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B23/NPM is a major nucleolar phosphoprotein that has a critical role in cell proliferation and cell death. Here, we show that it forms a complex with Akt on growth factor (GF) stimulation in both the cytoplasm and the nucleus, for which Akt activation is indispensable. The C terminus of B23 (239-294 residues) potently binds pleckstrin homology (PH) domain of Akt. Akt binding to B23 protects it from proteolytic degradation by caspase-3, leading to the up-regulation of cell survival. Interestingly, unsumoylated B23 K263R, but not wild-type B23, strongly interacts with Akt in the nucleoplasm in the absence of GFs. Furthermore, we show that Akt2, but not other isoforms, specifically regulates B23 sumoylation and protein stability. Also, nuclear Akt regulates the cell cycle progression activity of B23. Therefore, our findings support that nuclear Akt binds and stabilizes B23 in the nucleoplasm, and regulates its activities in cell survival and cell cycle. Akt/PKB | caspase-3 | stability | sumoylation

The role of the endoplasmic reticulum stress-regulated kinase, PERK, in mammary gland function was assessed through generation of a targeted deletion in mammary epithelium. Characterization revealed that PERK is required for functional maturation of milk-secreting mammary epithelial cells. PERK-dependent signaling contributes to lipogenic differentiation in mammary epithelium, and perk deletion inhibits the sustained expression of lipogenic enzymes FAS, ACL, and SCD1. As a result, mammary tissue has reduced lipid content and the milk produced has altered lipid composition, resulting in attenuated pup growth. Consistent with PERK-dependent regulation of the lipogenic pathway, Loss of PERK inhibits expression of FAS, ACL, and SCD1 in immortalized murine embryonic fibroblasts when cultured under conditions favoring adipocyte differentiation. These findings implicate PERK as a physiologically relevant regulator of the lipogenic pathway. lipid metabolism | SREBP1 | FAS | ACL | fatty acids

we develop a probabilistic method for analyzing global features of a cellular network under intrinsic statistical fluctuations, which is important when there are finite numbers of molecules. By making a self-consistent mean field approximation of splitting the variables in order to reduce the large number of degrees of freedom, which is reasonable for a not very strongly interacting network, we discovered that the underlying energy landscape of the mitogen-activated protein kinases (MAPKs) signal transduction network (with experimentally measured of inferred parameters such as chemical reaction rate coefficients in the network) is funneled toward a global minimum characterized by the nonequilibrium steady-state fixed point of the system at the end of the signal transduction process. For this system, we also show that the energy landscape is robust against intrinsic fluctuations and random perturbation to the inherent chemical reaction rates. The ratio of the slope versus the roughness of the energy landscape becomes a quantitative measure of robustness and stability of the network. Furthermore, we quantify the dissipation cost of this nonequilibrium system through entropy production, caused by the nonequilibrium flux in the system. We found that a lower dissipation cost corresponds to a more robust network. This least dissipation property might provide a design principle for robust and functional networks. Finally, we find the possibility of bistable and oscillatory-like solutions, which are important for cell fate decisions, upon perturbations. The method described here can be used in a variety of biological networks. funnel | stability | potential landscape | function

Protein kinase CK2 (also known as casein kinase 2) is present in the cytoplasm, nuclei, and several other organelles. In addition, this enzyme has been found bound to the external side of the cell membrane where it acts as an ectokinase phosphorylating several extracellular proteins. Previous experiments with transfection of HEK-293T cells demonstrated that expression of both subunits, CK2[alpha] (catalytic) and CK2[beta] (regulatory), was necessary for the appearance of the ectopic enzyme as an ectokinase. In this work, using deletion and point mutations of CK2[beta], it was possible to demonstrate that the region between amino acids 20 and 33 was necessary for the export of the enzyme as an ectokinase. Phenylalanines 21 and 22 and acidic residues in positions 26-28 are involved in the structural aspects that are required for export. However, the region encompassing amino acids 20-33 of CK2[beta] is not sufficient to make the carboxyl half of this subunit functional in bringing CK2 to the ectokinase locus. In cells transfected with only CK2[beta], it was demonstrated that 3-4% of the subunit is exported to the cell medium, but the subunit is not bound to the external membrane. casein kinase 2 | export of proteins | shedding

