Bentley Varghese

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway†

ABSTRACT The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.

Regulation of Prolactin Receptor Levels and Activity in Breast Cancer

From its traditional identity as a hormone involved in growth and differentiation of mammary epithelium and in lactation, to having a pertinent role in the development of mammary carcinoma, the peptide hormone/cytokine prolactin (PRL) has emerged as a versatile signaling molecule. There has been significant progress in our understanding of the fine working of PRL in the past several years. Notably, much effort has been concentrated on the mediator of PRL action, namely, the prolactin receptor (PRLr). The causal link between increased PRLr expression and breast cancer is being increasingly appreciated. Considering that the level of the receptor on the surface is a critical determinant of signaling output in response to PRL, the uncovering of regulatory elements that control receptor expression becomes important. The principle focus of this review is on the regulation of PRLr expression and activity in breast cancer with a brief overview of different isoforms of PRLr, their expression, signaling capabilities and the biological outcomes of PRL/PRLr signaling.

Ligand-Stimulated Downregulation of the Alpha Interferon Receptor: Role of Protein Kinase D2§

ABSTRACT Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells, regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor growth. This cytokine is often used to treat cancers and chronic viral infections. The extent of cellular responses to IFN-α is limited by the IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1 (IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron phosphorylation, which is induced by the ligand. Here we report identification of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor. Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates its interaction with IFNAR1. Expression and kinase activity of PKD2 are required for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and increases the efficacy of its antiviral effects. The mechanisms of the ligand-inducible elimination of IFNAR1 are discussed, along with the potential medical significance of this regulation.

Impaired Turnover of Prolactin Receptor Contributes to Transformation of Human Breast Cells

Signaling by polypeptide hormone prolactin (PRL) is mediated by its cognate receptor (PRLr). PRLr is commonly stabilized in human breast cancer due to decreased phosphorylation of residue Ser349, which when phosphorylated recruits the betaTrcp E3 ubiquitin ligase and facilitates PRLr degradation. Here, we show that an impaired PRLr turnover results in an augmented PRL signaling and PRL-induced transcription. Human mammary epithelial cells harboring degradation-resistant PRLr display accelerated proliferation and increased invasive growth. Conversely, a decrease in PRLr levels achieved by either pharmacologic or genetic means in human breast cancer cells dramatically reduced transformation and tumorigenic properties of these cells. Consequences of alteration of PRLr turnover for homeostasis of mammary cells and development of breast cancers, as well as the utility of therapies that target PRLr function in these malignancies, are discussed.

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.

Basal ubiquitin-independent internalization of interferon alpha receptor is prevented by Tyk2-mediated masking of a linear endocytic motif

Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates down-regulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFNα receptor chain 1 (IFNAR1) receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyr-based motif from the recognition by the AP50 subunit of AP2. These data delineate a long-sought ligand- and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as the regulation of the cell surface density of this receptor chain.

Prolactin Inhibits Activity of Pyruvate Kinase M2 to Stimulate Cell Proliferation

Mitogenic and prosurvival effects underlie the tumorigenic roles of prolactin (PRL) in the pathogenesis of breast cancer. PRL signaling is mediated through its receptor (PRLr). A proteomics screen identified the pyruvate kinase M2 (PKM2), a glycolytic enzyme known to play an important role in tumorigenesis, as a protein that constitutively interacts with PRLr. Treatment of cells with PRL inhibited pyruvate kinase activity and increased the lactate content in human cells in a manner that was dependent on the abundance of PRLr, activation of Janus kinase 2, and tyrosine phosphorylation of the intracellular domain of PRLr. Knockdown of PKM2 attenuated PRL-stimulated cell proliferation. The extent of this proliferation was rescued by the knock-in of the wild-type PKM2 but not of its mutant insensitive to PRL-mediated inhibition. We discuss a hypothesis that the inhibition of PKM2 by PRL contributes to the PRL-stimulated cell proliferation.