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Query: UNIPROT:P06889 (Mol)
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In this study, we have developed several Chinese Hamster ovary (CHO) cell clones stably expressing various deletion mutant forms of the rabbit prolactin receptor (rbPRL-R) to better define the domains of the receptor involved in JAK2 kinase interaction, STAT5 activation, and to assess the role of tyrosine phosphorylation of the PRL-R in signal transduction. We observed that the box 1 region of the receptor was critical for productive interaction with JAK2 and its tyrosine phosphorylation after PRL stimulation. However, this region appeared to require the presence of additional cytoplasmic domain region(s), such as box 2, to exert its complete effect. In addition, we found that a mutant form lacking the 141 C-terminal residues lost the capacity to be tyrosine phosphorylated in response to PRL but remained able to activate JAK2 kinase and STAT5 transcription factor, indicating that it contained the minimal sequence required for STAT5 activation. The absence of tyrosine phosphorylation of this C-terminal rbPRL-R mutant upon PRL stimulation indicated that the phosphorylation of the PRL-R normally occured in the last 141 animo acids (aa) containing three tyrosines and was not absolutely necessary for induction of these early events in PRL signal transduction. Transfectant cell lines expressing wild type (WT) PRL-R and this C-terminal mutant form were able to induce CAT activity upon PRL stimulation when transiently transfected with the ovine-beta-lactoglobulin promoter, containing STAT5 recognition sites, fused to the CAT reporter gene. The comparison between transcriptional activity of these two receptor forms leads to the conclusion that the C-terminal region of the rbPRL-R, containing the physiological sites for tyrosine phosphorylation, is probably responsible for an amplification of the PRL signal to milk protein genes.
Mol Cell Endocrinol 1997 Mar 28
PMID:Prolactin signal transduction to milk protein genes: carboxy-terminal part of the prolactin receptor and its tyrosine phosphorylation are not obligatory for JAK2 and STAT5 activation. 909 11

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the beta c subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1-luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1-luc. A dominant-negative form of Raf-1 inhibited the induction of DUB-1-luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5.
Mol Cell Biol 1997 Jun
PMID:JAK2 is required for induction of the murine DUB-1 gene. 915 35

Several different Janus kinases (JAKs) and signal transducers and activation of transcription (STATs) have been implicated in mediating the biological responses induced by PRL, based on their ligand-dependent tyrosine phosphorylation and activation. However, these criteria alone do not prove that a particular JAK or STAT is essential for signal transduction. We have used mutant cell lines defective in JAK1, JAK2, or STAT1 to examine their roles in PRL-dependent signaling. JAK2 is absolutely required for PRL-dependent phosphorylation of the receptor, activation of STATs, and induction of beta-lactoglobulin. Wild type, but not kinase-negative JAK2, restores all responses to PRL in JAK2-defective cells, suggesting that JAK2 function, not merely the protein, is required. In contrast, JAK1, which is phosphorylated in response to PRL, is not required for any of these functions. Although STAT1 homodimers do form in response to PRL, no defect in PRL-dependent signaling is apparent when STAT1 is missing, suggesting that STAT5, which is strongly activated in response to PRL, is primarily responsible for driving the expression of PRL-responsive genes.
Mol Endocrinol 1997 Jul
PMID:JAK2 and STAT5, but not JAK1 and STAT1, are required for prolactin-induced beta-lactoglobulin transcription. 921 64

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.
Mol Cell Biol 1997 Nov
PMID:Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. 934 27

Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100ng/mal IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 microg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAK1 and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.
Blood Cells Mol Dis 1997 Aug
PMID:Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation. 941 Apr 69

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces various functions, including the proliferation and differentiation of a broad range of hematopoietic cells. We previously reported that at least two distinct pathways are involved in human GM-CSF receptor signaling; both require the box 1 region of the common beta subunit (beta c). This region is essential for the activation of JAK2, which is necessary for all the biological functions of GM-CSF. The activation of JAK2 by GM-CSF leads to rapid tyrosine phosphorylation of cellular proteins, including the beta c. However, the significance of beta c phosphorylation with regard to the regulation of signaling molecules and the expression of GM-CSF functions is less well understood. Here we investigated the role of the cytoplasmic tyrosine residues of the beta c by using a series of beta c mutants expressed in murine BA/F3 cells. A mutant beta c with all eight cytoplasmic tyrosines converted to phenylalanine (Fall) activated JAK2 but not SHP-2, MAPK cascades, STAT5, or the c-fos promoter in BA/F3 cells, and it did not effectively induce proliferation. Adding back each tyrosine to Fall revealed that Tyr577, Tyr612, and Tyr695 are involved in the activation of SHP-2, MAPK cascades, and c-fos transcription, while every tyrosine, particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired growth was also restored, at least partly, by any of the tyrosines. These results provide evidence that beta c tyrosines possess distinct yet overlapping functions in activating multiple signaling pathways induced by GM-CSF.
Mol Cell Biol 1998 Feb
PMID:Definition of the role of tyrosine residues of the common beta subunit regulating multiple signaling pathways of granulocyte-macrophage colony-stimulating factor receptor. 944 70

