Cytokine Signaling Proteins

Cytokines are a loosely defined set of secreted factors that control a variety of important biological responses related to hematopoiesis and immune function. There are over 40 members of this family of small («15-30 kDa) glycoproteins, and many of them display functional redundancy [2]. Cytokines bind receptors that are characterized by sequence and structural similarities in their extracellular regions and the lack of catalytic domains in their intracellu-lar portions [3]. The intracellular regions of these receptors are associated with the Janus tyrosine kinases (JAKs), which are activated upon cytokine binding to the receptor. The JAKs then phosphorylate and activate a family of intracellu-lar proteins known as STATs (signal transducers and activators of transcription), which translocate to the nucleus and activate gene expression. This signaling pathway, referred to as the JAK-STAT pathway, is notable for the direct and rapid transmission of the signal from the cell surface to the nucleus. The essential steps in the JAK-STAT pathway are outlined in Fig. 1.

Cytokine receptors signal as oligomers, ranging from dimers to tetramers [4]. These receptors have been broadly classified into two subgroups (type I and type II cytokine receptors), based on patterns of conserved amino acid residues within their extracellular domains [5,6]. Type I cytokine receptors include receptors for interleukins, colony-stimulating factors, and hormones; type II receptors include the receptors for interferons and interleukin-10 [7]. Cytokine receptors have also been classified into subgroups based on the use of shared subunits [8]. There are three subfamilies of cytokine receptors that share common signal transducing receptor subunits within the family: (1) interleukin-6 (IL-6) subfamily of receptors, which have a common gp130 subunit; (2) granulocyte-macrophage colony-stimulating factor (GM-CSF) subfamily, which have a common P subunit (Pc); and (3) the interleukin-2 (IL-2) subfamily of receptors that share a y subunit (yc). In each case, the multi-subunit receptor consists of one or more ligand-specific subunits and a common subunit that is essential for signal transduction. Thus, different cytokines can bind to distinct receptors that share a signal transducer.

The four mammalian JAKs are JAK1, JAK2, JAK3, and Tyk2. The JAKs have two tandem kinase-like domains, one of which is a functional catalytic tyrosine kinase domain and is located at the C-terminal end of the protein. Immediately upstream of this functional kinase domain is a non-functional pseudokinase domain [3] that possesses many of the sequence motifs of tyrosine kinases but lacks several residues that are essential for kinase activity.

Figure 1 An overview of the JAK-STAT signalling pathway. The binding of erythropoietin (Epo) to its homodimeric cell surface receptor (EpoR) leads to a conformational change resulting in the apposition and transphosphorylation of receptor-associated JAK2 molecules. JAK2 phosphorylates EpoR on tyrosine residues, creating docking sites for the SH2 domains of STATs. The receptor-bound STAT5 molecules are phosphorylated by JAK2, leading to their dimerization via reciprocal SH2-phosphotyrosine interactions. Dimeric STAT5 molecules translocate to the nucleus, where they bind DNA and activate gene transcription.

Figure 1 An overview of the JAK-STAT signalling pathway. The binding of erythropoietin (Epo) to its homodimeric cell surface receptor (EpoR) leads to a conformational change resulting in the apposition and transphosphorylation of receptor-associated JAK2 molecules. JAK2 phosphorylates EpoR on tyrosine residues, creating docking sites for the SH2 domains of STATs. The receptor-bound STAT5 molecules are phosphorylated by JAK2, leading to their dimerization via reciprocal SH2-phosphotyrosine interactions. Dimeric STAT5 molecules translocate to the nucleus, where they bind DNA and activate gene transcription.

The cytokine receptors and the JAK kinases are associated constitutively, and this requires an «60-amino-acid membrane proximal domain in the signaling receptor. This domain contains two sequence motifs, referred to as box 1 and box 2, that are conserved in most cytokine receptors [9]. It is believed that ligand binding to the extracellular portion of the receptor causes the two JAK molecules associated with the intracellular region of the receptors to come into apposition, such that they are now able to phosphorylate each other. This phosphorylation occurs at tyrosines in the activation loop of the functional kinase domain, in the (E/D)YY motif that is conserved in all JAKs [10]. This transphosphorylation process is essential for the activation of the tyrosine kinase.

Some examples of the various receptor types and associated JAKs are shown in Fig 2. The simplest signaling complex is that of the homodimeric hormone receptors, such as the erythropoietin receptor (EpoR) and JAK2. Structural studies on the unliganded and ligand-bound extracellular domains of the EpoR have revealed some surprises and changed the way we view ligand-mediated tyrosine kinase activation. Contrary to the idea that receptor activation is brought about by ligand-induced oligomerization, the unliganded EpoR

extracellular domain crystallizes as a dimer [11]. This dimer, however, is in an open scissor-like conformation, in which the transmembrane domains are separated by «70 A, suggesting that the receptor-associated JAKs would be too far from each other to allow transphosphorylation. Structures of liganded receptors have shown how a single ligand molecule binds two Epo receptors, with two distinct surfaces of the ligand making contact with equivalent binding regions on the two receptors. This causes a conformational switch in the molecule, resulting in the transmembrane regions coming closer together so that they are now separated by only «30 A [12-14]. This would bring the two JAK molecules sufficiently close to each other so as to promote transphosphorylation. It is not yet certain if this mechanism is applicable to all cytokine receptors, as there are no structures of unliganded cytokine receptors other than for EpoR.

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