13 Cytokine Receptors, JAK/STAT, PI3K|Tulane

Cytokine Receptors, JAK/STAT, PI3K and TGFb/SMAD signaling pathway

Central themes in the activation of RTKs

• Ligands induce dimerization of receptors and activation of kinase by cis- or trans-phosphorylation of activation lip.
• Recruitment of adaptor proteins to p-Tyr (RTKs) via SH2 domain and activation of RAS through SOS
• Activation of MAP kinase pathway and activation of transcription via phosphorylation of transcription factors.

Receptors-associated Kinases

1. Receptors That Activate Protein Tyrosine Kinases
   • RTK (receptor tyrosine kinase): Intrinsic kinase activity
   • Cytokine Receptors (no intrinsic enzyme activity) Activate Tightly Bound JAK Protein Tyrosine Kinases that activate STATs.
2. Receptor Ser/Thr Kinases and SMAD signaling

Ser, Thr, Tyr phosphorylation and phosphomimetic aa

Part I: Cytokine Receptors and the JAK- STAT pathway

Cytokines

Cytokines are a family of relatively small, secreted signaling molecules that control cell growth and differentiation, especially the cells in the immune system.

interferons (IFNs) and interleukins (IL)

• Cytokines are critical for generation of all immune cells
• Erythropoietin and formation of red blood cells
  • Low blood oxygen levels
  • activates HIF1a (hypoxia inducible factor)
    in the kidney interstitial fibroblasts

low blood oxygen levels → activates HIF1a (hypoxia inducible factor) in the kidney interstitial fibroblasts

Blood doping in sports: Epo

Dr. Francesco Conconi; University of Ferrara
Lance Armstrong

• All cytokines and cytokine receptors have similar structures.
• Cytokine receptors have two subdomains comprised of seven conserved beta-strands (sheets) which are critical for cytokine- receptor interaction.
• Epo binds to two identical Epo receptors (homodimer).
• Cytokine receptors do not have intrinsic kinase activity.

Cytokine Receptor Activation

Cytokine receptors can form homodimers, heterodimers or oligomers with 3 or more subunits.

Cytokine receptors have NO intrinsic enzymatic activity. Rather, the receptors are associated with cytoplasmic kinases called JAK kinase. JAK is tightly bound to the cytosolic domain of all cytokine receptors. In the absence of cytokines, the JAK kinases are not activated.

JAK Protein Tyrosine Kinases

The JAK (Just Another Kinase, Janus) family are cytoplasmic protein tyrosine kinases, which are physically associated with the intracellular domains of cytokine receptors. There are four JAK in mammalian cells: JAK1, JAK2, JAK3, TYK2

The domain structure of JAKs

JH2, pesudo-kinase domain phospholate it’s self to cpntrol the JH1 kinase domain’s activity.

JAK kinases have two kinase domains (JH1 and 2) and do not have SH2 or SH3 domains. There are seven well-conserved JAK homology domains (JH).

JH1 is the kinase domain, which contains two tyrosines that can be phosphorylated after ligand stimulation. JH2 is considered catalytically inactive. However, a recent study showed that JH2 is a dual-specific kinase which keeps JAK inactive. The JH6 and JH7 domains mediate the binding of JAKs to receptors.

There are four mammalian JAKs (JAK1, 2, 3, TYK2) that are associated with specific cytokine receptors.

Cytokine Receptor Activation

Activated cytokine receptors signal through JAK-family Kinases

Binding of ligand triggers receptor dimerization and the activated receptor dimers interact through their intracellular domains which brings the associated-JAKs closer and allows rapid transphosphorylation and activation of the receptor- associated JAKs.

Activated JAKs then phosphorylate critical tyrosine residues on the receptor, which leads to recruitment of specific STATs through their SH2 domains followed by single tyrosine phosphorylation of the bound STAT.

SH2 domain is phospho-tyrosine-binding domain, which directly binds to phospho-tyrosine residues. The unique amino acid sequence of each SH2 domain determines the specific phospho- tyrosine residues it binds.

STAT proteins

(Signal Transducers and Activators of Transcription)

STAT proteins, a family of latent cytoplasmic transcription factors, are the principal players of JAK-STAT pathway.

