1-31-06

Eukaryotic Signaling

General Principles

Signaling Cells

Signal molecules

-most often secreted from signaling cell into the extracellular (EC) space

-some can diffuse through the plasma membrane

-often act at very low concentrations (≤10^-8 M)

-some can remain bound to the surface of the signaling cell (contact-dependent signaling)

-important during development and in immune responses

Target Cell

Receptor

-binds the signal molecule

-bind signal molecules with high affinity (K_a≥10⁸ L/mole)

-most of the time receptors are TM proteins that bind a ligand and a triggering of a cascade follows from ligand binding

-sometimes the receptors are in the target cell, so the signal molecule enters the cell to activate it-in such cases, the signal molecule has to be very small

Distance over which signal molecules act

-many of the same types of signaling molecules are used among the following signaling types, it is a matter of the speed and selectivity to which signals are delivered to the targets

-contact dependent-explained above

-paracrine signaling-signals are released into the EC space and act locally on neighboring cells

-synaptic signaling-neurons transmit signals electrically along their axons and release neurotransmitters at synapses, which are often located far away from the cell body of the neuron-this type of signaling is very fast, is not so dilute, and the signals have a low affinity for their receptors

-endocrine signaling-endocrine cells secrete hormones into the bloodstream and these hormones are then distributed widely throughout the body-this type of signaling is relatively slow and signals act at low concentrations, so the receptors have a high affinity for their ligands

-autocrine signaling-cells secrete signal molecules that can bind back to its own receptors-most effective when performed simultaneously by neighboring cells of the same type-development and cancer utilizes this signaling mechanism

Gap junctions

-specialized cell-cell junctions formed between two cells

-cytoplasms of cells connected by narrow water-filled channels

-coordinate the activities of neighboring cells

-allow exchange of small intracellular signaling molecules (Ca²⁺, cyclic AMP), but not proteins or nucleic acids

-can visualize gap junctions in tissues with intracellular electrodes or with the microinjection of water-soluble dyes

G-protein linked receptors

-largest class of cell surface receptors

-found in all eucaryotes

-~1/2 of all known drugs work through these receptors

-all G-protein linked receptors have a similar structure

-single polypeptide chain that passes through the plasma membrane 7 times

-EC binding to receptor leads to a conformational change

-this change leads the receptor to activate trimeric GTP-binding proteins (G proteins), which are bound to the cytoplasmic face of the membrane and act as relays between the receptor and other enzymes or ion channels

-G proteins are composed of 3 protein subunits: a, b, g

-a

-in unstimulated state, this subunit has GDP bound and the G protein is inactive

-when stimulated, GDP is released and GTP binds in its place and the protein dissociates into two activated components, a and the bg complex

-the dissociated a subunit adopts a new shape and interacts with target proteins

-GTPase, which hydrolyzes its bound GTP to GDP, and then reassociates with bg to reform an inactivate G protein

-GTPase activity is inefficient with only the a subunit, so binding with another protein enhances the GTPase activity

-the other protein can either be:

- the target protein or

-enhancing proteins, known as regulator of G protein signaling (RGS) or GTPase activating proteins (GAPs)

~25 RGS in the human genome

-bg

-this complex does not change shape after dissociation, but the surface masked by the a subunit becomes available to interact with other target proteins

cAMP

-normal concentration inside the cell is ~10^-7M

-EC can increase [cAMP] up to 20-fold in seconds

-cAMP is synthesized by the plasma membrane bound adenlyl cyclase

-adenlyl cyclase is large multipass TM protein with its catalytic domain on the cytosolic side of the membrane

~8 isoforms in mammals, which are regulated by G proteins and Ca²⁺

-continuously destroyed by one or more cAMP phosphodiesterases that hydrolyze cAMP to 5’AMP

-all receptors that act via cAMP are coupled to stimulatory G protein (G_s)

- G_s activates adenlyl cyclase and increase [cAMP]

-inhibitory G protein (G_i) inhibits adenlyl cyclase by directly regulating ion channels

-both G_s and G_i are targets for cholera toxin; cholera toxin catalyzes the transferof ADP ribose from intracellular NAD⁺ to the a subunit of G_s, and such ribosylation changes the a subunit so that it can no longer hydrolyze its bound GTP and results in the a subunit remaining in the active state to continuously stimulate adenlyl cyclase

-pertussis toxin catalyzes ADP ribosylation of the a subunit of G_i leading to the a subunit not being able to interact with receptors due to the bound GTP

-cholera and pertussis toxins are used as tools to determine whether a cell’s response to a signal is mediated by G_s or G_i

cAMP-dependent Protein Kinase (PKA)

-activation of ion channels

-catalyzes the transfer of the terminal phosphate group from ATP to specific serines or threonines of target proteins

-found in all animal cells

-may account for the effects of cAMP in cells

-inactive state-consists of a complex of two catalytic subunits and two regulatory subunits

-binding cAMP to the regulatory subunits alters the conformation and leads to dissociation of these subunits

