LIN-9 phosphorylation on threonine-96 is required for transcriptional activation of LIN-9 target genes and promotes cell cycle progression.

Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins.

ARF-induced downregulation of Mip130/LIN-9 protein levels mediates a positive feedback that leads to increased expression of p16Ink4a and p19Arf.

The ARF-MDM2-p53 pathway constitutes one of the most important mechanisms of surveillance against oncogenic transformation, and its inactivation occurs in a large proportion of cancers. Here, we show that ARF regulates Mip130/LIN-9 by inducing its translocation to the nucleolus and decreasing the expression of the Mip130/LIN-9 protein through a post-transcriptional mechanism. The knockdown of Mip130/LIN-9 in p53(-/-) and Arf(-/-) mouse embryonic fibroblasts (MEFs) mimics some effects of ARF, such as the downregulation of B-Myb, impaired induction of G2/M genes, and a decrease in cell proliferation.

Deletion of the p107/p130-binding domain of Mip130/LIN-9 bypasses the requirement for CDK4 activity for the dissociation of Mip130/LIN-9 from p107/p130-E2F4 complex.

Mip130/LIN-9 is part of a large complex that includes homologs of the Drosophila dREAM (drosophila RB-like, E2F, and Myb) and C. elegans DRM complexes. This complex also includes proteins such as Mip40/LIN-37, Mip120/LIN-54, and LIN-52.

Mip/LIN-9 can inhibit cell proliferation independent of the pocket proteins.

Progression through the G1-phase of the cell cycle requires that cyclin D and CDK4 phosphorylate pRB and the other pocket proteins, p107 and p130. Cyclin E and CDK2 further phosphorylate pRB to complete its inactivation and allow the cell to enter S-phase. These phosphorylation events lead to the inactivation of the antiproliferative effect of the pocket proteins.

Mammalian Mip/LIN-9 interacts with either the p107, p130/E2F4 repressor complex or B-Myb in a cell cycle-phase-dependent context distinct from the Drosophila dREAM complex.

Mammalian Mip/LIN-9 is a cell cycle regulatory protein that is negatively regulated by CDK4/cyclin D. It has been demonstrated that Mip/LIN-9 collaborates with B-Myb during S and G(2)/M in the induction of cyclins A and B, and CDK1. The ortholog of Mip/LIN-9 in Drosophila, Mip130, is part of a large multisubunit protein complex that includes RBF, repressor E2Fs and Myb, in what was termed the dREAM complex.

Mip/LIN-9 regulates the expression of B-Myb and the induction of cyclin A, cyclin B, and CDK1.

Members of the novel family of proteins that include Drosophila Mip130, Caenorhabditis elegans LIN-9, and mammalian LIN-9 intervene in different cellular functions such as regulation of transcription, differentiation, transformation, and cell cycle progression. Here we demonstrate that LIN-9, designated as Mip/LIN-9, interacts with B-Myb but not with c-Myb or A-Myb. Mip/LIN-9 regulates the expression of B-Myb in a post-transcriptional manner, and its depletion not only decreases the level of the B-Myb protein but also affects the expression of S phase and mitotic genes (i.

A mutant allele of BARA/LIN-9 rescues the cdk4-/- phenotype by releasing the repression on E2F-regulated genes.

It has been proposed that C. elegans LIN-9 functions downstream of CDK4 in a pathway that regulates cell proliferation. Here, we report that mammalian BARA/LIN-9 is a predominantly nuclear protein that inhibits cell proliferation.

The presumptive phosphatidylserine receptor is dispensable for innate anti-inflammatory recognition and clearance of apoptotic cells.

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells.

EVI1 abrogates interferon-alpha response by selectively blocking PML induction.

EVI1 is an oncogene frequently associated with chronic and acute myeloid leukemia. In hematopoietic cells, EVI1 impairs several pathways including proliferation, differentiation, and apoptosis. Interferon-alpha (IFN-alpha) is a powerful cytokine that controls the immune response and limits the expansion of several tissues including bone marrow.

Myc-ARF (alternate reading frame) interaction inhibits the functions of Myc.

The tumor suppressor protein ARF (alternate reading frame) inhibits MDM2 to stabilize and activate the functions of p53. Here we provide evidence for an additional activity of ARF that attenuates cell cycle progression independently of p53 activation. We show that ARF interacts with c-Myc independently of MDM2 or p53.

Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines.

The regulation of cell growth is one of the most important effects of type I interferons (IFNs). This response may involve a cytostatic effect or the induction of apoptosis depending on the cell context. Often the growth-inhibitory response of type I IFNs is studied in tumor cell lines carrying mutations of tumor suppressor genes, and therefore, the growth-inhibitory effect can be influenced by inactivation of these important regulators of cell proliferation.