There are seven mammalian STAT proteins (STAT 1, 2, 3, 4, 5A, 5B, 6)

• STAT1: INF response
• STAT2: Development
• STAT3: EGF and IL-6, Development
• STAT4: IL-12
• STAT5s: prolactin, growth hormones and many ILs
• STAT6: IL-4

STAT proteins

(Signal Transducers and Activators of Transcription)

STAT2 STAT4 STAT6	STAT1 STAT3 STAT5a STAT5b
Development of T cells IFNg signaling	Expressed in different tissues Involved in IFN signaling Development of mammary gland Embryogenesis Control cell cycle and apoptosis (cancer)

All STAT proteins share six structural regions:
Interaction and dimerization
JAKs

structure:
SH2 domain + DAN binding domain, Linker domain, phosphated Trannsactivation domain

The tyrosine residue within the C terminus is phosphorylated when the molecule is activated. Tyr phosphorylation is critical for dimerization and nuclear localization.

STAT Activation

1. Recruitment of STAT by p-Tyr of receptors by activated JAK
2. and 3. the formation of activated STAT dimers.
3. Phosphorylated STAT dimers translocate into nucleus and activate transcription of specific genes.

Specificity of JAK-STAT signaling

STAT heterodimers, homodimers, dimers with coactivators/corepressors

Conundrum:
There are more than 50 different cytokines but only 4 JAKs and 7 STATs.
How is the specificity to different cytokines achieved with only few JAKs and STATs? (e.g. IL6, a proinflammatory cytokine, that utilizes gp130, promotes activation of STAT3, while IL-10, an anti-inflammatory cytokine that does not utilize gp130, also activates STAT3.)

Specificity of JAK-STAT signaling

Heterodimerization of STAT proteins

Potential Mechanisms by which STAT heterodimers function

Specific interaction between STATs and cytokine receptors
Tissue-specific STAT protein expression and STAT heterodimerization
Tissue-specific epigenetic modification of chromatin

Erythrocytes Mammary gland
Epo Receptor Prolactin Receptor
JAK activation JAK activation
Milk genes
STAT5 activation STAT5 activation
BCL-XL Transcription of milk proteins
BCL-XL
Cell survival
Production of milk

Negative Regulation of JAK–STAT pathway

Signaling from cytokine receptors is terminated by the phosphotyrosine phosphatase SHP1 and several SOCS and PIAS proteins.

1. Phosphotyrosine phosphatases: PTPs and N-PTPs.
2. SOCS (Suppressor Of Cytokine Signaling).
3. PIAS (the protein inhibitor of activated STAT).

Negative Regulation by Phosphotyrosine Phosphatases

Protein tyrosine phosphatases (PTPs) in the cytoplasm (SH2- domain-containing PTP) , such as SHP1, SHP2, CD45, PTP1B, TC45 inactivate JAKs.

Nuclear protein tyrosine phosphatases

Nuclear protein tyrosine phosphatases (N-PTPs), such as SHP2, PTP1B, and TC45 dephosphorylate of STATs to complete the cycle of activation/inactivation.

The unphosphorylated dimer associates with the nuclear export factor, chromosome region maintenance 1 (CRM1), for transport back to the cytoplasm where it can be reactivated. The STATs are stable throughout this cycle.

Domain structure of SOCS proteins (Suppressors of Cytokine Signaling)

1. SOCS proteins contain an SH2 domain that is flanked by a variable amino-terminal domain and a carboxy-terminal SOCS box. The SOCS box can bind to elongins B and C, which are known components of a ubiquitin E3 ligase complex.
2. The SOCS family of proteins has eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7.

Negative Regulation by SOCS Proteins

SOCS are the target genes of tyrosine phosphorylated STATs; which form a classical negative feedback loop that switches off the activity of JAKs.

(1) Binding of SOCS to phosphotyrosine residues on activated receptor or JAK blocks binding of other signaling proteins (such as STATs).
(2) The SOCS box can also target proteins such as JAK for degradation by the ubiquitin- proteasome pathway.

The domain structure of PIAS proteins (Protein Inhibitor of Activated STAT)

PIAS proteins are E3 Sumo-protein ligases and interact with STATs and many other transcription factors. Thus, PIAS proteins act as transcriptional co-regulators.

There are four mammalian PIAS genes:
PIAS1
PIAS2
PIAS3
PIAS4

Negative Regulation by PIAS Proteins

a. PIAS1 and PIAS3 block the DNA- binding activity of STAT dimers.
b. PIASX (PIAS2) and PIASY (PIAS4) might act as transcriptional co- repressors of STAT by recruiting other co-repressor proteins such as histone deacetylase (HDAC).
c. PIAS proteins can promote the conjugation of small ubiquitin- related modifier (SUMO) to STAT1.

JAK-STAT signaling

JAK-STAT signaling regulates many cellular processes including:

1. innate and adaptive immune function.
2. Development.
3. cell proliferation, differentiation and apoptosis.