-the reg. subunits localize the kinase inside the cell by binding to PKA anchoring proteins, which bind to the reg subunits and to the membrane

-PKA anchoring proteins can also bind other kinases and phosphatase to form a signaling complex

-released catalytic subunits phosphorylate substrate proteins

-some genes have cAMP response elements (CREs) to which CRE-binding proteins (CREBs) binds

-CREBs get phosphorylated by PKA on a single serine and recruits the transcriptional coactivator CREB-binding protein (CBP) -the association of both the CREB and the CBP together bind to the CREB-binding element on the DNA

-if the serine is mutated, CBP cannot associate with CBP and gene transcription cannot happen in response to the increase [cAMP]

Protein phosphatases

-dephosphorylate the proteins phosphorylated by PKA

-4 types of serine/threonine phophatases

-protein phosphatase I

- protein phosphatase IIA

- protein phosphatase IIB

- protein phosphatase IIC

-all have a homologous catalytic subunit complexed with one or more of a large set of reg. subunits

-protein phosphatase I-dephosphorylating proteins phosphorylated by PKA, inactivates CREB by removing its activating phosphate and by turning off the transcriptional response caused by a rise in cAMP

- protein phosphatase IIA-broad specificity and is the main phosphatase that reverses the phosphorylations catalyzed by serine/threonine kinases

- protein phosphatase IIB = calcineurin-activated by Ca²⁺ and abundant in the brain

- protein phosphatase IIC-minor phosphatase unrelated to others

Phospholipase C

-trimeric G proteins link activated receptors to phospholipase C

-activation of phospholipase C leads to an increase in Ca²⁺ in the cytosol

- Ca²⁺ is more widely used as an intracellular mediator than cAMP

- phospholipase C acts on inositol phospholipid (a phosphoinositide), phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], which rests on the inner half of the plasma membrane bilayer

-receptors that operate through the inositol phospholipid signaling pathway mainly activate G_q, which activates phospholipase C-b

- phospholipase C-b cleaves PI (4,5)P₂ to generate inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol and the signaling pathway splits into two branches at this step

- IP₃ is a small, water-soluble molecule that leaves the PM and diffuses rapidly into the cytosol

-binds to and opens IP₃-gated Ca²⁺-release channels in the ER membrane

- Ca²⁺stored in the ER is release through the open channels resulting in a quick rise in Ca²⁺ in the cytosol

-several mechanisms initiate this Ca²⁺ response

- IP₃ rapidly dephosphorylated by phosphatases to form IP₂

-IP₃ is phosphorylated to IP₄

- Ca²⁺ entering the cytosol is rapidly pumped out to the exterior of the cell

- diacylglycerol remains embedded in the membrane, where it has 2 potential signaling roles

o can be further cleaved to release arachidonic acid, which can either act as a messenger or be used in synthesis of other small messengers, known as eicosanoids (prostaglandins are eicosanoids and participate in pain and inflammatory responses and anti-inflammatory drgs such as aspirin, ibuprofen and cortisone inhibit their synthesis)

o activate the serine/threonine protein kinase, protein kinase C (PKC), which is Ca²⁺ dependent

§ initial rise in cytosolic Ca²⁺ induced by IP₃ alters PKC so that it translocates from the cytosol to cytoplasmic face of the PM

§ PKC is then activated by Ca²⁺, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine (see 15-36 in Alberts on page 860)

§ PKC then phosphorylates target proteins

-Ionomycin or A23187 can mimick the effects of IP₃ to allow Ca²⁺ to move to the cytosol from EC fluid

-phorbol esters can mimick the effects of diacylglycerol by binding to PKC directly

-lymphocytes can be stimulated to proliferate in culture when treated with a Ca²⁺ ionophore and a PKC activator, but not when treated with either reagent alone

- Ca²⁺ in the cytosol is normally ~10^-7M while its conc in the EC fluid (~10^-3M) and in the ER lumen is high

- Ca²⁺ entering the cytosol increases in conc ~10-20 fold

3 main types of Ca²⁺ channels mediate Ca²⁺ signaling

-voltage dependent Ca²⁺ channels-in the PM and open in response to membrane depolarization and allow Ca²⁺ to enter activated nerve terminals and trigger neurotransmitter secretion

-IP₃-gated Ca²⁺ release channels-allow Ca²⁺ to escape from ER to cytosol when inositol phospholipid signaling pathway activated

-ryanodine receptors-react to a change in PM potential to release Ca²⁺ from the sacroplasmic reticulum and stimulate contraction of muscle cells

- Ca²⁺ in cytosol kept low in resting cells by a Ca²⁺ pump on the PM that uses ATP hydrolysis to pump Ca²⁺ out of the cytosol

- Ca²⁺ pump in ER also keeps cytosolic Ca²⁺ low

- Low affinity, high-capacity Ca²⁺ pump in the inner mitochondrial membrane returns the [Ca²⁺] to normal after a signal-takes up Ca²⁺ from cytosol during oxidative phosphorylation

