Chapter 16, Growth Factor and Cytokine Signaling Pathways That Control Gene Expression

bolded terms and pathways defined

Differentiation

A population of cells slowly acquiring discrete fates and becoming specialized mature cells

Processes like this control embryonic development of all metazoans

“Given the role of gene transcription in mediating these critical aspects of development, is should come as no surprise that altered activity of these pathways is responsible for many human diseases including cancer, diabetes, and immune-system disorders.”

special transcription factors

that alter expression of a set of target genes that promote the needed change in cell behavior

stimulate or inhibit transcription of specific genes by binding to other DNA regulatory elements, called cis-regulatory elements (CREs aka enhancers). They regulate assembly or function of the RNA polymerase complex and in doing so control the on/off state of the target genes.

• each specific transcription factor has affinity for a unique DNA sequence and therefore only bind CREs that have these sequences in them

• For each specific transcription factor there are hundreds of target genes in the genome with CREs they can bind

General transcription factors

are required to transcribe all genes. they associate with RNA polymerase on the promoter of all genes to initiate transcription.

epigenetic state

transduction pathways that activate the same specific transcription factor in different cell types may promote expression of different target genes

½ major reasons for this

epigenetic state:

• not all regions of the genome are euchromatin (open chromatin) and therefore can be transcribed

• Heterochromatin (closed chromatin) is tightly packed due to covalent modifications to histone proteins and the DNA itself

• Regions of euchromatin vs heterochromatin vary from cell type to cell type - dependent on their developmental history

therefore, the same specific transcription factor activated in different cell types has different target genes it can bind and regulate.

Combinatorial control

transduction pathways that activate the same specific transcription factor in different cell types may promote expression of different target genes

½ major reasons for this

combinatorial control:

• Most CREs are bound and regulated by multiple distinct specific transcription factors activated by different transduction pathways

• A gene can be regulated only in cells that activate the right combination of specific transcription factors to binds its CRE

therefore, the same specific transcription factor activated in different cell types may regulate a different set of target genes based on the combination of other specific transcription factors also activate in that cell.

Protein-tyrosine kinase

family of kinases which phosphorylate specific tyrosine residues on target proteins

Receptor tyrosine kinase (RtK)

Cell surface reseptors that transduce signals from growth factors.

General features:

• a single pass ⍺-helix transmembrane domain

• an extracellular ligand binding domain

• a cytosolic tyrosine kinase domain that is inactive when the receptor is not ligand bound

• a cytosolic activation loop: switch that activates the kinase domain

requires ligand induced/ mediated/ dimerization

Growth factors

Ligands that stimulate cell growth and proliferation

An extracellular polypeptide molecule that binds to a cell-surface receptor, triggering an intracellular signaling pathway generally leading to cell proliferation

Activation Loop

A region in most protein-tyrosine kinases, containing a tyrosine residue that, when phosphorylated, increases kinase activity

Ligand Induced Dimerization

Ligand binding induces RTK receptors to dimerize: A conformational change in the extracellular domain promotes molecular complementarity and dimerization.

bings two cytosolic domains together

basal kinase activity allows each RTK to auto-cross phosphorylate: they phosphorylate one another’s activation loop

this induces conformational change that triggers full kinase activity

homodimerization

between two identical proteins

heterodimerization

two similar but distinct proteins

auto-cross phosphorylate

basal kinase activity allows each RTK to auto-cross phosphorylate: they phosphorylate one another’s activation loop

Ligand mediated dimerization

Activated RTKs then auto-cross phosphorylate one another on multiple specific tyrosine residues within their cytosolic domains

these phosphotyrosines serce as docking sites for adapter proteins that transduce the signal to the cell interior

there are multiple phosphotyrosines on fully activated RTKs

Fibroblast

A common type of connective tissue cell that secretes collagen and other components of the extracellular matrix; migrates and proliferates during wound healing and in tissue culture

Fibroblast Growth Factor (FGF)

ligands induce FGFR dimerization

Fibroblast Growth Factor Receptor

Family of RTKs that transduce signals during development that regulate cell proliferation, migration, differentiation, and survival

major example of ECM involvement in cell-cell signaling:

• FGF binding to FGFRs require association with the negatively charged heparan sulfate GAGs of ECM proteoglycans

Epidermal Growth Factors (EGFs)

family of growth factors that promote cell proliferation

Human epidermal growth factor

HER 1-4

ligands binding to HERs promotes extension of dimerization and subsequent dimerization and activation via auto-cross phosphorylation

FGF and EGF and proliferation

FGF and EGF pathways promote proliferation

both sets of ligands and the pathways they activate/ promote proliferation

critical during embryogenesis and wound healing

mutations that inappropriately activate FGF and EGF pathways are major initiators of tumor formation and the onset of cancer

