Jak kinase

Besides the cytokine receptors that lack intrinsic kinase activity but have associated JAK kinases, STAT proteins can be activated by a variety of G-protein coupled receptors and growth factor receptors with intrinsic tyrosine kinase activity (for example EGF, PDGF, CSF-1, and angiotensin receptor). Increasing evidence suggests a critical role for STAT family members in oncogenesis and aberrant cell proliferation. Constitutively activated STATs have been found in many transformed cell lines and a wide variety of human tumor entities. Numerous non-receptor tyrosine kinases and viral oncoproteins, such as v-Src, v-Abl, v-Sis, and v-Eyk, have been identified to induce DNA-binding activity of STAT proteins. 

Suppressors of cytokine signaling are a family of cytokine-inducible proteins that inhibit JAK kinases. 

Cross-reactivity with JAK kinases is a recurring theme with pyrimidine-carboxamides [81,82], but compound 20 exemplifies a Syk-selective series [81]. Pyrimidine amide 20 has an IC50 of 6nM against Syk, and broad screening (at 300 nM concentration) demonstrated significant selectivity for Syk over 270 kinases. In Ramos cells, 20 inhibited BCR-induced phosphorylation of BLNK, a direct substrate of Syk, with an EC50 of 500-750 nM and Ca2+ flux with an EC50 = 117 nM. Compound 20 potently... 

The structurally related pan-JAK kinase inhibitor pyridone PI (15) has been evaluated in a mouse model of PV induced through the chronic administration of EPO. Administration of EPO (10 U/g) every other day for 5 days resulted in progressive polycythemia and splenomegaly. Administration of 15 at 100 mg/kg/day prevented EPO-induced... 

Examples of receptor-associated tyrosine kinases are given in Table 8.1. Most of the associated protein tyrosine kinases belong to the family of Src kinases (see 8.3) and the Jak kinases (see 11.1.3). 

Another family of protein kinases involved in signal transduction via cytokines includes the Janus kinases (Jak kinases). At least four different Jak kinases are known in mammals (Jakl, Jak2, Jak3 and Jak4). A characteristic feature of the structure of Jak kinases is the occurrence of two tyrosine kinase domains (Fig. 11.5). However, only... 

Fig. 11.5. Domain structure of the Jak kinases. JHl is the catalytic tyrosine kinase domain. JH2 shows similarity to a tyrosine kinase domain. The domains A—E are homologous elements of the Jak kinase family. JH Janus kinase homology region.

It is assumed that the Jak kinases bind to a cytoplasmic section of the receptor, which is in the vicinity of the membrane and contains two conserved sequence elements, Box 1 and Box 2. Binding of the Jak kinases leads to their activation, a process linked to mutual phosphorylation of the Jak kinases (Fig. 11.6). Activation of the Jak kinases may take place in a homodimeric receptor complex or it may also occm in hetero-oligomeric complexes. The circumstances are comphcated in that two (or more) Jak kinases may associate at an activated receptor. Two different Jak kinases are required for signal transduction via interferon receptors (see Fig. 11.8). Furthermore, the different Jak kinases are specific for the corresponding receptors. 

Starting from the activated Jak kinases, a signaling pathway leads directly to transcription factors that are phosphorylated by the Jak kinases on tyrosine residues and activated for stimulation of transcription (review Horvath and Darnell, 1997). These transcription factors belong to a class of proteins known as Stat proteins (Stat = signal transducer and activator of transcription). At least seven different Stat proteins are known (Statl-4, StatSa, StatSb, Stat6). The first Stat proteins, Statl and Stat2, were foimd in association with signal transduction via interferon y. 

The Stat proteins are found in a latent form in the cytosol and are activated by cytokine receptors and their associated kinases. On binding of the cytokine to the receptor and activation of the Jak kinase, the Stat proteins are recruited, via their SH2 domains, to the receptor-kinase complex and are then phosphorylated by the Jak kinase on a conserved Tyr residue at the C-terminus. 

Fig. 11.6. Model of activation of Jak kinases. The Jak kinases (Jakl and Jak2 are shown as examples here) are attributed a two-fold function in signal transduction via cytokine receptors. On binding to the activated cytokine receptor, the Jak kinases are activated and phosphorylation of the Jak kinases takes place, probably by a trans mechanism (dashed arrow). The Jak kinases also catalyze Tyr phosphorylation of the cytoplasmic domain of the receptor (solid arrow). The phosphotyrosine residues serve as attachment points for adaptor proteins or other effector proteins.