Plant recognition of pathogens leads to rapid activation of MPK3 and MPK6, two Arabidopsis mitogen-activated protein kinases (MAPKs), and their orthologs in other species. Here, we report that synthesis of camalexin, the major phytoalexin in Arabidopsis, is regulated by the MPK3/MPK6 cascade. Activation of MPK3/MPK6 by expression of active upstream MAPK kinase (MAPKK) or MAPKK kinase (MAPKKK) was sufficient to induce camalexin synthesis in the absence of pathogen attack. Induction of camalexin by Botrytis cinerea was preceded by MPK3/MPK6 activation, and compromised in mpk3 and mpk6 mutants. Genetic analysis placed the MPK3/MPK6 cascade upstream of PHYTOALEXIN DEFICIENT 2 (PAD2) and PAD3, but independent or downstream of PAD1 and PAD4. Camalexin induction after MPK3/MPK6 activation was preceded by rapid and coordinated up-regulation of multiple genes encoding enzymes in the tryptophan (Trp) biosynthetic pathway, in the conversion of Trp to indole-3-acetaldoxime (IAOx, a branch point between primary and secondary metabolism), and in the camalexin biosynthetic pathway downstream of IAOx. These results indicate that the MPK3/MPK6 cascade regulates camalexin synthesis through transcriptional regulation of the biosynthetic genes after pathogen infection. defense signaling | fungal resistance | camalexin biosynthesis | Botrytis cinerea

Multiple signaling pathways are engaged by the type I and II IFN receptors, but their specific roles and possible coordination in the generation of IFN-mediated biological responses remain unknown. We provide evidence that activation of Akt kinases is required for IFN-inducible engagement of the mTOR/p70 S6 kinase pathway. Our data establish that Akt activity is essential for up-regulation of key IFN-[alpha]- and IFN-[gamma]-inducible proteins, which have important functional consequences in the induction of IFN responses. Such effects of the Akt pathway are unrelated to regulatory activities on IFN-dependent STAT phosphorylation/activation or transcriptional regulation. By contrast, they reflect regulatory activities on mRNA translation via direct control of the mTOR pathway. In studies using Aktl and Akt2 double knockout cells, we found that the absence of Akt kinases results in dramatic reduction in IFN-induced antiviral responses, establishing a critical role of the Akt pathway in IFN signaling. Thus, activation of the Akt pathway by the IFN receptors complements the function of IFN-activated JAK--STAT pathways, by allowing mRNA translation of IFN-stimulated genes and, ultimately, the induction of the biological effects of IFNs.

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PARI/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease. Alzheimer's disease | cell signaling | chaperone | MARK2/PAR1

Articles in recent years have described two separate and distinct NF-[kappa]B activation pathways that result in the differential activation of p50- or p52-containing NF-[kappa]B complexes. Studies examining tumor-necrosis factor receptor-associated factors (TRAFs) have identified positive roles for TRAF2, TRAF5, and TRAF6, but not TRAF3, in canonical (p50-dependent) NF-[kappa]B activation. Conversely, it recently was reported that TRAF3 functions as an essential negative regulator of the noncanonical (p52-dependent) NF-[kappa]B pathway. In this article, we provide evidence that TRAF3 potently suppresses canonical NF-[kappa]B activation and gene expression in vitro and in vivo. We also demonstrate that deregulation of the canonical NF-[kappa]B pathway in TRAF3-deficient cells results from accumulation of NF-[kappa]B-inducing kinase (NIK), the essential kinase mediating noncanonical NF-[kappa]B activation. Thus, our data demonstrate that inhibition of TRAF3 results in coordinated activation of both NF-[kappa]B activation pathways. lymphotoxin-[beta] receptor | p50 | tumor-necrosis factor receptor-associated factor 3 (TRAF3)

Inositol pyrophosphates, also designated inositol diphosphates, possess high-energy [beta]-phosphates that can pyrophosphorylate proteins and regulate various cellular processes. They are formed by a family of inositol hexakisphosphate kinases (IP6Ks). We have created mice with a targeted deletion of IP6K1 in which production of inositol pyrophosphates is markedly diminished. Defects in the mutants indicate important roles for IP6K1 and inositol pyrophosphates in several physiological functions. Male mutant mice are sterile with defects in spermiogenesis. Mutant mice are smaller than wild-type despite normal food intake. The mutants display markedly lower circulating insulin. inosito | phosphate kinase | inositol polyphosphate | knockout mouse

Elevated expression and activation of the focal adhesion kinase (FAK) occurs in a large proportion of human breast cancers. Although several studies have implicated FAK as an important signaling molecule in cell culture systems, evidence supporting a role for FAK in mammary tumor progression is lacking. To directly assess the role of FAK in this process, we have used the Cre/IoxP recombination system to disrupt FAK function in the mammary epithelium of a transgenic model of breast cancer. Using this approach, we demonstrate that FAK expression is required for the transition of premalignant hyperplasias to carcinomas and their subsequent metastases. This dramatic block in tumor progression was further correlated with impaired mammary epithelial proliferation. These observations provide direct evidence that FAK plays a critical role in mammary tumor progression. breast cancer | Cre recombinase | transgenic

Circadian clocks are composed of central oscillators, input pathways that transduce external information to the oscillators, and output pathways that allow the oscillators to temporally regulate cellular processes. Little is known about the output pathways. In this study, we show that the Neurospora crassa osmosensing MAPK pathway, essential for osmotic stress responses, is a circadian output pathway that regulates daily rhythms in the expression of downstream genes. Rhythmic activation of the highly conserved stress-activated p38-type MAPK [Osmotically Sensitive-2 (OS-2)] by the N. crassa circadian clock allows anticipation and preparation for hyperosmotic stress and desiccation that begin at sunrise. These results suggest a conserved role for MAPK pathways in circadian rhythmicity. circadian output pathway | circadian rhythm | Neurospora crassa | osmotic stress phosphorelay

Activation of macrophages and subsequent 'killing' effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-[gamma] and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the unknown pathways of LM killing independent of reactive oxygen intermediates and reactive nitrogen intermediates. One differentially expressed gene, PKC[delta], had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKC[delta] during listeriosis, we infected PKC[delta]-deficient mice with LM. PKC[delta]-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKC[delta]-deficient macrophages demonstrated that, despite elevated levels of proinflammatory cytokines and NO production, increased escape of LM from the phagosome into the cytoplasm and uncontrolled bacterial growth occurred. Taken together these data identified PKC[delta] as a critical factor for confinement of LM within macrophage phagosomes. microarray | phagosomal escape | bacterial killing

The regulation of neurotrophin (NT) secretion is critical for many aspects of NT-mediated neuronal plasticity. Neurons release NTs by activity-regulated secretion pathways, initiated either by neurotransmitters and/or by existing NTs by a positive-feedback mechanism. This process depends on calcium release from intracellular stores. Little is known, however, about potential pathways that down-regulate NT secretion. Here we demonstrate that nitric oxide (NO) induces a rapid down-regulation of brain-derived neurotrophic factor (BDNF) secretion in cultured hippocampal neurons. Similar effects occur by activating a downstream target of intracellular NO, the soluble guanylyl cyclase, or by increasing the levels of its product, cGMP. Furthermore, down-regulation of BDNF secretion is mediated by cGMP-activated protein kinase G, which prevents calcium release from inositol 1,4,5-trisphosphate-sensitive stores. Our data indicate that the NO/cGMP/protein kinase G pathway represents a signaling mechanism by which neurons can rapidly down-regulate BDNF secretion and suggest that, in hippocampal neurons, NT secretion is finely tuned by both stimulatory and inhibitory signals.

The cellular phenomenon of hippocampal long-term potentiation (LTP) is initiated and stabilized by the activation of protein kinase C (PKC). The identification of the PKC involved in the cellular phenomenon is affected by employing a peptide substrate. The substrate is characterized by its specificity for PKC associated with the phosphorylation site of neurogranin/RC3, NG-(28-43). An analysis of the neuropeptide, NG-(28-43), an endogenous specific PKC substrate, corroborates the hypothesis of PKC activation during the induction and sustenance of LTP.

The type I regulatory (RI) and catalytic subunits of the Chinese hamster type I protein kinase (PKA) were coexpressed inside the same bacterial cell to facilitate the quick examination of mutants of RI and C which obstruct PKA activation by cAMP. Bacterial development is impaired by the activation of PKA by endogenous cAMP, as seen in the case of the BL21(DE3) strain of Escherichia coli.

FKBP25, a cloned 25-kDa FKBP family member, is regionalized to the nucleus, and casein kinase II phosphorylates a glutathione S-transferase fusion protein of FKBP25 (GST-FKBP25). When the GST-FKBP25 fusion protein was incubated with cell lysates or purified casein kinase II, a stable FKBP25/casein kinase II complex developed. A key nuclear substrate of casein kinase II was related to the GST-FKBP25/casein kinase II complex.

Anti-CD3 and anti-Fyn immunoprecipitates have PI 3-kinase activity as evaluated by TLC and HPLC. Free and receptor-bound Src-related tyrosine kinase p59fyn(T) bound to the lipid kinase. A unique process of interaction with PI 3-kinase has been evolved by Src-related kinases. PI-3 primarily bound to the SH3 region of Fyn. PI 3-kinase is a target of SH3 regions and probably will significantly influence the signals obtained from the T-cell antigen receptor zeta/CD3-p59fyn complex, because Fyn SH3 directly bound to the p85 subunit of PI 3-kinase, and an enhancement in the identification of Fyn SH3-related PI3-kinase activity is caused by anti-CD3 crosslinking.

Direct phosphorylation, serine or threonine residues are used in the transduction of the protein kinase signals from the extracellular ligands in the hematopoietic and other systems. The cloning of partial cDNAs encoding four putative protein kinases is made by the lineage differentiation potential of the murine embryonic stem cell system. Highly conserved residues Asp-Phe-Gly in domain VII of the protein kinase family are bound in three of the four genes. Downstream effectors of cytokines, which regulate self-renewal and lineage commitment in embryogenesis are received by the genes.

A study of the influence of dimerization and binding to DNA on the phosphorylation of Fos and Jun by many protein kinases revealed that distinct protein kinases can differentiate between Fos and Jun proteins in the form of heterodimers, monomers and homodimers, and between DNA-bound and non-DNA-bound proteins, indicating that these distinct forms of Fos and Jun can be identified and controlled separately by phosphorylation. Fos and Jun develop dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and control gene expression.

Six splice variants of the NR1 receptor gene of N-methyl-D-aspartate (NMDA) expressed in Xenopus oocytes have been electrophysiologically characterized. The N-terminal of the variants is involved in agonist and polyamine binding regions or indirectly alters their structuring. Subunit composition influences operational characteristics of NMDA receptors.

Analyses reveal that the processing of the herpes simplex virus regulatory protein alpha22, mediated by the UL13 protein kinase, influences the accumulation of a subset of alpha and gamma mRNAs and proteins of infected cells. The results indicate that UL13 encodes a protein kinase or phosphotransferase whose expression is delayed in the replicative life cycle, and which indirectly or directly phosphorylates ICP22.

The entire primary structure of human erythroid dematin whose sequence includes a homologue of the 'headpiece' sequence discovered at the C terminus of villin has been identified. The headpiece is necessary for villin's stimulation of actin redistribution and microvillar growth. The widely evident expression of dematin transcripts in human tissues indicates that dematin and its homologues may replace villin in villin-regulative tissues to control actin realignment by a phosphorylation-controlled process.