signaling)
neighboring cells (paracrine signaling)
many animal cell types produce and secrete members of the TGF-B superfamily in an ________ form that is stored attached to cell-surface molecules or in the ECM
inactive form
release of the active form from the matrix by protease digestion or inactivation of an inhibitor
leads to a quick activation of signaling molecules already in place
how did researchers identify the receptor TGF-B1 bound to
radiolabeled purified TGF-B1 with iodine 125
Cross-linking
purification revealed 3 different receptors
what are the 3 TGF-B receptor proteins called
RI, RII, RIII
RIII (B-glycan)
most abundant TGF-B receptor
cell surface transmembrane proteoglycan
binds and concentrates mature TGF-B molecules near the cell surface to aid their binding to RII
RI and RI receptor proteins are _____ transmembrane proteins with ______/_______ kinases as part of their cytosolic domains
dimeric, serine/threonine kinases
RII exhibits constitutive ______ activity
kinase
binding of TGF B to RI and RII causes what
induction of complexes containing 2 copies of each RI and RII
RII phosphorylates serine and threonine residues in RI to activate RI kinase activity
3 types of Smads
R-smads (receptor regulated – Smads2 and 3)
co-Smads (Smad4)
I-Smads (inhibitory Smads)
what does the MH1 domain of R-Smads contain
a nuclear localization signal that is hidden in the inactive form
phosphorylation of serines in R-Smads by RI allows binding of an importin to the NLS, which allows transport to the nucleus
within the nucleus, R-Smads are continuously being _________ by…
dephosphorylated by a nuclear phosphatase
______ feedback loops regulate TGF-B/Smad signaling
negative feedback loops
in most signaling pathways, the response to a growth factor or other molecule decreases with _____
time (desensitization)
which two cytosolic proteins downregulate TGF-B/Smad signaling
SnoN and Ski
block transcription activation by a bound Smad complex by deacetylating histones
makes cells resistant to growth-inhibitory effects of TGF-B
what are the Wnts and Hedgehogs?
evolutionarily conserved families of signaling proteins
play key roles in developmental pathways
induce expression of genes required for a cell to acquire a new identity or fate
similarities of Wnt and Hedgehog pathays 4 things
bind receptors that are like GPCR, but don’t activate G-proteins
in resting state, key TFs are ubiquitinated and targeted for proteolytic cleavage
activation of each pathway involves disassembly of cytosolic protein complexes, deubiquitination, and release of an active TF
the kinase GSK3 is used
NF-kB pathway controlled by ubiquitination
an inhibitor of a TF is deactivated by ubiquitination
in resting state, the TF NF-kB is in the cytosol bound to an inhibitor
stress-inducing conditions cause deubiquitination and degradation of the inhibitor, allowing cells to activate gene transcription
another function of polyubiquitination other than signaling things for degradation by proteases is….
forming a scaffold to assemble a key signal transduction complex
Wnt signaling triggers the release of a TF from a ______ protein complex
cytosolic
proto-oncogene
a normal gene whose inappropriate expression promotes cancer
ex. Wnt-1
Wnt means
wingless
what does the Wnt pathway control
brain development
stem cells
development of osteoblasts
Wnt proteins are secreted extracellular signaling molecules that are modified by addition of a ______ group near their N-termini
palmitate (hydrophobic)
tethers Wnt proteins to the membrane, limiting their action to nearby cells
2 cell-surface receptors Wnt acts through
Frizzled (Fz) - 7 transmembrane helices and directly binds Wnt
co-receptor LRP - associated with Fz
B-catenin (vertebrates) / Armadillo (flies)
major players in Wnt signaling
acts as a transcriptional activator and membrane-cytoskeleton linker protein
Wnt pathway
Wnt binds to Fz and the LRP co-receptor
kinases phosphorylate LRP and Axin binds
phosphorylation of B-catenin is blocked, allowing it to accumulate
B-catenin translocates to the nucleus and binds to TF as a co-activator
Hedgehog (Hh) signaling relieves ______ of target genes
repression
the Hh pathway involves ______ of an intracellular complex containing a TF
disassembly
unlike Wnt, the Hh protein undergoes distinctive ___-_________ processing
post-translational processing
do the two Hh membrane receptors move between the plasma membrane and intracellular vesicles?
yes
what happens to cells that receive a high or small amount of Hh
high: turn on genes to form certain structures
low: turn on different genes to form different structures
morphogens
signals that induce different cell fates depending on their concentration in target cells
how is Hh formed
from a precursor protein with autoproteolytic activity
cholesterol and palmitoyl is added, making it hydrophobic to limit its diffusion in the membrane
Hh pathway in flies
Hh binds to Patched (Ptc) to stop inhibition of Smo
Smo moves to the membrane, gets phosphorylated
Ci (a TF) moves to nucleus and recruits CREB-binding activator protein to express target genes
primary cilium
a single immotile cilium
intraflagellar transport proteins (ITF)
move proteins and particles from the base of a cilium to the tip
Hh pathway in vertebrates
Hh binding triggers movement of Smo to the ciliary memb and the KIF7 motor protein up the microtubule to the ciliary tip
Gli (a TF) accumulates and is transported down the cilium and released into cytosol –> nucleus
degradation of an inhibitor protein activates the _____ TF
NF-kB TF
in the inactive state, the NF-kB TF is…
in the cytosol bound to an inhibitor
activation of the NF-kB signaling pathway involves what
ubiquitination and degradation of the inhibitor, releasing the TF
rapidly activated in mammalian immune system cells in response to bacterial or viral infection
toll like receptors NF-kB
activated when bacterial or fungal cell walls bind toll-like receptors on the cell surface
NF-kB pathway
kinase phosphorylates inhibitor of NF-kB, which gets ubiquitinated and degraded by proteasomes
nuclear localization signal in NF-kB dimer is revealed, and it is transported to nucleus
NF-kB activates many genes
what types of genes does NF-kB activate
those encoding cytokines and chemokines
causes immune cells to infiltrate the site of infection
depending on the specific E3 ubiquitin ligase, ubiquitin forms multiple types of ______ that have different structures and functions
polymers
polyubiquitin chains linked to activated IL-1 receptor forms a scaffold that…
brings the TAK1 kinase near I-kB kinase complex and phosphorylates it
Signal is transmitted this way
When is NF-kB pathway activated
In response to viral or bacterial infection
Stress
