Luqin Deng

A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway

JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

Prolactin stimulates ubiquitination, initial internalization, and degradation of its receptor via catalytic activation of Janus kinase 2

Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.

The Role of Prolactin Receptor in GH Signaling in Breast Cancer Cells

GH and prolactin (PRL) are structurally related hormones that exert important effects in disparate target tissues. Their receptors (GHR and PRLR) reside in the cytokine receptor superfamily and share signaling pathways. In humans, GH binds both GHR and PRLR, whereas PRL binds only PRLR. Both hormones and their receptors may be relevant in certain human and rodent cancers, including breast cancer. GH and PRL promote signaling in human T47D breast cancer cells that express both GHR and PRLR. Furthermore, GHR and PRLR associate in a fashion augmented acutely by GH, even though GH primarily activates PRLR, rather than GHR, in these cells. To better understand PRLRs impact, we examined the effects of PRLR knockdown on GHR availability and GH sensitivity in T47D cells. T47D-ShPRLR cells, in which PRLR expression was reduced by stable short hairpin RNA (shRNA) expression, were compared with T47D-SCR control cells. PRLR knockdown decreased the rate of GHR proteolytic turnover, yielding GHR protein increase and ensuing sensitization of these cells to GHR signaling events including phosphorylation of GHR, Janus kinase 2, and signal transducer and activator of transcription 5 (STAT5). Unlike in T47D-SCR cells, acute GH signaling in T47D-ShPRLR cells was not blocked by the PRLR antagonist G129R but was inhibited by the GHR-specific antagonist, anti-GHR(ext-mAb). Thus, GHs use of GHR rather than PRLR was manifested when PRLR was reduced. In contrast to acute effects, GH incubation for 2 h or longer yielded diminished STAT5 phosphorylation in T47D-ShPRLR cells compared with T47D-SCR, a finding perhaps explained by markedly greater GH-induced GHR down-regulation in cells with diminished PRLR. However, when stimulated with repeated 1-h pulses of GH separated by 3-h washout periods to more faithfully mimic physiological GH pulsatility, T47D-ShPRLR cells exhibited greater transactivation of a STAT5-responsive luciferase reporter than did T47D-SCR cells. Our data suggest that PRLRs presence meaningfully affects GHR use in breast cancer cells.

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.

Growth Hormone-induced JAK2 Signaling and GH Receptor Down-regulation: Role of GH Receptor Intracellular Domain Tyrosine Residues

GH receptor (GHR) mediates important somatogenic and metabolic effects of GH. A thorough understanding of GH action requires intimate knowledge of GHR activation mechanisms, as well as determinants of GH-induced receptor down-regulation. We previously demonstrated that a GHR mutant in which all intracellular tyrosine residues were changed to phenylalanine was defective in its ability to activate signal transducer and activator of transcription (STAT)5 and deficient in GH-induced down-regulation, but able to allow GH-induced Janus family of tyrosine kinase 2 (JAK2) activation. We now further characterize the signaling and trafficking characteristics of this receptor mutant. We find that the mutant receptors extracellular domain conformation and its interaction with GH are indistinguishable from the wild-type receptor. Yet the mutant differs greatly from the wild-type in that GH-induced JAK2 activation is augmented and far more persistent in cells bearing the mutant receptor. Notably, unlike STAT5 tyrosine phosphorylation, GH-induced STAT1 tyrosine phosphorylation is retained and augmented in mutant GHR-expressing cells. The defective receptor down-regulation and persistent JAK2 activation of the mutant receptor do not depend on the sustained presence of GH or on the cells ability to carry out new protein synthesis. Mutant receptors that exhibit resistance to GH-induced down-regulation are enriched in the disulfide-linked form of the receptor, which reflects the receptors activated conformation. Furthermore, acute GH-induced internalization, a proximal step in down-regulation, is markedly impaired in the mutant receptor compared to the wild-type receptor. These findings are discussed in the context of determinants and mechanisms of regulation of GHR down-regulation.

Interruption of Growth Hormone Signaling via SHC and ERK in 3T3-F442A Preadipocytes upon Knockdown of Insulin Receptor Substrate-1

Insulin receptor substrate-1 (IRS-1) is a docking protein tyrosine phosphorylated in response to insulin, IGF-1, GH, and other cytokines. IRS-1 has an N-terminal plekstrin homology domain (which facilitates membrane localization), a phosphotyrosine-binding domain [which associates with tyrosine-phosphorylated insulin receptor or IGF-1 receptor (IGF-1R)], and tyrosine residues that, when phosphorylated, bind signaling molecules. The role of IRS-1 in GH signaling is uncertain. We previously reported that IRS-1 and Janus kinase 2 associate independently of tyrosine phosphorylation via IRS-1s N terminus and that IRS-1 reconstitution greatly enhances GH-induced ERK, but not STAT5, activation. We now use GH-responsive 3T3-F442A preadipocytes to study the influence of IRS-1 on GH action. We stably transfected cells with vector only (Control) or a vector encoding IRS-1 short hairpin RNA [knockdown (KD)] and compared representative clones. Immunoblotting confirmed more than 80% knockdown of IRS-1 in KD cells. GH caused characteristic Janus kinase 2 and STAT5 activation in both Control and KD cells, but ERK activation was dramatically reduced in KD cells in GH time course and dose-response experiments. Notably, GH-induced Src homology collagen (SHC) activation and SHC-Grb2 association in KD cells were also markedly diminished compared with Control cells. Subcellular fractionation revealed that IRS-1 in Control cells was largely cytosolic, but the component isolated with plasma membranes was highly enriched in lipid raft membranes (LR). In KD cells, GH-induced ERK activation in the LR fraction was particularly diminished compared with Control cells. These data suggest that LR-enriched IRS-1 contributes substantially to GH-induced ERK activation in LR in 3T3-F442A fibroblasts. Furthermore, our results are consistent with IRS-1 residing upstream of SHC in the GH-induced ERK-signaling pathway.