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. In mammalian cells, Mip130/LIN-9 specifically associates with the p107/p130-E2F4 repressor complex in G0/G1 and with B-Myb in S-phase. However, little is known about how the transition occurs and whether Mip130/LIN-9 contributes to the repressor effect of p107/p130. In this report, we demonstrate that Mip130/LIN-9, Mip40/LIN-37, Mip120/LIN-54, and Sin3b form a core complex, the Mip Core Complex or LIN Complex (MCC/LINC), which is detectable in all phases of the cell cycle. This complex specifically recruits transcriptional repressors such as p107, p130, E2F4 and HDAC1 in G0/G1, and B-Myb in S-phase. Importantly, we provide strong evidence that the transition between repressors and activators of transcription is mediated by CDK4, through the phosphorylation of the pocket proteins, p107 and p130. The requirement for CDK4 activity is bypassed by the deletion of the first 84 amino acids (Mip130/LIN-9(Delta84)), since this mutant is unable to interact with p107/p130 in G0/G1, while maintaining its association with B-Myb. Importantly, the Mip130/LIN-9(Delta84) allele rescues the low expression of G1/S genes observed in CDK4(-/-) MEFs demonstrating that Mip130/LIN-9 contributes to the repression of these E2F-regulated genes in G0/G1.

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. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.

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.e. cyclin A, CDK1, and cyclin B). The critical role of Mip/LIN-9 on the expression of S and G(2)/M genes is further supported by the finding that coexpression of Mip/LIN-9 and B-Myb results in the activation of cyclin A and cyclin B promoter-luciferase reporters, and both proteins are detected on the cyclin A and B promoters. Interestingly, although Mip/LIN-9 promoter occupancy peaks earlier than B-Myb, the highest levels of expression of cyclins A and B correlate with the maximum binding of B-Myb to these promoters. These data support the concept that Mip/LIN-9 is required for the expression of B-Myb, and both proteins collaborate in the control of the cell cycle progression via the regulation of S phase and mitotic cyclins.

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. The pocket proteins are the main targets of CDK4, and its unregulated activity can contribute to carcinogenesis. Mip/LIN9 is a recently described protein with growth suppressor, as well as growth promoting effects due to its ability to stabilize B-Myb and induce genes required for S phase and mitosis. The finding that a mutation that deletes the first 84 amino acids of Mip/LIN-9 corrects the defects of the CDK4 knockout mouse suggests that it should have a growth repressor effect that is blocked by CDK4. However, overexpression of cyclin D only partially blocks the inhibitory effect of Mip/LIN-9 on cell proliferation. Here, we performed experiments to further understand the antiproliferative effect of Mip/LIN-9 within the context of the pocket proteins. Our results suggest that there is a pocket protein-independent mechanism of the Mip/LIN-9 antiproliferative effect since it can be observed in cells with ablation of the three members of the family, and in NIH3T3 cells expressing the adenovirus E1A-12S protein. Altogether, the independence from the pocket proteins and the partial blockade of the antiproliferative effect produced by expression of cyclin D suggest that the role of Mip/LIN-9 downstream of CDK4 may be more closely related to the activation of B-Myb and the induction of S/M genes. Importantly, the regulatory effect of CDK4 is not due to direct phosphorylation of Mip/LIN-9 by this kinase or even CDK2, suggesting an indirect mechanism such as phosphorylation of the pocket proteins.

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. Taking advantage of this property of fibroblasts, we generated, PSR(+/+), PSR(+/-), and PSR(-/-) fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells.

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. More importantly, we demonstrate that BARA/LIN-9 also acts downstream of cyclin D/CDK4 in mammalian cells since (i) its antiproliferative effect is partially blocked by coexpression of cyclin D1, and (ii) a mutant form that lacks the first 84 amino acids rescues several phenotypic alterations observed in mice null for cdk4. Interestingly, mutation of BARA/LIN-9 restores the expression of E2F target genes in CDK4 null MEFs, indicating that the wild-type protein plays a role in the expression of genes required for the G1/S transition.

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. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that includes the IFN-alpha receptor 2/beta-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin.

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. In this report, we explored the role of the ARF-p53 pathway in the growth-inhibitory effect of type I IFNs. We found that p53 is only induced in cells that express p14(ARF) (p19(ARF) in mouse cells). Surprisingly, mouse embryonal fibroblasts that are null for p19(ARF) or P53, even after transformation with oncogenic RAS, respond as well as wild type to the growth-inhibitory effect of type I IFNs. Similarly, human ARF(-/-) U2OS and P53(-/-) SAOS-2 cells show a significant decrease in cell proliferation. However, only SAOS-2 or U2OS reconstituted with inducible p14(ARF) undergo apoptosis in response to IFN beta treatment, and this effect was not inhibited by expression of dominant negative p53. These data suggest that (i) at least in specific cell types, the induction of apoptosis by type I IFNs requires an ARF pathway that is p53-independent and (ii) the cytostatic and pro-apoptotic effects of type I IFNs employ different pathways.

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. We demonstrate that two regions of Jak1 are necessary for the interaction with cytokine receptors. First, a common N-terminal region that includes Jak homology (JH) domain 7 and the first 19 aa of JH6, and, second, a C-terminal region (JH6-3) that was different for distinct receptors. The contribution of the two different regions of Jak1 to cytokine receptor binding was also variable. Deletion of JH7-6 impaired the association of IL-2R beta and IL-4R alpha chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-alpha R beta L or IL-10R alpha. Interestingly, regardless of the effect on receptor binding, removal of JH7-6 completely abrogated kinase activation, indicating that this domain is required for ligand-driven kinase activation and, thus, for proper signaling through cytokine receptors.

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. On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor beta (IL2Rbeta), IFNgammaRalpha/IFNGR1, and IL10Ralpha/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jak1. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFNalphaRbetaL/IFNAR2 and IL2Rbeta subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFNgammaRalpha/IFNGR1 (SILLPKS) and IL10Ralpha/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8-10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38-58 amino acids from the membrane).