How Is Phosphorylation Level Determined?

The JAK-STAT pathway is a newly discovered signal transduction pathway shared by multiple cytokines. Its mode of action has been basically clarified, but the regulation of its role and its relationship with other cytokine signalling pathways remain to be further studied.

JAK-STAT pathway

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The JAK-STAT pathway is a newly discovered signal transduction pathway shared by multiple cytokines. Its mode of action has been basically clarified, but the regulation of its role and its relationship with other cytokine signalling pathways remain to be further studied.

Chinese name

JAK-STAT pathway

Foreign name

Mitogen activated

Basic mode of action

Evoked corresponding receptor formation

Explanation

A newly discovered intracellular signaling pathway

How cells respond specifically to various extracellular regulatory signals, that is, the study of cell signaling mechanisms, has been a hot topic in recent years. There are two basic pathways known to transmit cytokine stimulation signals in most cells: the Ras-MAPK pathway and the JAK-STAT pathway. The former was found in studying the signaling of multiple growth factors, which may be mainly related to the transmission of cell proliferation signals; the latter was found in the signaling studies of interferons, and it is now known that this pathway is used in almost all cytokine signaling Play an important role [1,2]. JAK, or Janus Kinase, is a non-receptor tyrosine protein kinase (PTK). Members of this family have 7 homologous regions (JH1 ~ 7), of which JH1 region is a kinase region and JH2 region is a pseudokinase region. Unlike other PTKs, there is no Src homology region 2 (SH2) structure in JAK, because it can catalyze tyrosine phosphorylation of cytokine receptors connected to it and phosphorylate a variety of signals containing specific SH2 regions The molecule thus activates it, so it is called Janus-

The basic mode of action of this pathway is: the ligand induces the corresponding receptor to form a dimer or oligomer, which causes JAK interaction with the receptor to interact with it and activates by its own tyrosine phosphorylation; at the same time it catalyzes the receptor to generate tyrosine Acid phosphorylation, followed by these phosphorylated tyrosines as "anchors", attracts a variety of signal molecules containing a specific SH2 region and activates them, and extracellular signals are introduced into the nucleus through the action of these molecules. However, with the deepening of research, it is found that this pathway is not so simple, its regulatory mechanism is fine and diverse, and it has intricate links with other signaling pathways. Although work in this area has just begun, the results achieved have raised people's understanding of signal transmission to a new level.

1 MAPK and JAK-STAT pathways

MAPK is mitogen activated protein kinase, which is a downstream signaling molecule in the Ras pathway. It has serine / threonine protein kinase activity and can activate C-Fos, C-Jun and other transcription regulators to form AP-1 acts in the nucleus, activating specific genes to transmit signals. MAPK activation requires that specific Tyr and Ser residues in the molecule be phosphorylated simultaneously [3]. It was previously thought that although the Ras pathway and the JAK pathway coexist in cytokine signaling, they are independent of each other. The former is related to the C-terminal sequence of the type I cytokine receptor, while the latter is bound to the proximal membrane end of the cytoplasmic region of the receptor [4,5]. Deletion of the receptor C-terminal sequence does not activate the Ras pathway, while the JAK-STAT pathway is not affected.

Recent findings suggest that MAPK may be in a critical position to regulate these two pathways. Initially, it was found that the activation of JAK was accompanied by the activation of MAPK, and that JAK activation alone was not enough to enable STAT to exert its transcriptional activation activity to the maximum extent. It was then confirmed that Stat3 not only phosphorylated Tyr residues, but also phosphorylated Ser residues [6], and that phosphorylated Ser residues were necessary for binding to regulatory regions on DNA; There are specific sequences on the protein that can be recognized and phosphorylated by MAPK [7]. Similar to Stat3, Stat1 must also be phosphorylated by Ser residues to be fully activated. The sequence containing this Ser residue is exactly the above-mentioned specific sequence that can be recognized and phosphorylated by MAPK, and the peptide Ser sequence containing this sequence was successfully phosphorylated with MAPK in vitro [8].

In vitro experiments show that in cells stimulated with type I IFN, MAPK interacts with INFR- chain to be activated, and then combines with Stat1 and promotes its activity [9]. When the action of MAPK is blocked, the activity of STAT to promote gene transcription is reduced. It can be inferred from the above phenomenon that STAT is one of the natural substrates of MAPK, and there is a bypass or regulation mode of MAPK-STAT signaling in cells. It has recently been discovered that in growth hormone-stimulated male rat hepatocytes, Stat5 serine phosphorylation exerts a different transcription regulating activity than before [10], and this effect is probably also mediated by MAPK.