Adapter proteins

Adapter proteins physically link one protein to another protein by binding to both of them. Adapter proteins directly or indirectly (via additional adapters) connect cell-adhesion molecules or adhesion receptors to elements of the cytoskeleton or to intracellular signaling proteins

the phosphotyrosines on RTK dimers are bound by these transducting proteins

Docking domain

Adapter protein families share conserved docking domain that recognizes phosphotyrosines within specific amino acid sequences

this provides specificity that determines which pathways are regulated by each type of RTK

SH2 domain (Src Homology)

Most well-studied docking domain

there are over 100 human proteins that possess SH2 domains (the SH2 family)

Sub-family members have subtle differences in the SH2 domain that generates unique specificity for phosphotyrosines and therefore which receptor that SH2 proteins can bind

Attenuating the signal after RTK activation

Once signal transduction occurs the cell need to terminate signaling.

Receptor mediated endocytosis and lysosomal degradation prevents overstimulation of the process the RTK regulates

receptors are mono-ubiquitinated promoting endocytosis and lysosomal degradation in multivesicular bodies

EX) acivated HER receptors are ubiquitinated by a specific E3-ubiquitin ligase called c-Cbl

• loss of function of c-Clb prevents appropriate attenuation of HER receptor signaling and is a driver of several forms of cancer the protein’s acronym name stands for Casitas B-Cell Lymphoma

Ras/MAPK signal transduction pathway

one of the most common cell proliferation pathways

almost all RTKs activate the Ras/MAP kinase pathway

functions by activating a MAP kinase cascade

uses the small monomeric G-protein Ras as the effector switch (Ras= Rat Sarcoma virus)

Activated Ras stimulates a MAPK cascade that controls cell cycle progression, proliferation and/or differentiation

Health relevance

• most human cancers have gain of function mutations in an RTK, Ras, or a kinase in the pathway

• Cells with mutants Ras constantly divide because the pathway is always active, even through there is no growth factor activating a receptor

Canonical

• Ligand (FGF, EGF, PDGF, etc) induces receptor dimerixation and auto cross-phosphorylation

• Activated RTK bound by adapter protein Grb2 via its SH2 domain

• Grb2 recruits the Ras GEF called SOS

• SOS induces Ras to exchange GDP for GTP, activating it

• Activated Ras recruits and acivates the first kinase in the cascade (the MEKK)

MAP kinase cascade

A series of three kinases that consecutively phosphorylate and activate the next kinase in the cascade

consist of three serine/ threonine kinases activated downstream of the receptor

• MEKK

• MEK

• MAP Kinase

  • terminal MAPK phosphorylates and activates several cytoplasmic enzymes that perform an immediate response

  • it translocates into the nucleus and phosphorylates specific transcription factors that regulate expression of genes needed for the response.

they phosphorylate each other in that order

microtubule-associated protein (MAP)

any protein that binds to microtubules and regulates their stability

MAP kinase

mitogen activated protein kinase

any of a family of protein kinases that are activated in response to cell stimulation by many different growth factors and that mediate cellular responses by phosphrylating specific transcription factos and other target proteins

Ras protein

A monomeric member of the GTPase superfamily of switch proteins that is tethered to the plasma membrane by a lipid anchor and functions in intracellular signaling pathways; activated by ligand binding to receptor tyrosine kinases and some other cell-surface receptors

MEKK

The Ras MAPK cascade

• The MEKK is called Raf (Rapidly Amplifying Fibrosarcoma)

• In unstimulated cells Raf is bound to an inhibitory protein called 14-3-3

• Activated Ras binds and releases Raf from 14-3-3

• Raf dimerizes and autocross phosphorylates

MEK

The Ras MAPK cascade

• Activated Raf phosphorylates the next kinase, MEK (MAP Kinase of ERK Kinase)

• Activated MEK phosphorylates the terminal MAPK, ERK (Extracellularly regulated kinase)

• ERK dimerizes, phosphorylates cytoplasmic proteins and translocates into the nucleus

• the pathway is silenced when Ras hydrolyzes GTP to GDP (often aided by a GAP)

MAPK

RTK activation as a mechanism for signal transduction: The Ras MAPK pathway

critical targets of MAPK:

• the cytoplasmic kinase p90^R5K

• And the nuclear specific transcription factor Ternary Complex Factors (TCF)

• p90^R5K translocates into the nucleus and phosphorylates and activates the TCF co-factor Serum Response Factors (SRF)

• cooperatively, TCF and SRF bind enhancers of early response genes- whose protein products push a cell from G1 of the cell cycle into S phase

