Biol 266 : Unit 8-2

Some receptors are activated by extremely low concentrations of ligands (10^-9 – 10^-11 M). These ligands are generally called _____________ (e.g. EGF) but also include _______________(e.g. insulin). The response is usually slow (hours) since they alter gene expression.

growth factors ; hormones

The response of receptors that are activated by extremely low concentrations of ligands is usually ____________ since they alter gene expression.

The response involves ___________________ of a downstream target or of the receptor itself.

Many such receptors are tyrosine kinases (receptor tyrosine kinases, RTKs) but some are serine/threonine kinases.

slow ; phosphorylation

Many receptors that are activated by extremely low concentrations of ligands are __________ kinases (receptor, RTKs) but some are _____________ kinases.

tyrosine ; serine/threonine

RTKs usually have a _______membrane spanning domain in contrast to G-protein-coupled receptors which have ______. Ligand binding often induces ____________ of the receptors, followed by ________________ (one receptor phosphorylates tyrosine residues of a neighboring receptor)

single ; 7 ; aggregation ; autophosphorylation

______ is small GTP binding protein. When bound to GDP it is inactive, when bound to GTP it is active.

Ras

___________ is an SH2 domain-containing protein (binds to phosphotyrosine residues). Mediates attachment of Sos to the activated EGF receptor.

GRB2

_______________ is a nucleotide exchange factor for Ras, activates the GTPase.

Sos

The Ras-Raf-MAP kinase pathway :

________ binds to its receptor, initiating a cascade of events leading to the activation of the small GTP binding protein ________.

1. __________ of the EGF receptors.

2. ________________ of the receptors on tyrosine residues in the cytosolic tail.

The GRB2-Sos complex binds to the activated receptor through the ________ domain of GRB2. Inactive Ras can then associate with ______.

Sos then activates Ras by releasing its GDP and assisting in its uptake of GTP.

Activated Ras binds to a complex of the kinase _______ and the protein __________ (an inhibitor of Raf kinase activity).

Binding to activated Ras relieves the _________ of 14-3-3 and allows for Raf ________________ and subsequent activation.

When Ras hydrolyses its GTP to GDP and Pi, it releases activated ________.

Activated Raf then phosphorylates and activates a downstream kinase called _________.

Activated MEK then phosphorylates and activates downstream kinases called _____________ (aka ERK1 and ERK2)

EGF ; Ras ; Aggregation ; Autophosphorylation ; SH2 ; Sos ; Raf ; 14-3-3 ; inhibition ; phosphorylation ; Raf ; MEK ; MAP kinases

Many targets of MAP kinase are transcription factors. The Ras-Raf MAP kinase pathway can activate ~160 genes called __________________ (needed for the cell to enter and progress through the cell cycle). The result of this signaling pathway is cell ________________, _______________ and ____________.

early response genes ; proliferation ; differentiation ; survival

All these genes contain a cis-acting DNA sequence, called ____________. The SRE binds to unphosphorylated forms of two transcription factors called _______________. Once phosphorylated, these transcription factors can activate transcription.

serum-response element (SRE) ; TCF and SRF

The three kinases Raf, MEK and ERK1/2 can be generally classified as follows:

The most downstream kinase of the three (ERK1/2) is called the________________.

MAP kinase (or MAPK)

The three kinases Raf, MEK and ERK1/2 can be generally classified as follows:

Since MEK is the kinase that phosphorylates the MAPK, it is called a _______________________.

MAPK kinase (or MAPKK)

The three kinases Raf, MEK and ERK1/2 can be generally classified as follows:

Since Raf is the kinase that phosphorylates the MAPKK, it is called a _____________________.

MAPKK kinase (or MAPKKK)

Growth Factor → ______ → _______ → ________ → Growth, Survival and Differentiation

A-Raf ; Mek1/2 ; ERK1/2

Mechanisms for silencing the cascade include :

1. _______________________ (mutation of Ras to a form that cannot hydrolyse GTP is a common mutation found in cancer cells).

2. ______________________ of the MAPK (one gene whose transcription is upregulated by ERK1 is MKP-1, a phosphatase that acts on ERK1).

