JAK/STAT signaling pathway

JAK/STAT signaling pathway

Cytokine-induced multimerization of class I and class I cytokine receptors activates a JAK/STAT signal-transduction pathway.

Signaling molecule or ligands: Cytokines (IL 2 -15 except IL -1 and IL -8, Interferons, growth hormone, Prolactin erythropoietin etc)

What are cytokines?

Cytokines form a diverse group of small soluble proteins that, when secreted by cells, act in an autocrine, paracrine, or endocrine fashion to induce a great variety of responses, including the immune response, cell proliferation, growth, differentiation, apoptosis (programmed cell death)and chemotaxis (the movement of a motile cell along a concentration gradient of a specific substance).

Many cytokines signal through the JAK-STAT pathway, although not all do. (For instance, TNF-alpha receptors have a completely different structure and signal transduction pathway).

Receptor for JAK/STAT signaling: Cytokine receptors class I and Class II

Figure 1: Structure of class I and Class II cytokine receptor

Cytokine receptors have an extracellular domain for ligand binding, cytosolic domain for associating with JAK and a transmembrane helix.

Major components in the pathway: Receptor, JAK, STAT

Activation mechanism of Cytokine receptor (class I and class II)

  • Ligand binding to extracellular domain of the receptor causes a conformational change that promotes formation of a functional dimeric receptor.
  • It will bring together two intrinsic or associated kinases, which then phosphorylate each other on a tyrosine residue in the activation lip.
  • Phosphorylation causes the lip to move out of the kinase catalytic site, thus allowing ATP or a protein substrate to bind.
  • The activated kinase then phosphorylates other tyrosine residues in the receptor’s cytosolic domain.
  • The resulting phosphotyrosines function as docking sites for various signal-transduction proteins.
Figure 2: Activation of cytokine receptors

JAK –STAT Signal-transduction pathway triggered by ligand (Epo) binding to the erythropoietin receptor (EpoR)

Epo: Erythropoietin

(Cytokine secreted by kidney cells, prevents apoptosis and promotes proliferation and differentiation of erythroid progenitor cells in the bone marrow). An excess of erythropoietin or mutations in its receptor that prevent down-regulation result in production of elevated numbers of red blood cells.

Figure 3: Overview of signal-transduction pathways triggered by ligand binding to the erythropoietin receptor (EpoR), a typical cytokine receptor

Step 1: Binding of ligand to its receptor and dimerization

  • Erythropoietin binds simultaneously to the extracellular domains of two EpoR monomers on the cell surface.
  • Binding of Epo causes the receptors to dimerize, which brings the receptor-associated JAKs into close proximity, where they can phosphorylate each other.

Step 2: Activation of JAK (Janus kinase)

 What are JAK?

JAK are family of intracellular, nonreceptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway.

There are 4 JAK proteins: JAK1, JAK2, JAK3 and TYK2

  • Receptor dimerization activates tyrosine kinase activity of JAK.
  • The JAKs then phosphorylate each other on tyrosine residues located in regions called activation lip, through a process called transphosphorylation, which increases the activity of their kinase domains.

Step 3: Phosphorylation of tyrosine residues in the receptor

  • Kinase domain will further phosphorylate several tyrosine residues on the receptor’s cytosolic domain, creating binding sites for proteins possessing SH2 domains.

Step 4: Binding of STAT to phosphorylated receptor

  • The phosphotyrosine residues on the receptor proteins are binding sites for STAT
  • The STAT proteins are considered as latent transcription factors. “Latent” means that they are always present in the cytoplasm, and waiting to be activated by JAK.

What are STAT proteins?

 STAT: Signal Transducer and Activator of Transcription

  • Members of the STAT protein family are intracellular transcription factors that mediate many aspects of cellular immunity, proliferation, apoptosis and differentiation.
  • They are primarily activated by membrane receptor-associated Janus kinases (JAK)
  • There are 7 STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6
  • All STAT proteins contain an N terminal SH2 domain that binds to a phosphotyrosine in the receptor’s cytosolic domain, a central DNA-binding domain, and a C-terminal domain with a critical tyrosine residue.