We previously reported that a single intraperitoneal injection of prolactin (PRL) in female adult rats rapidly and transiently activates mitogen-activated protein kinase (MAPK) in the liver (Piccoletti et al., (1994) Biochem. J. 303, 429-423). Here we analysed the PRL signalling pathway that accounts for MAPK activation. We found that total liver MAPK kinase-1 phosphorylating activity and Raf-1 activity significantly increase after PRL treatment, following a time course that accounts for the activation of MAPK. We also identified a significant increase in the phosphotyrosine content of the 52 kDa Shc protein, accompanied by an increase in Shc coimmunoprecipitated Grb2, which suggests the Ras involvement by PRL. We found that Janus kinase (JAK)2 tyrosine kinase, which appears constitutively associated with the PRL receptor expressed in the liver, is activated and associated with Shc proteins after in vivo PRL treatment. Taken together our data provide evidence that in vivo PRL activates the Shc Ras Raf MAPK cascade in the liver by the involvement of JAK2 and suggests the possibility that the liver short form of PRL receptor plays a role in triggering this signalling pathway.
Mol Cell Endocrinol 1997 Dec 12
PMID:Signal transduction pathway of prolactin in rat liver. 948 13

Abnormal erythropoietin (EPO)-independent cell growth is induced after infection of erythroid progenitor cells with a polycythemic strain of Friend virus (FVp). Binding of its Env-related glycoprotein (gp55) to the EPO receptor (EPOR) mimics the activation of the EPOR with EPO. We investigated the gp55-EPOR signaling in erythroblastoid cells from mice infected with FVp and in cells of FVp-induced or gp55-transgenic-mouse-derived erythroleukemia cell lines, comparing it with the EPO-EPOR signaling in EPO-responsive erythroblastoid cells. While the Janus protein tyrosine kinase JAK2 and the transcription factor STAT5 became tyrosine phosphorylated with the EPO stimulation in EPO-responsive erythroblastoid cells from anemic mice, JAK1 and STAT5 were constitutively tyrosine phosphorylated in all of these FVp gp55-induced erythroblastoid or erythroleukemic cells. Moreover, this constitutively tyrosine-phosphorylated STAT5 was unable to bind to its specific DNA sequences and did not translocate to the nucleus. Nuclear translocation and DNA binding of this STAT5 species required EPO stimulation. These findings clearly indicate that the FVp gp55-EPOR signaling is distinct from the EPO-EPOR signaling and suggest that STAT5 may not play an essential role in the transmission of the cell growth signals in FVp gp55-induced erythroleukemia cells.
Mol Cell Biol 1998 Mar
PMID:Erythropoietin and Friend virus gp55 activate different JAK/STAT pathways through the erythropoietin receptor in erythroid cells. 948 32

We have recently demonstrated that two different forms of IL-4R exist; classical or alternative. The classical IL-4R is predominantly expressed in hematopoietic cells and consist of IL-4R and IL-2Rgammac (gammac) chains. On the other hand, alternative form of IL-4R is predominantly expressed in non-hematopoietic cells and consists of IL-4R and IL-13Ralpha' chains. Moreover, the alternative form of IL-4R is also utilized as a functional component IL-13R complex. It has been shown that the phosphorylation and activation of JAK3 tyrosine kinase is crucial for IL-4 activation of STAT6 in hematopoietic cells. However, we have recently demonstrated that non-hematopoietic cells lack JAK3 expression. We also demonstrated that in these cells, STAT6 activation is mediated through JAK1 and JAK2 tyrosine kinases instead. Furthermore, our results show that IL-4 and IL-13 signals are transmitted through the alternative form of IL-4R in these cells. Thus, major differences exist between hematopoietic and non-hematopoietic cells with regard to structure and signal transduction through IL-4R and IL-13R systems.
Int J Mol Med 1998 Mar
PMID:Structure of and signal transduction through interleukin-4 and interleukin-13 receptors (review). 985 61

STAT5b (signal transducer and activator of transcription 5b) is a key mediator of the effects of plasma GH pulses on male-specific liver gene expression. STAT5b is activated in liver cells in vivo by physiological pulses of GH and then is rapidly deactivated. Investigation of the cellular events involved in this activation/deactivation cycle using the rat liver cell line CWSV-1 established that a brief exposure to GH and the associated activation of JAK2 (Janus kinase 2) tyrosine kinase activity are both necessary and sufficient to initiate all of the downstream steps associated with STAT5b activation by tyrosine phosphorylation and the subsequent deactivation of both JAK2 kinase and STAT5b. JAK2 signaling to STAT5b at the conclusion of a GH pulse could be sustained by the protein synthesis inhibitor cycloheximide or by the proteasome inhibitor MG132, indicating that termination of this JAK2-catalyzed STAT activation loop requires synthesis of a labile or GH-inducible protein factor and is facilitated by the proteasome pathway. This factor may be a phosphotyrosine phosphatase, since the phosphatase inhibitor pervanadate both sustained GH pulse-induced JAK2 signaling to STAT5b and blocked the rapid deactivation of phosphorylated STAT5b (t(1/2) = 8.8 +/- 0.9 min) seen in its absence. Finally, the serine kinase inhibitor H7 blocked down-regulation of JAK2 signaling to STAT5b in a manner that enabled cells to respond to a subsequent GH pulse without the need for the approximately 3-h interpulse interval normally required for full recovery of GH pulse responsiveness. Termination of GH pulse-induced STAT5b signaling is thus a complex process that involves multiple biochemical events. These are proposed to include the down-regulation of JAK2 signaling to STAT5b via a cycloheximide- and H7-sensitive step, proteasome-dependent degradation of a key component or regulatory factor, and dephosphorylation leading to deactivation of the receptor-kinase signaling complex and its STAT5b substrate via the action of a phosphotyrosine phosphatase.
Mol Endocrinol 1999 Jan
PMID:Termination of growth hormone pulse-induced STAT5b signaling. 989 11


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