Fig. 11.7. Model of activation of Stat proteins. The Stat proteins are phosphorylated (at Tyr701 for Statl) as a consequence of binding to the receptor-Jak complex, and Stat dimers are formed. The dimerization is mediated by phosphotyrosine-SH2 interactions. In the dimeric form, the Stat proteins are transported into the nucleus, bind to corresponding DNA elements, and activate the transcription of neighboring gene sections. In the figure, activation of Stat proteins is shown using the IL-6 receptor as an example (according to Taniguchi, 1995). Other Jak kinases and Stat proteins may also take part in signal conduction via IL-6, in addition to the Jak kinases and Statl shown.

Fig. 11.8. Scheme of signal transduction via interferon a. The receptor for interferon a (IFNa) binds and activates the Jak kinases Jakl and Tykl. These phosphorylate the Stat factors Statl and Stat2, leading to formation of Statl-Stat2 heterodimers. The heterodimers are transported into the nucleus and bind to a corresponding DNA element known as ISRE (interferon stimulated response element). Another protein, p48, is also involved in transcription activation of the interferon regulated gene. 

At the level of the receptor tyrosine kinase complex, many different combinations are possible since the structure of the cytokine receptors is very variable and there are various (at least 4) Jak kinases. 

Sakatsume M, Igarashi K-I, Winestock KD, Garotta G, Larner AC, Finbloom DS. The Jak Kinases differentially associate with the a and b (accessory factor) chains of the interferon-g receptor to form a functional receptor unit capable of activating ST AT transcription factors. J Biol Chem 1995 270 17528-17534. 

A variation on the basic theme of receptor Tyr kinases is seen in receptors that have no intrinsic protein kinase activity but, when occupied by their ligand, bind a soluble Tyr kinase. One example is the system that regulates the formation of erythrocytes in mammals. The cytokine (developmental signal) for this system is erythropoietin (EPO), a 165 amino acid protein produced in the kidneys. When EPO binds to its plasma membrane receptor (Fig. 12-9), the receptor dimerizes and can now bind the soluble protein kinase JAK (Janus kinase). This binding activates JAK, which phosphory-lates several Tyr residues in the cytoplasmic domain of the EPO receptor. A family of transcription factors, collectively called STATs (signal transducers and activators of transcription), are also targets of the JAK kinase activity. An SH2 domain in STATS binds (P)-Tyr residues in the EPO receptor, positioning it for this phosphorylation by JAK. When STATS is phosphorylated in re-... 

W8. Watanabe, S., Itoh, T., and Arai, K., Roles of JAK kinases in human GM-CSF receptor signal transduction. J. Allergy Clin. Immunol. 98, S183—S191 (1996). 

The intracellular domains (IDs) of the receptors share essentially no amino acid sequence identity. However, they can be placed into two groups based on the size of their domains. The IDs of the six human HCRIIs identified thus far are composed of 200-300 amino acids (IDl), while a second group of four receptors have short C-terminal tails (IDs) of 60-100 amino acids. Both IDl and IDs domains provide recruitment sites for JAK kinases, while only the IDl chains provide STAT recruitment sites. Together they activate intracellular pathways required to complete the signal transduction cascade originated by the cytokine-receptor interaction. Several informative reviews on JAK/STAT interactions have recently been published (Kerr et al, 2003 Kisseleva et at, 2002 O Shea et at, 2002). 

Thus, the IFN signaling model represents a paradigm for the functioning of many other cytokine receptors that utilize the JAK/STAT pathway for intracellular signal transduction (Ihle, 1996). The importance of individual members of the JAK kinase family and individual STAT proteins varies depending on the specific cytokine involved, but the basic principles remain the same. 

Figure 8. Schematic of membrane receptors lacking intrinsic enzymatic activity. Intracellular signaling is initiated by coupling of the receptor to an intracellular kinase (e.g JAK kinase). These receptors mediate cytokine signaling (prosurvival protective and maladaptive pathways leading to apoptosis). JAK/STAT signalling is involved in the cellular response to ischemia. Activation of STAT3 reduces ischemia induced apoptosis, whereas activation of STAT1 has the opposite effect.

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