The WD motif-containing protein RACK-1 functions as a scaffold protein within the type I IFN receptor-signaling complex.

The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-alpha receptor 2/beta subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation.

Contribution of the Box 1 and Box 2 motifs of cytokine receptors to Jak1 association and activation.

Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the beta chain of type I interferon receptor (IFNalphaRbetaL/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1.

Two distinct domains within the N-terminal region of Janus kinase 1 interact with cytokine receptors.

The interaction between receptors and kinases of the Janus kinase (Jak) family is critical for signaling by growth factors, cytokines, and IFNs. Therefore, the characterization of the domains involved in these interactions is pivotal not only in understanding kinase activation but also in the development of drugs that mimic or inhibit signaling. In this report, we have characterized the domains of Jak1 required to associate with distinct cytokine receptor subunits: IFN-alpha R beta L, IFN-gamma R alpha, IL-10R alpha, IL-2R beta, and IL-4R alpha.

Phosphatidylinositol 3-kinase confers resistance to encephalomyocarditis and herpes simplex virus-induced cell death through the activation of distinct downstream effectors.

The Janus kinase/STAT pathway has emerged as the paradigm of IFN-induced protection from viral infections. However, the possible participation of other signaling proteins in this protection is not clearly understood. In this report, we demonstrate that activation of phosphatidylinositol 3-kinase (PI3K) by either serum factors or IFNs blocks cell death induced by encephalomyocarditis virus (EMCV) and HSV.

The WD motif-containing protein receptor for activated protein kinase C (RACK1) is required for recruitment and activation of signal transducer and activator of transcription 1 through the type I interferon receptor.

An obligatory step in the activation of Signal Transducers and Activators of Transcription (STATs) by cytokines is their docking to specific receptors via phosphotyrosines. However, this model does not address whether STATs pre-associate with their corresponding receptor or exist free in the cytoplasm before receptor activation. In this report, we demonstrate that pre-association of STAT1 with the receptor is required for type I interferon (IFN) signaling.

Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor.

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346.

Role of the cytoplasmic domains of the type I interferon receptor subunits in signaling.

Type I interferons are imperative in maintaining a defense against viral infection. These cytokines also play an important role in the control of cell proliferation. These effects are triggered by ligand binding to a specific cell surface receptor.

Structure-function study of the extracellular domain of the human type I interferon receptor (IFNAR)-1 subunit.

Despite accumulating information about the different effector molecules and signaling cascades that are invoked on interferon-alpha (IFN-alpha) binding to the type 1 IFN receptor, little is known about the specifics of the binding interactions between the ligand and the receptor complex. The IFN-alpha/beta receptor (IFNAR)-2 subunit of the IFN receptor is considered the primary binding chain of the receptor, yet it is clear that both receptor subunits, IFNAR-1 and IFNAR-2, cooperate in the high-affinity binding of IFN to the receptor complex. Earlier results from our laboratory suggested that an association of IFNAR-1 with membrane Galalpha1-4Gal-containing glycolipids facilitates receptor-mediated signaling.

Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells.

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.

Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha.

We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells.

The proximal tyrosines of the cytoplasmic domain of the beta chain of the type I interferon receptor are essential for signal transducer and activator of transcription (Stat) 2 activation. Evidence that two Stat2 sites are required to reach a threshold of interferon alpha-induced Stat2 tyrosine phosphorylation that allows normal formation of interferon-stimulated gene factor 3.

The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation.

Expression of type I interferon receptor and its relation with other prognostic factors in human neuroblastoma.

Expression of type I interferon receptor (IFN-R) has been found in several normal tissues and in malignant neoplasms, mainly those with epithelial differentiation. In order to analyze the immunohistochemical expression of type I IFN-R we studied 79 cases of neuroblastoma. Results of expression of type I IFN-R were statistically correlated with histopathology, stage, bcl-2 and PCNA expression, N-myc amplification and apoptosis.

Interferon alpha activates the tyrosine kinase Lyn in haemopoietic cells.

We investigated whether the src-family tyrosine kinase Lyn is involved in the generation of interferon alpha (IFN alpha) signals in haemopoietic cells. In vitro kinase assays using IFN alpha-sensitive cells of B-cell origin demonstrated the presence of IFN alpha-dependent kinase activity in anti-Lyn immunoprecipitates. Further studies demonstrated that Lyn associates via its src homology 2 (SH2) domain with the Janus family tyrosine kinase Tyk-2.

Jak2 is essential for signaling through a variety of cytokine receptors.

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis.