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Biochemistry Signal Transduction 1
Terms in this set (35)
DNA Binding Domain (DND)
A highly conserved domain that contains two zinc fingers that bind to specific regions of DNA termed "hormone response elements (HRE)". In addition, it interacts with itself, stabilizing receptor dimerization.
Ligand Binding Domain (LBD)
A highly conserved α-helical globular domain that binds to a specific steroid hormones, which stimulates its "transcriptional activation function (AF2)". Like the DBD, it also interacts with itself, stabilizing receptor dimerization.
N-Terminal Domain (NRD)
A highly variable sequence/structure that contains the "transcriptional activation function (AF1)", which synergizes with AF2 of the LBD. Together, AF1/2 bind coregulators for chromatin remodeling.
Type 1 Hormone Receptor
Located inside the cytoplasm bound to heat shock proteins (HS70, HS90) that hold intermediate state protein, when the hormone binds to the ligand binding domain, it triggers a conformation change where the heat shock proteins bind together and homo-dimerize.
Because its located in the cytoplasm it must translocate to the nuclease. (may have something to do with the hinge region)
Both Type 1 and Type 2 once bound to DNA they both recruit co-regulators for the activation of mRNA transcription.
Ex.) receptors for estrogen,
androgen, progesterone, and glucocorticoid.
Type 2 Hormone Receptor
Located inside the nucleus, therefore the signal must get to the nuclease. When the hormones binds in the nuclease,
Ex.) Receptors for various forms
of vitamin A, vitamin D, and thyroid hormones.
Note: Type II receptors most often associate as
hetero-dimers, thus requiring two different signaling molecules to elicit a transcriptional response
Coregulators (mech slide)
The majority of transcriptional coregulators are histone modifying enzymes.
(i) "Coactivators" cause release of histones
from DNA (e.g., HATs).
(ii) "Corepressors" cause condensation of
nucleosomes (e.g., HDATs).
Important to understand that transcription of estrogen will for example, have down regulatory effects on some genes and up-regulate others.
A number of steroid hormones, vitamins, and assorted other "ligands" diffuse into cells and specifically bind their target receptor. This induces enhances DNA binding to "hormone response elements" and leads to recruitment and activation of transcriptional "coregulators".
G Protein Coupled Receptors (GPCR)
800> different genes for regulation.
A large family of cell surface protein receptors with seven α-helices that span the lipid bilayer in alternating fashion.
adenylyl cyclase (AC)
Contains six TM α-helices, followed by a cytoplasmic "C1 domain", another
six TM α-helices, and the final cytoplasmic
"C2 domain". The C1/C2 domains associate to form the active site and catalyze production of "3',5'-cyclic AMP (cAMP)" and PPi from ATP.
cAMP is a type of "second messenger
Transmits message from extracellular to intracellular. It is composed of three subunits α, β- and γ.
α-subunit binds GTP and hydrolysis it to GDP (modulating it's function)
GDP= OFF: GTP=ON.
The active α-subunit with GTP bound then dissociates from the GPCR/βγ complex and associates and activates the trans- membrane (TM) enzyme, "adenylyl cyclase"
cAMP-dependent protein kinase (PKA)
Background: Extracellular signal is converted into second messenger which then activates a Protein Kinase.
Consists of a two catalytic subunits which are held inactive by the (R1/R2) regulatory subunits. When cAMP comes along, it binds to the R1/R2 subunits which dissociate and releases the now activate PKA catalytic subunits.
cAMP response element binding protein (CREB)
The activated PKA then translocates to the nuclease (through transporter.) Inside the nuclease, it phosphorylates and activates c-AMP Response Element Binding Protein (CREB.)
which causes CREB to dimerize, bind the "cAMP response element (CRE)", and recruit/activate various transcriptional coregulators (chromatin modelers or remodeler) which activate or de-activate transcription.
The DNA binding domain is a "basic leucine zipper (bZIP)". CREB regulates expression of numerous and different genes in different organs. Perhaps, it is most noted for its "role in learning and memory"!
Activated PKA that remains in the cytoplasm phosphorylates PDE, which changes conformation and activates phosphodiesterase' ability to hydrolyze cAMP back into AMP.
Thus, the R subunits can again bind and sequester active PKA subunits, attenuating the downstream signaling events and resulting in the inactivation of PKA.
Notes from online: Phosphodiesterase Inhibitors in turn block the inactivation of PKA by R subunits. Ex) PD5 inhibitor (Viagra) and Caffeine (weak and non-selective)
Example of a Tumor Suppressor
Non-CREB effects of c-AMP
Aside from CREB-dependent transcriptional activation, PKA is most recognized for its role in controlling enzyme activities related to
(i) Starvation: Low blood sugar is counteracted by "glucagon" action on cAMP.
(ii) Fight-or Flight: Rapid increased heart rate and respiration, with production of needed glucose
fuel is stimulated by catecholamine action on cAMP production.
Catecholamines: "epinephrine, "norepinephrine", and "dopamine".
Wnt Canonical Pathway.
Involved in embryogenesis and development. Shutoff after development, however is resurrected in cancer cells. 7 Re
First recognized as a component of cell adhesion complexes (cytoskeletal protein) however, it was later found to play significant role in transcriptional regulation in other cells. One of first examples of a "moonlighting" protein (i.e., same gene product acting with different functions)
Destruction Complex (GSK3)
Numerous proteins β-Cat and enzymes that sequester the transcriptional regulator, β-catenin, and target it for degradation by phosphorylation via"glycogen synthase kinase-3 (GSK3)", which signals it for ubiquitination by E3 ligase and is subsequently degraded.
