Identification of Small Molecule Activators of the Janus Kinase/Signal Transducer and Activator of Transcription Pathway Using a Cell-Based Screen

We identified orthologues of all mammalian Janus kinase (JAK) and signal transducer and activator of transcription (STAT) genes in teleostean fishes, indicating that these protein families were already largely complete before the teleost tetrapod split, 450 million years ago. In mammals, the STAT repertoire consists of seven genes (STAT1, -2, -3, -4, -5a, -5b, and -6). Our phylogenetic analyses show that STAT proteins that are recruited downstream of endocrine hormones (STAT3 and STAT5a and -5b) show a markedly higher primary sequence conservation compared with STATs that convey immune signals (STAT1-2, STAT4, and STAT6). A similar dichotomy in evolutionary conservation is observed for the JAK family of protein kinases, which activate STATs. The ligands to activate the JAK/STAT-signalling pathway include hormones and cytokines such as GH, prolactin, interleukin 6 (IL6) and IL12. In this paper, we examine the evolutionary forces that have acted on JAK/STAT signalling in the endocrine and immune systems and discuss the reasons why the JAK/STAT cascade that conveys classical immune signals has diverged much faster compared with endocrine JAK/STAT paralogues.

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The Fas ligand as a cell death factor and signal transducer

Signal Transduction ◽

10.1002/sita.200300022 ◽

2003 ◽

Vol 3 (12) ◽

pp. 33-46 ◽

Cited By ~ 12

Author(s):

Andreas Linkermann ◽

Jing Qian ◽

Dieter Kabelitz ◽

Ottmar Janssen

Keyword(s):

Cell Death ◽

Fas Ligand ◽

Signal Transducer ◽

A Cell

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A common epitope is shared by activated signal transducer and activator of transcription-5 (STAT5) and the phosphorylated erythropoietin receptor: implications for the docking model of STAT activation

Blood ◽

10.1182/blood.v97.8.2230 ◽

2001 ◽

Vol 97 (8) ◽

pp. 2230-2237 ◽

Cited By ~ 33

Author(s):

Dwayne L. Barber ◽

Bryan K. Beattie ◽

Jacqueline M. Mason ◽

Melody H.-H. Nguyen ◽

Monique Yoakim ◽

...

Keyword(s):

Structural Features ◽

Sh2 Domain ◽

Erythropoietin Receptor ◽

Janus Kinase ◽

Signal Transducer ◽

Specific Antibodies ◽

Docking Model ◽

Receptor Interactions ◽

Cytokine Stimulation ◽

Carboxy Terminal

Abstract Erythropoietin (EPO) specifically activates the Janus kinase JAK2 and the transcription factor signal transducer and activator of transcription-5 (STAT5). All members of the STAT family are tyrosine phosphorylated in response to cytokine stimulation at a conserved carboxy-terminal tyrosine, Y694, in the case of STAT5. To determine structural features important for STAT signaling, we generated an activation-specific STAT5 antibody using a phosphopeptide containing amino acids 687 to 698 of STAT5 as antigen. This antibody specifically recognizes tyrosine- phosphorylated STAT5 but not nonphosphorylated STAT5. In immunoprecipitation reactions from cell lines and primary erythroblasts, 2 distinct polyclonal activation-specific STAT5 antibodies selectively immunoprecipitate the tyrosine phosphorylated EPO receptor (EPO-R) in addition to STAT5 under native and denaturing conditions. We propose that the activation-specific STAT5 antibody recognizes the 2 substrates to which the STAT5 SH2 domain interacts, namely, the tyrosine- phosphorylated EPO-R and STAT5 itself. Several studies have implicated EPO-R Y343, Y401, Y431, and Y479 in the recruitment of STAT5. Using a series of EPO-R tyrosine mutants expressed in Ba/F3 cells, we have shown that the activation-specific STAT5 antibody immunoprecipitates an EPO-R containing only 2 tyrosines at positions 343 and 401, confirming the importance of these tyrosines in STAT5 recruitment. These data uncover a novel aspect of STAT SH2 domain recognition and demonstrate the utility of activation-specific antibodies for examining the specificity of STAT–cytokine receptor interactions.

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