3. ____________of the receptor by :

a. receptor dephosphorylation

b. Endocytosis and delivery to the lysosome for degradation

c. Therapeutic method – antibody inhibition. Herceptin is an antibody that recognizes Her2 (aka EGF receptor) and is used to treat breast cancer.

GTP hydrolysis by Ras ; Dephosphorylation ; Inactivation

Mechanisms for silencing the cascade include :

Inactivation of the receptor by:

a. receptor ____________________

b. Endocytosis and delivery to the lysosome for _________________.

c. Therapeutic method – __________________. Herceptin is an antibody that recognizes Her2 (aka EGF receptor) and is used to treat breast cancer.

dephosphorylation ; degradation ; antibody inhibition

The insulin receptor is a heterotetramer, composed of 2 a and 2 b subunits.

The a subunits are extracellular and together form a binding pocket for insulin.

Binding of insulin changes the conformation of the a subunits. Since these subunits are covalently linked to the b subunits through a ______________, a conformational change in the b subunits takes place.

This activates its __________________ activity and it ___________________.

The autophosphorylated receptor binds adaptor proteins called _______________________.

These proteins are also phosphorylated by the receptor. ______________ can activate several pathways including Ras by binding to GRB2- Sos, and a phosphoinositide pathway by binding the lipid kinase PI3K.

__________ phosphorylates phosphatidylinositol-4- phosphate and phosphatidylinositol-4,5- bisphosphate to produce __________.

These are second messengers and recruit various proteins to the membrane.

disulfide bond ; tyrosine kinase ; autophosphorylates ; insulin receptor substrates (IRS1 and IRS2) ; IRS1 ; PI3K ; PIP2 and PIP3

PIP3 recruits two enzymes from the cytosol: ________ and _________. These enzymes bind through their PH domains.

AKT ; PDK1

PDK1 _____________ and activates _______. A second kinase called mTOR also contributes to the activation.

phosphorylates ; AKT

Activated __________ then dissociates from the membrane and is a major component of a number of different pathways leading to ___________________ (by inducing the translocation of the _______ transporter to the cell surface), and __________ synthesis.

AKT ; protein synthesis, glucose uptake ; GLUT4 ; glycogen

PI3K signaling can be terminated by a lipid phosphatase called _________ that converts _____ to ________.

PTEN ; PIP3 ; PIP2

G-protein coupled receptors activate _____________.

PLC-B

RTKs activate _______________.

PLC-y

RTKs can also signal through __________. This results in a rise in __________ and activates downstream molecules such as ________________.

IP3 ; Ca2+ ; PKC and CaM

The best studied of Signaling through proteolysis pathways is the __________________. This pathway is important for ________________________.

Notch-Delta pathway ; cell fate determination

Signaling through proteolysis :

1. Delta acts as the ________and Notch as the ___________. Both are _______________ proteins.

ligand ; receptor ; transmembrane

Signaling through proteolysis :

2,3. When the two interact, Notch is cleaved by two proteases:

a. ________________ – cleaves extracellularly

b. _______________ – cleaves within the membrane-spanning region and releases a fragment of the Notch intracellular domain.

ADAM10 ; Presenilin 1

Signaling through proteolysis :

4. This domain then translocates to the nucleus where it activates ____________.

transcription

______________________ — when receptors can activate similar pathways. Thus, different signals may lead to expression of similar genes. For example, two different receptors may lead to the recruitment of GRB2-Sos, activating Ras-Raf-MAPK pathway.

Convergence

________________ – when a single receptor can activate numerous pathways. For example, the insulin receptor activates PI3K, GRB2-Sos and other pathways.

Divergence

______________ – when part of one pathway influences part of another pathway. For example, activated PKA phosphorylates and inhibits the function of Raf, thereby blocking Raf signaling pathways.

Cross-talk

Methods used to dissect pathways include:

1. _______________________ methods (e.g. co-immunoprecipitation)

2. ____________________ to determine the key amino acids required for interaction and for signaling.

3. Using ______________ experiments with mutant cell lines to determine the order of the pathway.

protein-protein interaction ; Mutagenesis ; bypass