-Ca²⁺ fluorescent indicators-aequorin or fura-2 used in monitoring cytosolic Ca²⁺ in individual cells after inositol phospholipid signaling pathway has been activated

- Ca²⁺ initially stimulates more release of Ca²⁺, but as the Ca²⁺ conc gets high, then Ca²⁺ inhibits further release of Ca²⁺

Ca²⁺ binding proteins

-transducers of cytosolic Ca²⁺ signal

-calmodulin

-found in all euc cells

-can constitute up to 1% of the total protein mass

-multipurpose intracellular Ca²⁺ receptor

-has highly conserved, single polypeptide chain with 4 high-affinity Ca²⁺ binding sites

- 2 or more Ca²⁺ activates it to undergo a conformational change and this leads it to react in a switch like manner to increasing [Ca²⁺]

-has no enzymatic activity but acts by binding to other proteins

-when bound to a target molecule, calmodulin undergoes a change in conformation

- Ca²⁺ / calmodulin-dependent protein kinases (CaM kinases)

-phosphorylate serines and threonines

-CaM-kinase II

-found in all animal cells

-especially enriched in the nervous system

G-protein linked Receptor Desensitization

-depends on receptor phosphorylation by PKA, PKC or G-protein-linked receptor kinases (GRKs)

-high concentration of stimulating ligand for a prolonged period triggers desensitization

-3 general ways to desensitize

-receptors can become altered so that they no longer interact with G proteins (receptor inactivation)

-can be temporarily moved to the interior of the cell (receptor sequestration)

-can be destroyed in lysosomes after internalization (receptor downregulation)

-GRKs phosphorylate multiple serine and threonines on a receptor, which leads to the receptor to bind with high affinity to the arrestins

-arrestins can desensitize by 2 ways

-inactivates the receptor by preventing it from interacting with G proteins

-can serve as an adaptor protein to couple the receptor to clathrin coated pits to induce receptor mediated endocytosis

Enzyme linked receptors

-involved in responses to EC signal proteins that promote growth, proliferation, differentiation or survival of animal tissues

-usually act as local mediators at very low concs

-responses to them are slow, in the magnitude of hours

-TM proteins

-cytosolic domain has either an intrinsic enzyme activity or associates directly with an enzyme

-6 classes of these receptors

-receptor tyrosine kinases-phosphorylate specific tyrosines

-tyrosine-kinase-associated receptors-associate with intracellular proteins that have tyrosine kinase activity

-receptor like tyrosine phosphatases-remove phosphate groups from tyrosines of specific intracellular signaling proteins

-receptor serine/threonine kinases-phosphorylate specific serines or threonines on associated latent gene reg proteins

-receptor guanylyl cyclases-directly catalyze the production of cyclic GMP in the cytosol

-histidine kinase associated receptors-activate a “two-component” singaling pathway in which kinase phosphorylates itself on histidine and then immediately transfers the phosphate to a 2^nd intracellular signaling protein

-receptor tyrosine kinases

-phosphorylate specific tyrosines

-secreted growth factors and hormones act through these receptors

-EGF

-PDGF

-FGF

-HGF

-IGF-1

-VEGF

-M-CSF

-NGF

-ephrins

-largest class of membrane-bound ligands that act through these receptors, these receptors are known as Eph receptors

-ephrins and Eph receptors simultaneously acts as both ligand and receptor-this is called bidirectional signaling

-bidirectional signaling can keep cells in particular areas from mixing with cells in neighboring parts

-regulate cell adhesion and repulsion responses that guide the migration of cells and axons along specific pathways during animal development

-specifics of receptor binding with ligand in receptor tyrosine kinases

-binding signal protein to the ligand-binding domain activates the intracellular tyrosine kinase domain, sometimes the receptor has already formed a dimer/oligomer before binding, sometimes binding triggers dimerization/oligomerization of receptors-this rearrangement allows neighboring kinase domains of the receptor chains to cross-phosphorylate each other on multiple tyrosines (autophosphorylation)-the ligand has to simultaneously bind 2 adjacent receptor chains

-the kinase domain transfers a phosphate group from ATP to selected tyrosine side chains of the receptor proteins-these phosphorylated tyrosines within the kinase domain increases the kinase activity of the enzyme while the phosphorylated tyrosines outside the kinase domain creates high-affinity docking sites for intracellular proteins-the signaling proteins may also become phosphorylated on tyrosines

-the kinase domain then transfers the phosphate from the receptor to an intracellular signaling protein that subsequently binds with the phosphorylated receptor

-making dominant negative mutants of receptors

-transfect cells with DNA encoding a mutant form of the receptor that oligomerizes but has an inactive kinase domain

-coexpress the mutant receptors at a high level with normal receptors

-the mutant receptor oligomerizes with normal receptors, but does not phosphorylate the normal receptor

Phosphorylated tyrosines serve as docking sites for proteins with SH2 Domains

-phospholipase C-g (PLC-g) functions similarly to PLC-b to activate the inositol phospholipid signaling pathway and increase cytosolic Ca²⁺