Family of GPCRs that serve as cell surface receptors for Wnt signaling. A Cys-rich
(5 disulfide) extracellular region binds Wnt, in cooperation with its coreceptor, the "lipoprotein receptor related protein (LRP)"
A multidomain adaptor protein that associates with cytoplasmic loops of Wnt bound Fz receptor. Then, it binds and inhibits the Destruction Complex.
Wingless-related integration site (Wnt)
Diverse family of highly palmitoylated glycoproteins that cells secrete to initiate a developmental response. First messenger in the WNT canonical pathway.
Two Faced Roman God.
Extracellular signaling proteins that are secreted by cells, primarily for developmental and immunological responses.
Cytokine Receptor (CR)
Family of cell surface
receptors, composed of an (i) "extracellular
domain", (ii) a "transmembrane domain", and a
(iii) "cytoplasmic domain". On binding any number
of particular extracellular "cytokines", CR monomers associate into dimers, and likely higher order P structures, which results in Tyr phosphorylation
of their cytoplasmic domains by "Janus kinases (Jak)
Family of intracellular "nonreceptor tyrosine kinases" that are associate and activated by cytokine-bound receptors. First, they phosphorylate Tyr residues in the cytoplasmic domain of bound receptors. The "Stat" family of transcription factors bind to pTyr using their central "SH2 domain", which is next phosphorylated by Jak
Signal Transducer and activator of transcription (STAT)
signal transducer & activator of transcription (Stat): Family of transcription factors that bind pTyr in activated cytokine receptors and become pTyr phosphorylated by Jaks. This leads to dimerization (homo-/hetero-), nuclear transport, and DNA binding.
Jak/STAT pathway (kinase linked)
1.) Cytokine binds to CR on the extracellular cytokine binding domain leading to the dimerization of the CR and bringing together the JAKs attached to the cytoplasmic end of the receptors.
2.) JAK then phosphorylates the Cytoplasmic domain tail of the cytokine receptor and recruits Signal Transducer and Activator of Transcription (STAT) which binds to the phosphorylated region of the CR.
3.) JAKs phosphorylates STAT.
4.) Addition of phosphate to STAT causes them to also dimerize.
5.) STAT dimers transition into the nucleus where they can directly activate transcription.
Animation video: http://www.dnatube.com/video/4103/Cytokine-Binding-or-JAKSTAT-Signaling-Pathway
A mitogen is a chemical substance that encourages a cell to commence cell division, triggering mitosis.
1.) Ligand (mitogen) binds to extra cellular portion of membrane-bound Receptor Tyrosine Kinase (RTK) leading to the dimerization of two RTK subunits.
2.) Upon dimerization the inner portion of RTK domain catalyzes phosphorylation of itself and the other subunit.
3.) Next, the Growth Factor Receptor Bound Protein 2 (GRB2) Adaptor Protein, binds to the phosphorylated RTK.
4.) Protein SOS (Son of Sevenless) binds to the membrane bound protein RAS and connects to GRB2.
5.) Inactive RAS is bound to the nucleotide GDP, SOS catalyzes the exchange of GDP --> GTP which activates RAS protein.
6.) The activated Ras-GTP protein binds (effector protein) B-Raf protein, activating the phosphorylation and activation of Mek, which phosphorylates and activates Erk.
7.) Kinase kaskade leads to transcription factors of the AP family 1 (activator protein 1 ) ( transcription factors JUN and FOS)
8.) JUN and FOS are activated, form a heterodimer
Basic Leucine Zipper
and move to a cell nucleus then bind to an AP1 motif of DNA. Cell proliferation is activated.
Video Animation: http://www.dnatube.com/video/7040/MAPKinase-MAPK-signalling-pathway
Termination of MAP Kinase Pathway
RAS GTP is inactivated by the GPase Activating Protein (GAP)
GAP binds to RAS GTP and increases the weak innate GPAse activity of RAS by providing it with a domain that assists in the hydrolysis of GTP --> GDP.
GDP bound RAS is no longer activated and can no longer bind to B-RAF (effector protein). As a result the MapK signaling pathway is turned off.
MAP Kinase Mutation in Cancer
Occurs when the normal RAS protein loses its ability to be inactivated by the GTPase and thus the pathway is never turned off.
A normal gene that becomes a cancer causing agent due to hyperactivation by either (i) mutation or (ii) over-expression. A majority are enzymes in cell signaling pathways that drive cell growth, proliferation, and differentiation. These would qualify as potential "targets" for new "anti-cancer therapies".
Examples: receptors, kinases, G-proteins, and transcription factors
Tumor Supressor Gene (Anti-Oncogene)
A normal gene that contributes to cancer progression due to inactivation by either (i) mutation, (ii) deletion, or (iii) silencing. A majority are enzymes in cell signaling pathways that restrain cell growth, proliferation, and differentiation. Since these components are already inactivated, they most often
qualify as potential "targets" for new "anti-cancer therapies" because you don't want to inhibit something that prevents cancer.
Examples: phosphatases, GAPs, and transcription factors
1.) Extracellular signal such as a Growth Factor (Insulin) bind to Receptor Tyrosine Kinase (IRTK)
2.) Signal binds and causes dimerization of IRTK and on one end leads to the entire MAP Kinase pathway.
SH2 domains allow proteins containing those domains to dock to phosphorylated tyrosine residues on other proteins. SH2 domains are commonly found in adapter proteins that aid in the signal transduction of receptor tyrosine kinase pathways
Activated adenylcyclase converts ADP--> ATP
cAMP activates PKA by binding to regulatory subunits alters conformation and liberates the catalytic subunits to phoshorylate target proteins.
PKA enters the nuclease where they phosphorylate CREB. Which activates transcription.
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