The JAK-dependent or –independent STAT tyrosine phosphorylation

1. Tyrosine phosphorylation of STATs can also be stimulated by binding of growth factors to receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor or platelet-derived growth factor receptor. This activation may be direct or indirect. The latter case involves the recruitment of nonreceptor tyrosine kinases (NRTKs) such as Src-family tyrosine kinases.
2. Alternatively, the STATs can be tyrosine phosphorylated in a JAK-dependent manner after hormone- and chemokine- binding to G protein-coupled receptors (also known as seven-transmembrane receptors).

Unphosphorylated STAT proteins

• Recent evidence shows that unphosphorylated STATs can enter the nucleus via importin-α-dependent (STAT2, STAT3) or carrier-independent transport (STAT 1, 3, 5).
• In unstimulated cells, unphosphorylated STATs (STAT 1, 2, 3, 5) constitutively shuttle between the nucleus and cytoplasm.
• Unphosphorylated STAT1 and STAT3 have been found to activate transcription by binding to other transcription factors (IRF1 and NF-κB, respectively).

Part II: PI-3 kinase pathway

Generation of phosphatidylinositol 3-phosphates important in cancers

PI-3 kinase
(Phosphatidylinositol-3 kinase)

PLCg activates PKC via IP3 and DAG

PI-3 kinase

Class I phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3-hydroxyl group of the inositol ring of phosphoinositide (4,5) bisphosphate (PtdIns(4,5)2) to generate the lipid second- messenger phosphoinositide (3,4,5) trisphosphate (PtdIns(3,4,5)3), which binds to PH (pleckstrin homology) domains of effector proteins, inducing their plasma membrane translocation and activation.

PI3K is comprised of two subunits:

• Regulatory subunit
• Catalytic subunit

Classification and domain structure of mammalian PI3Ks

p85 Regulatory

p85 Regulatory subunit binds to p-Tyr of RTKs, Cytokine receptors or adaptor proteins and activates the p110 catalytic subunit PI-3 kinase

Receptor Tyrosine kinase
Cytokine receptor
Adaptor Proteins

Recruitment and activation of protein kinase B (PKB/AKT) in PI-3 kinase pathways

Activation of PI3K triggers diverse cellular responses

The PI-3 Kinase Pathway Is Negatively Regulated by PTEN Phosphatase

Signaling via the PI-3 kinase pathway is terminated by the PTEN phosphatase. The PTEN (phosphatase and tensin homolog deleted on chromosome 10) gene encodes a plasma membrane lipid phosphatase that is recurrently lost in various human cancers.

PTEN phosphatase:

• the major function: its ability to remove the 3-phosphate from PI 3,4,5-trisphosphate.
• also it can remove phosphate groups attached to serine, threonine, and tyrosine residues in proteins.

Phosphatidylinositol-3,4,5- trisphosphate generation

PTEN Phosphatase

The PTEN gene is deleted in multiple types of advanced human cancers. The resulting loss of PTEN protein contributes to the uncontrolled growth of cells.

Part III: TGF-β receptors and the SMAD signaling pathway

TGF-β Superfamily the multipotential cytokine

TGF-β was first identified for its ability to induce a malignant phenotype(anchorage-independent growth) in several cultured early stage cancerous mammalian cell lines.

Cancerous cells:

1. Loss of contact inhibition of cell growth (cultured in vitro).
2. Colony formation in soft agar(anchorage-independent growth).
3. Tumor formation in vivo.

In general, TGF- b signaling inhibits cell proliferation.

From Roberts et al., Cancer Research 1981; 41:2842-48

TGF β Superfamily

Transforming Growth Factor b (TGF b ) family comprises a large number of secreted and structurally related proteins with multiple roles in all major cell activities including developmental patterning, tissue differentiation and proliferation, and homeostasis.
The TGF b family is divided into distinct classes, based on different biological functions:

1. TGF- b (three human isoforms TGF- b 1, 2, and 3), known to potently prevent the proliferation of most normal (non-cancerous) mammalian cells; also to promote expression of cell-adhesion molecules and extracellular-matrix molecules, which play important roles in tissue organization.
2. Bone morphogenetic proteins (BMPs), known to induce the formation of bone and cartilage. Many BMPs are critical for development of many tissues.
3. Activins and inhibins are important for genital development.
4. Mullerian-inhibiting substance (MIS).

Structure of TGF β Superfamily

TGF-β is produced by many different cells in the body and inhibits growth both of the secreting cell (autocrine signaling) and neighboring cells (paracrine signaling).