-Src-a cytoplasmic tyrosine kinase that phosphorylates other signaling proteins on tyrosine

-phosphatidylinositol 3’-kinase (PI 3-kinase) generates specific lipid molecules in the plasma membrane to attract other signaling proteins there

-all of these share a highly conserved phosphotyrosine-binding domain

-SH2 domain (Src homology region or PRB for phosphotyrosine binding)-enables proteins to bind to proteins that have been phosphorylated on tyrosine

-SH3 domain-enables proteins to bind to praline rich motifs in intracellular proteins

-not all proteins that bind to activated tyrosine kinase via SH2 domains help to relay the signal onward

-c-Cbl decreases the signaling process, providing negative feedback, by coupling the activated receptor with ubiquitin, which promotes the internalization and degradation of the receptors (receptor downregulation)

-adaptor proteins-some proteins are composed almost entirely of SH2 and SH3 domains and function as adaptors to couple tyrosine-phosphorylated proteins to other proteins that do not have their own SH2 domains

Ras signaling

-Ras is a signaling protein that can change the nature of the signal and send a signal along multiple downstream pathways

-mutations that activate the Ras pathway stimulate cell division inappropriately and cause cancer

-adaptor proteins important in Ras signaling

-Ras proteins

-belong to the Ras superfamily of monomeric GTPases

-have a covalently attached lipid group that anchors these proteins to a membrane

-subfamilies

-Rho family-involved in relaying signals from cell-surface receptors to the actin cytoskeleton

-Rab family-involved in regulating the traffic of monomeric GTPases

-can inhibit RAs function by microinjecting neutralizing anti-Ras Abs or by expressing a dominant negative mutant Ras so that the cell proliferation or differentiation responses do not occur

-can stimulate cell proliferation or differentiation without ligand binding of the EC receptor by introducing the expression of a hyperactive Ras mutant

-Ras functions as a switch

-active when GTP bound

-inactive when GDP bound

-guanine nucleotide exchange factors (GEFs) promote the exchange of bound nucleotide by stimulating the dissociation of GDP and the uptake of GTP from the cytosol to activate Ras

-GTPase activating proteins (GAPs) increase the rate of hydrolysis of bound GTP by Ras to inactivate Ras

-hyperactive mutant Ras are resistant to GAP GTPase stimulation and are locked in the GTP-bound active state

-receptor tyrosine kinases can activate Ras by either activating a GEF or by inhibiting a GAP

-GEFs bind indirectly via their SH2 domains to activated receptor tyrosine kinases

-GAPs bind directly via their SH2 domains to activated receptor tyrosine kinases

-Ras adaptor proteins

-Grb-2 binds through SH2 domain to specific phosphotyrosines on activated receptor tyrosine kinases and through its SH3 domains to praline-rich motifs on a GEF called Sos

-Shc-binds to some activated receptor tyrosine kinases that do not display phosphotyrosines required for Grb-2 docking-binds to both the activated receptor and to Grb-2 to couple the receptor to Sos

-receptor-Shc-Grb-2-Sos-complex brings Sos into position to activate neighboring Ras by stimulating the exchange bound GP for GTP

Ras can activate the serine/threonine phosphorylation cascade

-cascade is highly conserved in euc cells

-MAP-kinase (mitogen activated protein kinase) is important in this cascade

-activated Ras can stimulate the conversion of short-lived signals into longer-lasting ones through serine/threonine phosphorylations

-MAP-kinase

-full activation requires the phosphorylation of both threonine and tyrosine, which are one amino acid (aa) apart

-MAP-kinase-kinase catalyzes the phosphorylations of both the threonine and the tyrosine on MAP-kinase

-MAP-kinase-kinase is called MEK in the Ras signaling pathway

-MAP-kinase-kinase-kinase catalyzes the phosphorylation of MAP-kinase-kinase

-MAP-kinase-kinase-kinase is called Raf in the Ras signaling pathway

-Raf is activated by activated Ras

-activated MAP-kinase enters the nucleus and phosphorylates one or more components of a gene regulatory complex

-once the gene regulatory complex is phosphorylated, immediate early genes are transcribed

-immediate early genes are turned on within minutes of the time that the cells are stimulated by an EC signal

-G₁ cyclins, genes involved in cell proliferation, are activated by the MAP-kinase pathway

-MAP-kinases are inactivated by dephosphorylation

Avoiding cross-talk between different parallel signaling pathways to ensure a specific response

-scaffold proteins are used by cells to prevent cross-talk between parallel signaling pathways

-scaffold proteins can bind all or some kinase in a specific module to form a complex

-at least 5 parallel MAP-kinase modules can operate in a mammalian cell

-modules can be composed of at least 12 MAP-kinases, 7MAP-kinase-kinases and 7MAP-kinase-kinase-kinases

-modules can be activated by cell stresses such as:

-UV irradiation

-heat shock

-osmotic stress

-stimulation by inflammatory cytokines

-scaffolding helps

-provide precision

-to create a large change in MAP-kinase activity in response to small changes in signal molecular concentration