It is still unclear how JAK activates MAPK, either directly or by activating Ras-MAPK pathway. How type I cytokine receptors activate Ras is also unclear, and is generally believed to be similar to growth factor receptors containing PTK activity. The latter first induces Shc phosphorylation, then recruits Ras through the SH-2 and SH-3 regions of Shc and Grb2, and activates Ras through Shc and mSos [11]. It is known that IL-2, IL-3, IL-5, IL-6, and EPO can all induce Shc phosphorylation, and interference with the binding of JAK to the receptor complex can significantly reduce Shc phosphorylation [12]. Recently, it has been found that this effect of EPO does not rely on the binding of EPOR to Shc, but instead binds the SH-2 region of Shc to phosphorylated tyrosine residues on JAK2 [13]. It has been confirmed that JAK2 can phosphorylate Shc, but whether other phosphorylated proteins are involved in Shc activation and recruitment remains unclear.

2 Negative feedback regulation of SH-PTP1

SH-PTP1 is a tyrosine phosphatase (PTPase1) containing SH2 region. PTPase is divided into two types: transmembrane type and non-transmembrane type. SH-PTP1 and 2 belong to the latter. SH-PTP2 participates in receptor-type PTK-mediated signaling and can be regarded as a positive signaling molecule; SH-PTP1 mainly inhibits PTK-mediated signaling. Unlike other widely expressed tyrosine phosphatases, SH-PTP1 (also known as PTP1C, HCP or SHP) is mainly expressed in hematopoietic cells. Initially, SH-PTP1 was found to bind to the IL-3R chain after the action of IL-3 [14]. After the action of SH-PTP1 antisense RNA, the level of phosphorylation of the IL-3R chain increased. Motheaten mice that do not normally express SH-PTP1 exhibit a variety of hematopoietic and immune abnormalities [15], and their CFU-E precursor cells are highly sensitive to EPO, often accompanied by systemic autoimmune diseases.

Both IL-3 and EPO are known to conduct signals mainly through JAK2 [3,4]. Not long ago, it was confirmed that the tyrosine at position 429 on EPOR was phosphorylated by JAK after the action of EPO, thus possessing the ability to bind SH-PTP1 and activate it [16]. The specificity of this effect depends on the specific amino acid sequence around this residue, namely PY-Leu-Tyr-Leu-Val-Val. An 11 peptide containing this sequence is sufficient to bind and activate SH-PTP1. Deletion of this tyrosine on EPOR can increase the sensitivity of cells to EPO by 5 to 10 times. The level of tyrosine phosphorylation of wild-type EPOR cells and JAK2 returned to normal soon after the action of EPO, and the cells expressing the tyrosine-mutated EPOR at the 429th position remained at a high level for a long time. IL-3R contains a sequence similar to the SH-PTP1 binding site in EPOR, suggesting that a similar negative feedback regulation mechanism may exist in IL-3 signaling.

Similarly, when IFN- acts, IFN- receptor can reversibly bind to SH-PTP1 [17]. Similarly, in macrophages of Moptheaten mice receiving this stimulation, JAK1 and Stat1 tyrosine phosphorylation levels were significantly higher than normal, while TYK2 and Stat2 activation levels, which are also important for type I IFN signaling, did not change significantly. It is known that in type I IFN signaling, JAK1 specifically activates Stat1 and Tyk1 specifically activates Stat2 [18]. Therefore, it can be considered that the above-mentioned phenomenon is caused by the selective action of SH-PTP1 on JAK1, and its specific mechanism deserves further study. Recently, it was found that, of the two SH2 regions of SH-PTP1, the N-terminal SH2 region can bind with its enzyme active region in the molecule to play a self-inhibiting role. When this region is combined with other molecules, SH-PTP1 is activated [19 ]; While the SH-region at the C-terminus has no significant effect on its activity, it only participates in its recruitment process.

3 Functions of JAK and / or STAT

STATs regulate a wide range of genes: IFN-regulated genes require Stat1 and 2; IL-6-induced acute-phase response genes require Stat3; Stat5 mediates prolactin-induced expression of multiple genes; Stat6 is induced by IL-4 Necessary for the conversion of immunoglobulin classes and the up-regulation of MHC class II antigens and immunoglobulin receptors. At present, Stats has nothing to do with the proliferative response caused by cytokines. IL-2, IL-4 and IL-6 can all cause mutant receptors not to activate STAT and trigger a proliferative response [11].

