Members of the cytokine/growth hormone/prolactin (PRL) receptor superfamily are associated with cytoplasmic tyrosine kinases of the Jak family. For the PRL receptor (PRLR), after PRL stimulation, both the kinase Jak2 and the receptor undergo tyrosine phosphorylation. To assess the role of tyrosine phosphorylation of the PRLR in signal transduction, several mutant forms of the PRLR in which various tyrosine residues were changed to phenylalanine were constructed and their functional properties were investigated. We identified a single tyrosine residue located at the C terminus of the PRLR to be necessary for in vivo activation of PRL-responsive gene transcription. This clearlyindicates that a phosphotyrosine residue in the cytoplasmic domain of a member of the cytokine/growth hormone/PRL receptor superfamily is directly involved in signal transduction.The prolactin receptor (PRLR) is a member of the cytokine growth hormone (GH)/PRL receptor superfamily (1). Members of this family do not contain any intrinsic catalytic domain (such as tyrosine or serine/threonine kinase) but have been shown to associate with cytoplasmic tyrosine kinases from the Jak family that are necessary for signal transduction (for review, see ref.2). For the PRLR, the receptor constitutively associates with the tyrosine kinase Jak2 (3-6). In addition, PRL-induced activation of Jakl has also been reported in BAF-3 cells (5). We have shown (4) that upon PRL stimulation in Nb2 cells, the associated kinase Jak2 is activated, resulting in the tyrosine phosphorylation of both the kinase and the receptor, and that phosphorylation of both Jak2 and PRLR was time-and dose-dependent in the Nb2 cell line. Moreover, in 293 cells (human embryonic kidney cells), transiently cotransfected with the cDNAs encoding Jak2 and PRLR, both the receptor and the kinase are basally tyrosine-phosphorylated, due to the high level of expression of Jak2. However, upon PRL stimulation, a significant increase in tyrosine phosphorylation of both Jak2 and the PRLR is observed (36). Recently, it has also been reported for several members of the family that upon ligand stimulation, the receptors themselves undergo tyrosine phosphorylation (7-12). The functional significance of this event still remains poorly understood. Phenylalanine replacement of the C-terminal tyrosine of the cytoplasmic domain of the interferon ry (IFN--y) receptor, a member of the cytokine receptor superfamily, results in a nonfunctional receptor (13). Recent studies have indicated that growth factors and cytokines, including PRL, may regulate gene expression through direct phosphorylation of a group of Src-homology domain 2 (SH2)-containing latent cytoplasmic transcription factors known as Stat proteins (14)(15)(16)(17)(18)(19)(20). It has been suggested that signal specificity may be achieved through the activation of Stat molecules that can transactivate different sets of genes. Such a transcription factor termed mammary gland factor (MGF or Stat5) has recently been implicated inThe publication ...

Cytokines that interact with receptors of the hematopoietin super-family have recently been reported to stimulate receptor-associated JAK tyrosine kinases, including PRL activation of JAK2. Unlike other tyrosine kinases, none of the JAK kinases has thus far been implicated in oncogenesis, and their involvement in growth signaling has not been established. Using the PRL-dependent pre-T-cell line Nb2, the present study provided a link between bivalent dimerization of a hematopoietin receptor and activation of its associated JAK kinase, and demonstrated a strong positive correlation between the mitogenic potency of a series of bivalent anti-PRL receptor antibodies and the degree of induced tyrosine phosphorylation of JAK2. Antibody bivalency was required for JAK2 phosphorylation. Monovalent anti-PRL receptor Fab fragments alone were inactive, but their activity could be partially restored by cross-linking with bivalent anti-Fab antibodies. Additional evidence for antibody-induced receptor dimerization was provided by a bell-shaped dose-response curve for the most potent receptor agonist, monoclonal antibody T6. This phenomenon is typically seen at pharmacological concentrations of bivalent ligands, when bound ligand molecules fail to adjoin a second receptor due to occupancy. The present study provided functional support for a model of PRL receptor triggering by ligand-induced receptor homodimerization and subsequent activation of the associated tyrosine kinase JAK2.

IntroductionDeregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFβ) in breast cancer progression.MethodsCyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion.ResultsWe found TGFβ to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFβ-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFβ-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFβ promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21.ConclusionsThus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFβ-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.