-avoids cross-talk

PI 3-Kinase

-signals cells to survive and grow

-phosphorylates inositol phospholipids

-activated by receptor tyrosine kinases and other cell surface receptors, e.g. G-protein linked ones

-catalyzes the phosphorylation of inositol phospholipids, phosphatidylinostiol (PI) at the 3 position of the inositol ring to generate the following two lipids:

-PI(3,4)P₂

-PI(3,4,5)P₃

-these two lipids serve as docking sites for intracellular singlaing proteins to bring the proteins together into signaling complexes

-these lipids are not cleaved by PLC

-these lipids remain in the PM until they are dephosphorylated by specific inositol phospholipid phosphatases that remove phosphate from the 3 position of the inositol ring

-mutations in such phosphatases cause cancer

-intracellular proteins bind to these lipids through their Pleckstrin homology (PH) domain

-mutations in the PH of BTK results in genetic immunodeficiency in humans and mice

-BCRs on B cells activate PI 3-kinase and the resulting inositol lipid docking sites recruit both BTK and PLC-g to the cytoplasmic face of the PM

-BTK phosphorylates and activates PLC-g

- PLC-g cleaves PI(4,5)P₂ to generate IP₃ and diacylglycerol to relay the onward signal

-if there is a mutant in BTK, the BTK cannot bind the lipid docking sites produced after receptor activation, so the receptors cannot signal the B cells to proliferate or survive, resulting in severe Ab production deficiency

-Several types of PI 3-kinases

-composed of

-one catalytic and one regulatory subunit

-reg subunit is an adaptor protein that binds phosphotyrosines on activated receptor tyrosine kinases through SH2 domains

-activated by receptor tyrosine kinases

-one catalytic and one reg subunit, but the reg subunit is different

-activated by the bg complex of a trimeric G protein when G-protein linked receptors are activated by their EC ligand

-the catalytic subunit in both cases has a binding site for activated Ras

-PI 3-kinase/Protein Kinase B Signaling Pathway

-PI 3-kinase signals cells to survive by indirectly activating protein kinase B (PKB or also called Akt)

- PI 3-kinase gets activated through a EC survival signal

-PKB gets directed to the PM through its PH domain to activated PI 3-kinase

-PKB binds to PI (3,4,5)P₃ on the cytosolic face of the PM

-then PKB alters its conformation and can be phosphorylated by PDK1, which is recruited to the membrane in the same way and is phosphatidyl inositol dependent

-once PKB is phosphorylated by PDK1, it returns to the cytoplasm and can phosphorylate BAD, which encourages cells to undergo apoptosis-phosphorylating BAD inactivates BAD to promote cell survival-PKB inhibits other cell death activators, too

-PI 3-kinase signals stimulate cell growth by increasing protein synthesis

-PI 3-kinase signaling leads to the activation of S6 kinase through PDK1

-S6 kinase phosphorylates the S6 subunit of ribosomes-this helps to increase the translation of mRNAs that encode ribosomal proteins and other proteins involved in translation

Include Figure 15-61

Receptors without tyrosine kinase domains

-some receptors depend on tyrosine phosphorylation, but lack a tyrosine kinase domain

-these receptors rely on cytoplasmic tyrosine kinases that associate with the receptors to phosphorylate target proteins, including even the receptor after the receptor has bound its ligand

-such cytoplasmic tyrosine kinases noncovalently associate with the receptors with the oligomerized receptor

-Src family tyrosine kinases are often associated with such receptors

-they are associated noncovalently with the receptor through their SH2 and SH3 domains while also having a covalently attached lipid chain that associates with the PM

-Lyn, Fyn, and Lck are associated with different receptors in lymphocytes

-integrins are associated with cytoplasmic tyrosine kinases

-integrins are receptors that bind to the EC matrix

-the EC matrix can be composed of laminin or fibronectin

-such binding to the EC matrix can affect the cell’s behavior

-integrins cluster at the site of matrix contact to form focal adhesions

-the integrins indirectly bind to the actin cytoskeleton at these focal adhesions

-focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is recruited to an integrin after EC matrix binding

-clustered FAK cross-phosphorylate each other to create a phosphotyrosine docking site where Src kinase binds

-Src and FAK phosphorylate each other and other proteins in the junction to signal to the cell that it has adhered to a suitable substratum so that the cell now has a place to grow, divide, migrate, etc.