Step 5: Phosphorylation of STAT by JAK

  • Once STAT binds to phosphorylated tyrosine on receptor, they are phosphorylated by JAKs (Phosphorylate tyrosine residues on STAT protein)
  • Phosphorylated STAT spontaneously dissociates from the receptor
  • Once phosphorylated, two STATs can then form a STAT dimer (The SH2 domain of each STAT binds the phosphorylated tyrosine of the opposite STAT) and they expose their NLS (nuclear localization signal).
  • The STAT dimer is an active transcription factor.  It travels to the nucleus where it binds to specific sequences in the DNA to induce transcription of target gene.

Movement of STATs from the cytosol to the nucleus

  • The phosphorylated dimer is actively transported to the nucleus via an importin α/β ternary complex (unphosphorylated STAT proteins also shuttle between the cytosol and nucleus)

To move from the cytosol to the nucleus, STAT dimers have to pass through nuclear pore complexes (NPCs), which are protein complexes present along the nuclear envelope that control the flow of substances in and out of the nucleus.To enable STATs to move into the nucleus, an amino acid sequence on STATs, called the nuclear localization signal (NLS), is bound by proteins called importins.Once the STAT dimer (bound to importins) enters the nucleus, a protein called Ran (associated with GTP) binds to the importins, releasing them from the STAT dimer.The STAT dimer is then free in the nucleus.

  • Different STATs activate different genes in different cells.
  • In erythroid progenitors, stimulation by erythropoietin leads to activation of STAT5.
  • The major protein induced by active STAT5 is Bcl-xL, which prevents the programmed cell death (apoptosis) of these progenitors, allowing them to proliferate and differentiate into erythroid cells.

Tyrosine phosphorylated and activated cytokine receptor is not only the binding site for STAT proteins.

It can activate various signal transduction pathway via different adaptors and signaling molecules.

Figure 4: Various outcome of Epo signaling

Two mechanisms for terminating signal transduction from the erythropoietin receptor (EpoR)

 Mechanism 1: Short-Term Regulation by SHP1 Phosphatase

SHP1 (protein tyrosine phosphatase) negatively regulates signaling from several types of cytokine receptors in several types of progenitor cells.

Example: Mutant mice lacking SHP1 phosphatase die because of excess production of erythrocytes and several other types of blood cells.

Domains of SHP1: Phosphatase catalytic domain, two SH2 domains.

  • When cells are not stimulated by a cytokine (are in the resting state), one of the SH2 domains physically binds to and inactivates the catalytic site in SHP1.
  • In the stimulated state, this blocking SH2 domain binds to a specific phosphotyrosine residue in the activated receptor.
  • The conformational change that occurs as a result of this interaction will unmask its phosphatase catalytic site and positions it near the phosphorylated tyrosine in the lip region of JAK2.
  • Removal of the phosphate from this tyrosine inactivates the JAK kinase, so that it can no longer phosphorylate the receptor or other substrates (eg: STATs) unless additional cytokine molecules bind to cell-surface receptors, initiating a new round of signaling.
Figure 5: JAK2 deactivation induced by SHP1 phosphatase

Mechanism 2: Long term regulation by SOCS proteins

They can act in two different ways

  • The SH2 domain in several SOCS proteins can binds to phosphotyrosines on an activated receptor or phosphotyrosine on JAK. This will prevent the binding of other SH2-containing signaling proteins like STAT and thus inhibiting receptor signaling.

Eg: SOCS -1 protein binds to the critical phosphotyrosine in the activation lip of activated JAK2 kinase and its catalytic activity is inhibited.

  • All SOCS proteins contain a domain called the SOCS box that recruits components of E3 ubiquitin ligases. As a result JAK2 becomes polyubiquitinated and then degraded in proteasome, thus permanently turning off all JAK2-mediated signaling pathways.
Figure 6: Signal blocking and protein degradation induced by SOCS proteins

Drugs targeting the JAK-STAT pathway

  • Drugs that target the JAK-STAT pathway are used to turn down the immune response.
  • One way to block the pathway is with a drug that blocks the cytokine receptor.

Eg: Basiliximab (binds to the IL-2 receptor, and is used to prevent transplant rejection)

  • JAK inhibitors are drugs that inhibit the kinase activity of JAK.

Eg: Ruxolitinib (Prevent abnormal proliferation of cells in the bone marrow)

      Tofacitinib (treatment of rheumatoid arthritis)

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