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Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers (1-3), and is closely associated with poor prognosis and chemo- or radiotherapeutic resistance (4). Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark (7). Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer., Using single live-cell imaging (8), we found that Akt activation fluctuated across the cell cycle, inversely correlating with Cdt1 abundance (9) (Fig. 1a and Supplementary Fig. 1a). Statistical analysis of [...]

Cyclin D1 is a component of the core cell cycle machinery (1). Abnormally high levels of cyclin D1 are detected in many human cancer types (2). To elucidate the molecular functions of cyclin D1 in human cancers, we performed a proteomic screen for cyclin D1 protein partners in several types of human tumours. Analyses of cyclin D1 interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process (3). We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation (4,5). These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be unaffected by cyclin D1 inhibition., To elucidate the molecular functions of cyclin D1 in human cancer cells, we set out to identify cyclin Dl-interacting proteins in four types of human tumours: mantle cell lymphoma (Granta [...]

A solution NMR spectroscopy to characterize the domain structure of siah-interacting protein (SIP), which includes a novel helical harping domain at the N-terminus flexible linked to a CS domain and an unstructured carboxy terminal SGS domain, is used. The principle role of the molecular structure of SIP appears to be in bringing two proteins into physical proximity and orchestrating the orientation required for polyubiquitination of beta-catenin in the intact SCF-type complex.

During development of the vertebrate CNS, the basic helix-loop-helix (bHLH) transcription factor Olig2 sustains replication competence of progenitor cells that give rise to neurons and oligodendrocytes. A pathological counterpart of this developmental function is seen in human glioma, wherein Olig2 is required for maintenance of stem-like cells that drive tumor growth. The mitogenic/gliomagenic functions of Olig2 are regulated by phosphorylation of a triple serine motif (S10, S13, and S14) in the amino terminus. Here, we identify a set of three serine/threonine protein kinases (glycogen synthase kinase 3α/β [GSK3α/β], casein kinase 2 [CK2], and cyclin-dependent kinases 1/2 [CDK1/2]) that are, collectively, both necessary and sufficient to phosphorylate the triple serine motif. We show that phosphorylation of the motif itself serves as a template to prime phosphorylation of additional serines and creates a highly charged “acid blob” in the amino terminus of Olig2. Finally, we show that small molecule inhibitors of this forward-feeding phosphorylation cascade have potential as glioma therapeutics. [ABSTRACT FROM AUTHOR]

β-Catenin, together with LEF1/TCF transcription factors, activates genes involved in the proliferation and differentiation of neuronal precursor cells. In mature neurons, β-catenin participates in dendritogenesis and synaptic function as a component of the cadherin cell adhesion complex. However, the transcriptional activity of β-catenin in these cells remains elusive. In the present study, we found that in the adult mouse brain, β-catenin and LEF1 accumulate in the nuclei of neurons specifically in the thalamus. The particular electrophysiological properties of thalamic neurons depend on T-type calcium channels. Cav3.1 is the predominant T-type channel subunit in the thalamus, and we hypothesized that the Cacna1g gene encoding Cav3.1 is a target of the LEF1/ β-catenin complex. We demonstrated that the expression of Cacna1g is high in the thalamus and is further increased in thalamic neurons treated in vitro with LiCl or WNT3A, activators of β-catenin. Luciferase reporter assays confirmed that the Cacna1G promoter is activated by LEF1 and β-catenin, and footprinting analysis revealed four LEF1 binding sites in the proximal region of this promoter. Chromatin immunoprecipitation demonstrated that the Cacna1g proximal promoter is occupied by β-catenin in vivo in the thalamus, but not in the hippocampus. Moreover, WNT3Astimulation enhanced T-type current in cultured thalamic neurons. Together, our data indicate that the LEF1/ β-catenin complex regulates transcription of Cacna1g and uncover a novel function for β-catenin in mature neurons. We propose that β-catenin contributes to neuronal excitability not only by a local action at the synapse but also by activating gene expression in thalamic neurons. [ABSTRACT FROM AUTHOR]

Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-1ike CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same β-sandwich fold as p23 but lacks the last highly conserved β-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATPγS. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-1ike domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies. [ABSTRACT FROM AUTHOR]