Cytokine Receptors

-subfamily of enzyme-lined receptors

-largest and most diverse class of receptors that rely on cytoplasmic kinases

-stably associated with the tyrosine kinases known as Janus kinases (Jaks)

-Jaks get activated by cytokine receptor binding to its ligand

-Jaks activate by phosphorylation the latent gene regulatory proteins called STATs that are located at the cell surface-STAT stands for signal transducers and activors of transcription

-all STATs have a SH2 domain that enables them to dock onto some activated receptor tyrosine kinases independent of Jak

-this and the fact that STATs, but not Jaks are made in C. elegans suggests that STATs evolved before Jaks

-cytokine or hormone binding causes STATs to migrate to the nucleus and activate gene transcription-

-Jak-STAT signaling pathway

-one of the most direct routes for signaling pathways leading to the nucleus

-initially discovered on the effects of interferons

-interferons bind to receptors on noninfected neighboring cells and induce cells to produce proteins that increase their resistance to viral infection

-Cytokine receptors are composed of 2 or more polypeptide chains

-some chains are specific to a cytokine receptor and some chains are shared among other cytokine receptors

-all cytokine receptors are associated with one or more Jaks

-four Jaks exist

-Jak1

-Jak2

-Jak3

-Tyk2

-IFN-a is associated with Jak1 and Tyk2

-there is a typ-o in the table in Alberts on p. 884, Jak2 is not associated with IFN-a

-IFN-g is associated with Jak1 and Jak2

-there are 7 known STATs

-each STAT SH2 domain recognizes specific docking sites on the receptor, so each cytokine has specific STATs that get recruited to it

-cytokine binding either induces the receptor chains to oligomerize or reorients the chains in a pre-formed oligomer

-cytokine binding brings the associated Jaks close enough to cross-phosphorylate each other and to increase the activity of their tyrosine kinase domains

-Jaks phosphorylate the tyrosines on the cytokine receptors, which creates phosphotyrosine docking sites for STATs and other signaling proteins

-the SH2 domain of the STAT then performs 2 functions

-1-mediates the binding of the STAT protein to a phosphotyrosine docking site on an activated cytokine receptor

-Jaks then phosphorylate the STATs on tyrosines leading to the STAT being released from the receptor

-2-the SH2 domain on the released STAT mediates its binding to a phosphotyrosine on another STAT to form a STAT homodimer or heterodimer

-the STAT dimer then moves to the nucleus and combines with other gene reg proteins and to bind to the specific cytokine DNA response element in various genes to stimulate transcription

-responses mediated by STATs are regulated by negative feedback

-STAT dimers can also activate genes that encode inhibitory proteins

-sometimes the inhibitor binds to both the activated cytokine cytokine receptor and the STAT proteins to block further STAT activation

-other times the inhibitor can block Jak function

-Jaks and STATS also have to be dephosphorylated at their phosphotyrosines by protein tyrosine phosphatases in order to fully turn off the response

Protein Tyrosine Phosphatases (PTPs) that act as Cell-Surface Receptors

-~30 PTPs in the human genome

-not structurally related to serine/threonine protein phosphatases

-occur in cytoplasmic and TM forms

-TM PTPs

-many TM PTPs exist, and many of the functions are unknown

-some thought to function as receptors and are called receptorlike tyrosine phosphatase (RTPs)

-all RTPs have a single TM segment and usu have 2 tyrosine phosphatase domains on the cytosolic side

-e.g. CD45

-found on the surface of all white blood cells

-involved in T and B cell activation by foreign Ags

-ligand presumed to bind to the EC domain of CD45, but the ligand has not been identified

-cell-adhesion RTPs

-mediate homophilic cell-cell binding in cell adhesion

-some TM PTPs serve as signaling ligans to activate receptors on a neighboring cell

-cyoplasmic PTPs

-with SH2 domains

-SHP-1-dephosphorylates activated Jak

-SHP-2

-these two can also help terminate responses mediated by some receptor tyrosine kinases

Enzyme Linked Receptors relying entirely on serine/threonine phosphorylation

-transforming growth factor-b (TGF-b)

-large family of structurally related, secreted, dimeric proteins

-superfamily = TGF-bs, activins and bone morphogenetic proteins (BMPs)

-act as hormones or as local mediators

-often secreted as inactive precursors that are subsequently activated by proteolytic cleavage

-in development, influence proliferation, differentiation, EC matrix production, cell death

- TGF-bs serve as graded morphogens to induce different responses in a developing cell depending on their concentration

-different amounts of active Smad (see below, but a complex formed later in the signaling process) in the nucleus induce different responses

-perhaps the DNA binding sites of the target genes have different affinities for the complexes

-in adults, involved in tissue repair and immune regulation

-these molecules all act through enzyme-linked receptors

-enzyme linked receptors interacting with the TGF-b superfamily

-single-pass TM proteins

-have serine/threonine kinase domain on cytosolic side

-2 classes of receptor serine/threonine kinases

-type I

-type II

-each member of the TGF-b superfamily interacts with a specific combination of these types of receptors

-both receptors are required for signaling

-after the receptor complex gets activated, the type I receptor directly binds and phosphorylates the Smad protein, which is a latent gene reg protein

-activated TGF-b and activin receptors phosphorylate Smad2 or Smad3 while activated BMP receptors phosphorylate Smad1, Smad5, or Smad8

-phosphorylated Smad dissociates from the receptor and binds to Smad4, which can form a complex with any of the other five activated Smads

-Smad complex then moves into the nucleus

-associates with other gene reg proteins and binds to specific sites in DNA and activate target genes

-feedback inhibition of the Smad pathway

-inhibitory Smads

-Smad 6

-Smad 7

-these inhibitory Smad target genes get activated by Smad complexes

-act as decoys

-bind to activated type-I receptors to prevent other Smads from binding there and to subsequently block the formation of active Smad complexes

-EC ligands that inhibit the Smad pathway

-IFN-g activates the Jak-STAT pathway

-the STAT dimmers induce the production of Smad7 to inhibit TGF-b signaling

-EC inhibitory proteins also bind to signal molecules to prevent them from activating their receptors on target cells

Receptor Guanylyl Cyclases (RGCs)

-single-pass TM receptors with intracellular guanylyl cyclase catalytic domain

-Mechanism for pathway

-receptor binds EC ligand

-the cyclase domain then gets activated

-cyclic GMP is then produced

-cyclic GMP binds to and activates PKG (cyclic GMP dependent protein kinase)

-PKG phosphorylates target proteins on serine or threonine

-signal molecules for RGCs

-natriuretic peptides-regulate salt and water balance and dilate blood vessels

Bacterial Chemotaxis and Two-Component Signaling

-Two-component signaling pathway, e.g. chemotaxis receptors of E.coli flagellum

-histidine-kinase associated receptors

-dimeric M proteins

-bind specific attractants and repellents on the outside of the PM

-the cytoplasmic tails are stably associated with CheW, an adaptor protein, and CheA, a histidine kinase

-these couple the receptors to the flagellar motor

-attractant binding inactivates the receptors

-repellent binding activates the receptors and activates CheA

-then, CheA then phosphorylates itself on a histidine

-then CheA immediately transfers the phosphate to an aspartic acid on a messenger protein CheY

-phosphorylated CheY dissociates from the receptor, diffuses into the cytosol, binds to the flagellar motor, and causes the motor to rotate clockwise so that the bacterium tumbles

-CheY with its intrinsic phosphatase activity dephosphorylates itself with the aid of CheZ

Signaling Pathways that depend on regulated proteolysis

-4 signaling pathways

-Notch-mediated

-Wnt-mediated

-Hedgehog- mediated

-NF-kB-dependent

-Notch

-most widely used signaling pathway in animal development

-controls cell fate choices during development by amplifyin and consolidating molecular differences between adjacent cells

-lateral inhibition

-one cell among a neighborhood of cells develops along one fate while the other cells in the neighborhood do not develop in the same way as the single cell

-depends on the signal molecule Delta, which gets displayed on the one cell that is differentiating from its neighbors

-when Delta binds with Notch on a neighboring cell, the neighboring cell gets the signal to not differentiate into the cell that has already committed to differentiating

-Deltalike ligand Serrate can also be used by the differentiating cell to determine cell fate within a neighborhood of cells

-characteristics of Notch and Delta

-both are single-pass TM proteins

-both require proteolytic processing to function

-unknown why Delta gets cleaved

-Notch cleavage activates Notch to alter gene expression in the nucleus

-Mechanisms of Notch signaling

-Notch binds Delta

-intracellular protease cleaves off the cytoplasmic tail of Notch

-the released tail moves into the nucleus and binds the gene regulatory protein CSL to convert it from a transcriptional repressor to a transcriptional activator

-transcription of Notch-response genes occurs

-these response genes have an inhibitory action by blocking the expression of genes that result in the one cell differentiating into a separate fate from its neighbors

-Notch undergoes 3 proteolytic cleavages

-during the normal biosynthesis of Notch, the protease furin acts in the Golgi apparatus to cleave the newly synthesized Notch protein

-this cleavage converts Notch to a heterodimer, which is then transported to cell surface as a mature receptor

-when Delta binds Notch, a second cleavage, mediated by another protease, occurs in the EC domain

-then the cleavage by presenilin-1 (PS-1) of the cytoplasmic tail of the activated receptor occurs-this cleavage is close to the PM

-Notch signaling is regulated by glycosylation

-the Fringe family of glycotransferases adds extra sugars to the O-linked oligosaccharide on Notch to alter the specificity of Notch for its ligands

Wnt pathway

-Wnt proteins

-secreted signal molecules acting as local mediators to control development

-receptors for Wnts are part of the Frizzled family of 7-pass TM proteins

-resemble G-protein-linked receptors in structure and some can signal through G proteins and the inositol phospholipid pathway

-require Dishevelled, a cytoplasmic signaling protein involved in G-protein independent pathways

-Dishevelled dependent pathway

-regulates the proteolysis of b-catenin (aka.Armadillo)

- b-catenin is involved in cell-cell adhesion and is a latent gene regulatory protein

-with Wnt signaling

-Wnts activate the disheveled by binding Frizzled and a co-receptor protein which is called LDL-receptor-related protein (LRP), but the exact mechanism for activation is not known

-inhibition of b-catenin phosphorylation and degradation occurs from Frizzled and LRP binding to Wnt protein