Scaffold proteins

Proteins that participate in intracellular signaling by binding several proteins in a signaling pathway and localizing them to a specific region of the cell

isolate distinct MAPK pathways in the same cell from one another

aids in specificity by tethering proteins of the pathway in specific spatial ortientations

ensures that activation of one MAPK pathway by a particular extracellular signal does not lead to inappropriate activation of other pathways containing shared components

Phosphoinositide signal transduction pathways

PI kinases add phosphate groups to specific -OH groups of inositol carbons

some GPCR pathways activate Phospholipase C which cleaves PIP₂ releasing IP₃ and DAG as second messengers

some RTK pathways activate phospholipase C_𝛾 which also cleaves PIP₂ releasing IP₃ and DAG

As with GPCR signaling IP₃ promotes Ca²⁺ release from the ER and DAG activates Protein Kinase C family proteins

PI₃ Kinase modify PIs to activate diverse downstream kinases

• some RTKs phosphorylate and activate phosphoinositide 3-kinases

• Active PI₃K phosphorylates the 3’ carbon on one of two phosphoinositide substates

  • PI 4-phosphate

  • PI 4,5-disphosphate

• yielding two distinct phosphatidylinositol 3-phosphates

  • PI 3,4 bisphosphate

  • PI 3,4,5 triphosphate

• These phosphorylated PIs activate a kinase family that transduces the signal: Protein Kinase B family

Protein Kinase B

Cytosolic enzyme that is recruited to the plasma membrane by signal-induced phosphoinositides and subsequently activated; also called Akt

Akt

A cytosolic serine/threonine kinase that is activated following binding to PI 3,4-bisphosphate and PI 3,4,5-trisphosphate; also called protein kinase B

Phosphoinositides

A group of membrane-bound lipids containing phosphorylated inositol derivates; some function as second messengers in several transduction pathways

Phosphoinositide 3-Kinase pathways (PI3K)

Protein Kinase B (Akt) binds PI 3,4-bisphosphate via a domain called the PH domain (plectin homology) which normally hides PKB’s kinase domain

Binding to PI3 exposes PKB Ser/Thr residues that are phosphorylated by another PI3,4-bP regulated kinase PDK1 (and a cytoplasmic PDK2, part of the MTOR complex)

ONce activated, PKB dissociates, and phosphorylates target proteins in a cell dependent manner. Principle celular functions are:

• Anti-apoptosis (survival)

• Pro-proliferation

Human Health Relevance:

• Negative regulation: PKB activity is inhibited by the phosphatase PTEN, which not only removes activating phosphates from PKB, but also from PI 3,4-bP

• PTEN is deleted in many types of advanced cancers which contributes to uncontrolled growth and evasion of the programmed cell death pathway

Cytokine receptors

Are non-covalently associated with cytoplasmic kinases what are activated following ligand binding. the term cytokine is interchangeable with growth factor.

Cytokine receptor signaling is also a major promoter of cell proliferation

Member of major class of cell-surface signaling receptors, including those for erythropoietin, growth hormone, interleukins, and interferons. Ligand binding leads to activation of cytosolic JAK kinases associated with the receptor, thereby initiating intracellular signaling pathways.

Erythropeietin (Epo)

A cytokine that triggers production of red blood cells by inducing the proliferation and differentiation of erythroid progenitor cells in the bone marrow.

Hormone released by our kidneys that triggers production of red blood cells (erythrocytes). It induces proliferation and differentiation of erythroid progenitor cells from stem cells in the bone marrow.

Health Relevance:

• Epo is often given to patients receiving cancer therapy that reduces production of red blood cells

Cytokine

Any of numerous small, secreted proteins (e.g., erythropoietin, G-CSF, interferons, interleukins) that bind to cell-surface receptors on blood and immune-system cells to trigger their differentiation or proliferation.

JAK/STAT Signaling Pathway

Example of cytokine receptor signaling

• Cytokine receptors do not have kinase domains but are non-covalently associated with members of a tyrosine kinase family JAKs (Janus Kinase)

• require ligand induced dimerization

• JAKs then phosphorylate receptor tyrosines generating docking sites for SH2 domain containing adapter proteins

• Like RTKs this allows for multiple pathways to be simultaneously activated

• all JAKs activate STAT proteins

• when STATs bind phosphotyrosine of activated receptor they are phosphorylated by the JAKs

• Phosphorylated STATs dissociate from receptor, homodimerize which exposes their nuclear localization signal, promoting entry into nucleus and gene activation

JAKs

Protein Tyrosine Kinases, like RTKs they auto-cross phosphorylate each other’s activation loop; enhancing their kinase activity.

STAT

A family of specific transcription factors that promote cell proliferation and inhibition of apoptosis

TGF-β/ Smad signaling pathway

Ligands form heterodimers or heterotrimers

Three required receptors:

• RIII: binds all concentrates ligands at cell surface

• They then transfer the ligands to RIII receptors, constitutively active Ser/Thr kinases that only phosphorylate class RI receptors (Constitutive means always on)

• RII ligand binding alters conformation that allows them to bind RI receptors in a heterotetramer- 2 copies each of RII and RI

• RII phosphorylates RI on conserved Ser/THr residues

• RI kinase is active once phosphorylated

• RI receptors phosphorylate Smad proteins: specifically, Smad2 or 3. This changes SMAD conformation and unmasks a nuclear localization signal

• The Smads then bind Smad4 (its called a co-SMAD) and translocate into nucleus

• In the nucleus the Smad complex binds enhancers and promotes transcription of target genes

• Often collaboration with other transcription factos (like TFE3 in this example).

Smads

Class of transcription factors that are activated by phosphorylation following binding of members of the transforming growth factor beta (TGFβ) family of signaling molecules to their cell-surface receptors

Transforming Growth Factor Beta (TGFβ)

A family of secreted signaling proteins that are used in the development of most tissues in most or all animals. Members of the TGFβ family more often inhibit growth than stimulate it. Mutations in TGFβ signal transduction components are implicated in human cancer, including breast cancer.

Transforming growth factor. Large superfamily of related signaling ligands (~3 dozen encoded by human genome)

main role is to inhibit cell growth and proliferation

Ski-mediated down-regulation of Smad

Negative feedback loops regulate TGF-β/ Smad signaling

Ski is a TGF-β transcriptional target (Ski= Sloan kettering Institute)

Ski protein directly represses Smad function by preventing Smad4 from binding phosphorylated Smad3

Ski and a related protein Sno were identified as cancer-promoting oncoproteins. Their expression is elevated in many cancers.

The growth-inhibitory activity of TGF-β is blocked in cancer when these repressors are over-expressed

Wnt signaling pathway

an example of pathways controlled by ubiquitination and protein degradation

Wnt pathway promotes cell proliferation and differentiation. In absence of Wnt ligand:

• nucleus: transcription factor called TCF (T Cell Factor) bind target gene enhancers with a transcriptional repressor called Groucho- target genes are silent.

• cytoplasm: β-catenin is bound by the destruction complex (Axin, APC, and the kinases CK1 and GSK3)

• GSK3 phosphorylates β-cateinin which targets β-catenin for ubiquitination and proteasomal degradation

• Wnt ligand binds its receptor Frizzled (and a co-receptor)

• THe activated receptor binds Axin of the Destruction Complex protein and the complex is disrupted

• β-catenin is no longer degraded- it becomes stabilized

• Free β-catenin translocates into nucleus and binds TCF, displacing repressor Groucho

• Bound to β-catenin TCF activates expression of the same genes it was previously repressing.

Human Health Relevance:

90% of human colon cancers have hyperactive Wnt signaling. This primarily happens via loss of function mutations in a gene encoding one of the three Destruction Complex proteins APC

APC

adenomatous polyposis coli

Dominant loss of function mutations in the APC gene prevent activity of the destruction complex

Morphogen

A signaling molecule whose concentration determines the fate target-differentiating cells

Morphogen Function

A signaling protein that is secreted by a cell or group of cells and diffuses away creating a concentration gradient of signaling molecules.

Wnt signaling is a good example

Cells receiving the signal respond differently to different concentrations of the morphogen.

This is a critical process that drives the establishment of different cellular fates across a field of cells with similar developmental potential.

Hedgehod (Hh) signaling

Hedgehog signal proteins also act as morphogens on nearby cells promoting proliferation and differentiation

3 human family members:

• Desert Hh

• Indian Hh

• Sonic Hh

Mutations in the Sonic Hh pathway are associated with a number of early congenital development defects.

Vertebrate Hedgehog (Hh) reception occurs only in primary cilia

In the absence of signal the Hh receptor Patched (Ptc) prevents a protein called Smoothened (Smo) from being inserted into the plasma membrane.

Smo remains in cytoplasm vesicles near the base of the primary cilium

A cytoplasmic protein complex phosphorylates a transcription factor Gli (glioblastoma) promoting its proteolytic cleavage into a transcriptional repressor- GliR

When Hh binds Ptc they are endocytosed and degraded by lysosomes

Smo enters the plasma membrane and is transported to tip of the primary cilium where Smo promotes dissociation of the Gli inhibiory complex

Gli is no longer cleaved Full length GliA (active) accumulates in the cytosol and translocates into the nucleus

GliA replases GliR and activates expression of the same genes GliR had been repressing.

Primary Cilium

The single nonmotile cilium present on almost all vertebrate cells that serves as a sensory organelle to detect extracellular signals.