It has been found that mutations in the JAK homologue in Drosophila can cause its cell transformation. HTLV-1 infection-induced cell transformation is also thought to be caused by JAK-STAT activation, and may be directly or indirectly activating this pathway in a receptor-like manner [20]. In v-src-transformed cells, constitutive Stat-3 tyrosine phosphorylation was observed [21], and whether this effect requires JAK mediation is unclear. JAK-family kinase activation was observed in this cell, but v-src was observed to directly activate Stat-3 in other systems. In mouse pre-B cells transformed by v-ab1, JAK1 and JAK3 showed constitutive kinase activity, and could activate the STAT activity usually induced by IL-4 and IL-7, but no two kinds of IL existed at this time. [twenty two]. The above evidence suggests that JAK and / or STATs activation may be related to cell transformation. EGF, CSF-1, and PDGF can all induce STAT activation, and the role of STAT in these systems is unclear. Recently, it was found that EGF activation of Stat-1, 3 does not depend on JAK, but acts through the kinase region in EGFR [23], although JAK1 is activated at this time. At present, it seems that the growth signal of EGF is mainly transmitted by the Ras pathway. STAT has little effect on the activation of known early genes such as c-fos, suggesting that there may be other unknown target genes.

It is known that JAK can directly or indirectly phosphorylate other signal molecules such as Vav, PLC-1, thereby activating other signaling pathways? [26]. IL-4-induced phosphorylation of insulin receptor substrates (IRS) 1, 2 also requires JAK activation [27]. And cytokines, such as EGF, PDGF, CSF-1, etc., which transmit signals through their corresponding receptor-type tyrosine protein kinases can also induce STAT activation. In summary, it can be seen that JAK and STAT each have a relatively independent role in addition to jointly constituting the JAK-STAT pathway to conduct signals.

4 Other molecules that regulate the JAK / STAT pathway

IFN- mainly transmits signals by inducing the formation of Stat-1 homodimer (also known as IFN- response factor, referred to as GAF). It is known that IFN- can inhibit the proliferation of Th2 cells and has no effect on Th1 cells. Recently, it was found that this is because Th1 cells lack the -chain of IFN- receptor (also called cofactor 1 / AF-1) and cannot induce GAF formation [24]. As mentioned above, the type I IFN-induced GAF formation is selectively regulated by SH-PTP1, suggesting that the JAK-STAT pathway is very finely regulated in vivo. Th1 / Th2 imbalances can cause a variety of diseases, and these discoveries make it possible to understand and intervene at the molecular level.

Recent studies have found that forskolin, an adenylate cyclase activator, can significantly inhibit IFN-induced phosphorylation of IFNR, JAK1, TRK2 and Stat1, Stat2 [29], and the formation of complexes that bind to IFN response elements; After the modification loses adenylate cyclase activation activity, it has no effect on the JAK-STAT pathway, suggesting that protein kinase A (requiring cAMP for activation) may have a regulatory effect on the JAK-STAT pathway.

5 Research Outlook

The structure of JAK has not yet been fully understood. In addition to the activation site, there are several unidentified phosphorylation sites that may be involved in recruiting other signaling molecules. The functional domain of JAK binding to the receptor's near-membrane region has not been determined, and the functions of the JAK homology region and pseudokinase region are yet to be studied. There are also many questions about STAT that are not clear: such as how STAT multimers are transferred into the nucleus, and whether IRF-1 and ICSBP, which belong to the same category as STAT, are combined with other STAT members to function. A variety of different JAK and STAT gene knock out mice will help answer these questions.

In addition, the relationship between JAK and STAT and disease and its applied research are also an attractive area. In this regard, a child with congenital JAK3 deficiency has been found to have symptoms similar to those of sexually combined severe immunodeficiency disease (XSCID), but is autosomal recessive [25]. Constitutive activation of STAT-related transcription factors in peripheral blood cells of patients with acute leukemia may be one of the causes of leukemia [28]. In addition, there may be related diseases with more complicated mechanisms, which need to be further studied.

In short, the study of the JAK-STAT pathway raises many more complex questions while answering previous questions. Research in this field, especially in the relationship between JAK-STAT and other signaling pathways, will definitely help to understand the major life mystery of signaling more comprehensively and deeply.