-Dishevelled is bound to casein kinase1 through some unknown mechanism of activation

-unphosphorylated b-catenin accumulates in the cytoplasm and nucleus

-the b-catenin in the nucleus binds an inhibitory complex, the LEF-1/TCF family proteins bound to Groucho-this inhibitory complex normally silences the target genes for Wnt signaling

- b-catenin displaces Groucho in the inhibitory complex, and the b-catenin functions as a coactivator to induce transcription of Wnt target genes

-c-myc is transcribed and c-Myc protein stimulates cell growth and proliferation

-without Wnt signaling, most of the cells b-catenin is located at the cell-cell adherens junctions where it is associated with cadherins (TM adhesion proteins)

-b-catenin helps link the cadherins to the actin cytoskeleton

-any b-catenin not associated with cadherins are rapidly degraded in the cytoplasm

-degradation complex of b-catenin composed of three proteins

-glycogen synthase kinase-3b (GSK-3b)- a serine/threonine kinase that phosphorylates b-catenin to mark it for ubiquitinylation

-adenomatous polyposis coli (APC) –tumor suppressor protein that degrades b-catenin by increasing the affinity of the degradation complex for b-catenin

-axin-a scaffold protein that holds the complex together

Hedgehog Signaling Pathway

-Hedgehog proteins (Hh)

-family of secreted signal proteins

-act as local mediators

-active form of protein is covalently coupled to cholesterol

-this helps to restrict Hh diffusion following secretion

-cholesterol added after Hh cleaves itself

-Hh proteins also modified by addition of fatty acid chain, which may be required for singaling activity

-3 genes encode hedgehog proteins

-sonic hedgehog-

-desert hedgehog

-indian hedgehog

-Receptors for Hh

-Patched

-may be a 12-pass TM protein

-binds Hh protein

-without Hh signal, Patched inhibits Smoothened activity

-Smoothened

-7-pass TM protein

-structure similar to Frizzled

-Hh signaling pathway

-without Hh signal

-gen reg protein, Cubitus interruptus (Ci), is proteolytically cleaved in proteasomes

-instead of being completely degraded, processed to smaller protein that accumulates in the nucleus which then acts as a transcriptional repressor of Hh responsive genes

-proteolytic processing of Ci done by multiprotein complex

-Fused-serine/threonine kinase

-Costal-anchoring protein that binds the complex to microtubules to keep Ci out of the nucleus

-Suppressor of Fused-adaptor protein

-with Hh signal

-Ci processing suppressed

-unprocessed Ci protein gets released from the complex and enters the nucleus

-activates transcription of Hh responsive genes

-wingless gets activated by Ci and helps pattern tissues

-patched gets activated and the increase in Patched on the cell surface inhibits further Hh signaling-form of negative feedback

-in vertebrates (above is in fruitflies)

-3 Hh proteins

-2 Patched proteins

-3-Ci like proteins (Gli1, Gli2, Gli3)

-unlike in inverts, vert Hh signaling stimulates the transcription of Gli genes

-mutants in patched result in excessive Hh signaling and therefore cancer, most frequent cause of skin cancer, so Patched may keep skin cell proliferation regulated

NF-kB Dependent Signaling Pathway

-latent gene reg proteins

-involved in most inflammatory responses to protect cells from stresses

-involved in intercellular signaling during normal vertebrate development

-EC signals that activate NF-kB are unknown

-in fruitflies the NF-kB dependent pathway is involved in dorsal-ventral pattern development and in defending the fly from infection

-vertebrate cytokines involved in inducing inflammatory responses that are dependent on NF-kB

-TNF-a

-IL-1

-both made by macrophages in response to infection/tissue injury

-both bind to cell-surface receptors that activate NF-kB

- NF-kB normally sequestered in an inactive form in the cytoplasm of almost all of our cells

- activated NF-kB activates the transcription of more than 60 known genes that participate in inflammatory responses

-5 NF-kB proteins in mammals

-RelA

-RelB

-c-Rel

- NF-kB1

- NF-kB2

-all of these form a variety of homodimers and heterodimers to activate its own set of genes

-inhibitory proteins

-IkB bind tightly to dimers and hold them in an inactive state within large protein complexes in the cytoplasm

-Mechanism of NF-kB dependent signaling

-TNF-a or IL-1 activate the dimers by triggering a pathway to phosphorylate, ubiquitylate, and degrade IkB

-ligand binding causes the cytosolic tails of the clustered receptors to recruit various adaptor proteins and cytoplasmic serine/threonine kinases

- IkB kinase kinase (IKKK) is likely recruited and directly phosphorylates IkB kinase (IKK)

- IkB kinase (IKK) phosphorylates IkB

-degradation of IkB exposes a nuclear localization signal on NF-kB

- NF-kB then moves into the nucleus and stimulates transcription of specific genes

-not all the signaling proteins recruited to the cytosolic tail of TNF-a receptor contribute to NF-kB activation

-some trigger a MAP-kinase cascade

-others activate a proteolytic cascade that leads to apoptosis