TGF β Receptor Activation

Signaling by all TGF β family members occurs via transmembrane serine/threonine kinases that upon ligand binding form a heteromeric complex of type II and type I receptors.
In the canonical pathway, signaling to the transcriptional machinery is transduced by a unique family of intracellular signaling mediators called SMADs.

TGF β Receptors I and II are Ser/Thr Kinases

1. TGF-β binds either RII (intrinsic kinase activity) or RIII.

2. Ligand bound receptor RII recruits and phosphorylates RI on Ser/Thr residues.

3. The activated RI phosphorylates SMAD2 or 3. Phosphorylation of SMAD2 or 3 leads to NLS unmasking.

4. Two phosphorylated SMAD2 or 3 proteins bind to SMAD4 with importin- b to form a large cytosolic complex.

5. and

6. After the entire complex translocates into nucleus, the importin dissociates by Ran-GTP protein.

7. TFE3 (a nuclear transcription factor) then associates with Smad2/3/4 complex to form an activation complex which binds to the regulatory sequence of a target gene.

8. This complex then recruits transcriptional co-activators and induces gene transcription.

9. The dephosphorylation of Smad2/3 by a nuclear phosphatase.

10. Smad2/3 recycles through a nuclear pore to the cytosol for the reactivation by another TGF b receptor complex.

SMADs

SMADs are intracellular proteins that transduce extracellular signals from TGF b ligands to the nucleus where they activate downstream gene transcription.

There are three classes of SMAD:

• The receptor-regulated Smads (R-SMAD): SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8/9.
• The common-mediator Smad (co-SMAD): only SMAD4 which interacts with R-SMADs to participate in signaling.
• The antagonistic or inhibitory Smads (I-SMAD): SMAD6 and SMAD7 which block the activation of R-SMADs and Co-SMADs.

The SMADs, which form a trimer of two receptor-regulated SMADs and one co-SMAD, act as transcription factors that regulate the gene expression.

The domain structure of SMADs and their interactions

The C-terminal SSXS motif of receptor-regulated Smads is directly phosphorylated by specific type I receptors.
This phosphorylation allows both the association of the receptor-regulated Smads with Smad4 and translocation into the nucleus.

N-terminal MH1 domain contains the specific DNA- binding segment and the nuclear-localization signal (NLS).Three serine residues near the C-terminus of R- Smad are directly phosphorylated by activated type I receptors. This phosphorylation induces NLS unmasking and allows both the association of the receptor- regulated Smads with Smad4 through the phosphoserine-binding sites in the MH2 domain and translocation into the nucleus.

Negative feedback regulation of TGF- b /Smad signaling pathway

1. Ski and SnoN, inhibiting transcription mediated by the
   Smad2/3/Smad4 complex. (Ski and SnoN are oncogenes).
2. I-Smad 7, inhibiting Smad activation.
3. TGIF (Transforming growth interacting factor), a potential
   repressor of TGF b signaling.

Model of Ski-mediated down-regulation of Smad transcription-activating function

1. Ski directly binds to Smad4 and P-R-Smads. Binding of Ski disrupts the normal interactions between Smad3 and Smad4 necessary for transcriptional activation.
2. Ski recruits N-CoR, which directly binds to mSin3A; in turn, mSin3A interacts with histone deacetylase (HDAC). HDAC-induced chromatin remodeling and transcription repression.

TGF b causes rapid degradation of Ski and SnoN but after few hours, SnoN expression is induced by Smad2/Smad4 complex, which then inhibits the Smad complex.

Negative feedback regulation of TGF- b /Smad signaling pathway

• Inhibitory Smad, Smad7, is a transcriptional target of Smad signaling, which acts in a negative feedback loop to inhibit Smad activation.
• Smad7 binds to the receptor and
  blocks the ability of activated type I receptors (RI) to phosphorylate R-Smad; and it also recruits the E3 ubiquitin ligases, Smurf 1/2 (Smad Ubiquitin Regulatory Factors 1/2), to mediate receptor degradation.

I-Smads and Smurfs regulate receptor turnover

Loss of TGF b signaling plays a key role in cancer development

• TGF- b signaling generally inhibits cell proliferation.
• Loss of various components of the signaling pathway contributes to abnormal cell proliferation and malignancy, for example, Inactivating mutations of either TGF b receptors or R-Smad or Smad4 proteins are resistant to growth inhibition by TGF b .
  1. Smad4, called DPC (deleted in pancreatic cancer).
  2. Mutations in Smad2 in several types of human tumors.
  3. loss of type I or type II TGF- b receptors in retinoblastoma, colon and gastric cancer, hepatoma, and some T- and B-cell malignancies.
  4. Overexpression of inhibitors of Smad signaling (e.g. Ski, SnoN).