Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.

The liver kinase B1 is a central regulator of T cell development, activation, and metabolism.

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4(+) and CD8(+) T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1-AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.

Vitamin D, d-dimer, Interferon γ, and sCD14 Levels are Independently Associated with Immune Reconstitution Inflammatory Syndrome: A Prospective, International Study

© 2015.To determine the immunological profile most important for IRIS prediction, we evaluated 20 baseline plasma biomarkers in Acquired Immunodeficiency Syndrome (AIDS) patients initiating antiretroviral therapy (ART). Patients were enrolled in a randomized, placebo-controlled ART initiation trial in South Africa and Mexico to test whether maraviroc could prevent IRIS. Participants were classified prospectively as having IRIS within 6. months of ART initiation. Twenty plasma biomarkers were measured at study enrollment for 267 participants. Biomarkers were tested for predicting IRIS with adjustment for covariates chosen through forward stepwise selection. Sixty-two participants developed IRIS and of these 21 were tuberculosis (TB)-IRIS. Baseline levels of vitamin D and higher d-dimer, interferon gamma (IFNγ), and sCD14 were independently associated with risk of IRIS in multivariate analyses. TB-IRIS cases exhibited a distinct biosignature from IRIS related to other pathogens, with increased levels of C-reactive protein (CRP), sCD14, IFNγ, and lower levels of Hb that could be captured by a composite risk score. Elevated markers of Type 1 T helper (Th1) response, monocyte activation, coagulation and low vitamin D were independently associated with IRIS risk. Interventions that decrease immune activation and increase vitamin D levels warrant further study.