Cyclin-dependent kinases (Cdks) are the catalytic sub-units of a family of mammalian heterodimeric serine/threonine kinases that have been implicated in the control of cell-cycle progression, transcription and neuronal function. Recent genetic evidence obtained with gene-targeted mice has shown that Cdk4 and Cdk6 are not needed for entry into the cell cycle after mitogenic stimuli and organogenesis; however, they are essential for the proliferation of some endocrine and hematopoietic cells. Cdk2 is also dispensable for the mitotic cell cycle. Indeed, mice without Cdk2 are normal except for their complete sterility: unexpectedly, Cdk2 is crucial for the first meiotic division of male and female germ cells. These findings have important implications both for our current understanding of the role of Cdks in regulating the mammalian cell cycle and for their potential use as therapeutic targets in cancer.
Nuclear factor-kappaB (NF-kappaB) is a transcription factor that has crucial roles in inflammation, immunity, cell proliferation and apoptosis. Activation of NF-kappaB mainly occurs via IkappaB kinase (IKK)-mediated phosphorylation of inhibitory molecules, including IkappaBalpha. Optimal induction of NF-kappaB target genes also requires phosphorylation of NF-kappaB proteins, such as p65, within their transactivation domain by a variety of kinases in response to distinct stimuli. Whether, and how, phosphorylation modulates the function of other NF-kappaB and IkappaB proteins, such as B-cell lymphoma 3, remains unclear. The identification and characterization of all the kinases known to phosphorylate NF-kappaB and IkappaB proteins are described here. Because deregulation of NF-kappaB and IkappaB phosphorylations is a hallmark of chronic inflammatory diseases and cancer, newly designed drugs targeting these constitutively activated signalling pathways represent promising therapeutic tools.
Although glycolysis is a biochemical pathway that evolved under ancient anaerobic terrestrial conditions, recent studies have provided evidence that some glycolytic enzymes are more complicated, multifaceted proteins rather than simple components of the glycolytic pathway. These glycolytic enzymes have acquired additional non-glycolytic functions in transcriptional regulation [hexokinase (HK)-2, lactate dehydrogenase A, glyceraldehyde-3-phosphate dehydrogenase (GAPD) and enolase 1], stimulation of cell motility (glucose-6-phosphate isomerase) and the regulation of apoptosis (glucokinase, HK and GAPD). The existence of multifaceted roles of glycolytic proteins suggests that links between metabolic sensors and transcription are established directly through enzymes that participate in metabolism. These roles further underscore the need to consider the non-enzymatic functions of enzymes in proteomic studies of cells and tissues.
Three originally distinct concepts - lipid rafts, detergent-resistant membranes (DRMs) and liquid-ordered (lo) lipid phases - are often confused in current literature; many researchers have assumed that all three names refer to the same chemico-biological entity. In fact, theoretical and experimental findings provide strong evidence against identifying DRMs with rafts and lo domains. Because much of what we think we know about lipid rafts is based on their unjustified identification as DRMs, functional domains in biological membranes might differ markedly from the generally accepted picture.
A basic mechanism by which individual proteins can increase network complexity is moonlighting, whereby a given protein fulfils more than one function. Traditionally, this phenomenon is attributed to separate binding surfaces of globular, folded proteins but we suggest that intrinsically unstructured proteins [IUPs) might provide radically different mechanisms. Eleven IUPs have been identified that suggest that the structural malleability of IUPs gives rise to unprecedented cases of moonlighting by eliciting opposing (inhibiting and activating) action on different partners or even the same partner molecule. Unlike classical cases, these proteins use the same region or overlapping interaction surfaces to exert distinct effects and employ non-conventional mechanisms to switch function, enabled by their capacity to adopt different conformations upon binding. Owing to the apparent functional benefits, we expect to see many more examples of this parsimonious use of protein material in complex metabolic networks.
Mediator was discovered because of its activity in a yeast RNA polymerase II (pol II) transcription system - it is needed for the system to respond to a transcriptional activator. Mediator is the central link in the enhancer -> activator -> Mediator -> pol II -> promoter pathway. The transduction of regulatory signals through this pathway is crucial for transcription of almost all pol II promoters in all eukaryote organisms.
Phosphoinositide 3-kinases (PI3Ks) generate lipids that control a wide variety of intracellular signalling pathways. Part of this diversity in PI3K actions stems from the broad range of protein effectors of the PI3K lipids. A further layer of complexity is added by the existence of multiple isoforms of PI3K. Gene-targeting studies in the mouse have recently uncovered key roles for specific PI3K isoforms in immunity, metabolism and cardiac function. Remarkably, some of these actions do not require PI3K catalytic activity. In addition, loss-of-expression of certain PI3K genes leads to increased PI3K signalling following insulin stimulation. PI3K gene targeting has, in many cases, led to altered expression of the non-targeted PI3K subunits, making it difficult to exclude that some of the reported phenotypes result from ' knock-on ' effects of PI3K gene deletion. Targeting strategies that take into account the complex interplay between members of the PI3K family will be crucial to gain a full understanding of the physiological roles of the isoforms of PI3K.
Formin proteins are potent regulators of actin dynamics. Most eukaryotes have multiple formin isoforms, suggesting diverse cellular roles. Formins are modular proteins, containing a series of domains and functional motifs. The Formin homology 2 (FH2) domain binds actin filament barbed ends and moves processively as these barbed ends elongate or depolymerize. The FH1 domain influences FH2 domain function through binding to the actin monomer-binding protein, profilin. Outside of FH1 and FH2, amino acid similarity between formins decreases, suggesting diverse mechanisms for regulation and cellular localization. Some formins are regulated by auto-inhibition through interaction between the diaphanous inhibitory domain (DID) and diaphanous auto-regulatory domain (DAD), and activated by Rho GTPase binding to GTPase-binding domains (GBD). Other formins lack DAD, DID and GBD, and their regulatory mechanisms await elucidation.
Most mitochondrial mRNAs in kinetoplastids require editing, that is, the posttranscriptional insertion and deletion of uridine nucleotides that are specified by guide RNAs and catalyzed by multiprotein complexes. Recent studies have identified many of the proteins in these complexes, in addition to some of their functions and interactions. Although much remains unknown, a picture of highly organized complexes is emerging that shows that the complex that catalyzes the central steps of editing is partitioned into distinct insertion and deletion editing subcomplexes. These subcomplexes coordinate hundreds of ordered catalytic steps that function to produce a single mature mRNA. The dynamic processes, which might entail interactions among multiprotein complexes and changes in their composition and conformation, remain to be elucidated.
Nucleotide pyrophosphatase/phosphodiesterase (NPP)type ectophosphodiesterases are found at the cell surface as type-I or type-II transmembrane proteins, but are also found extracellularly as secreted or shedded enzymes. They hydrolyze pyrophosphate or phosphodiester bonds in a variety of extracellular compounds including nucleotides, (lyso)phospholipids and choline phosphate esters. Despite their structurally related catalytic domain, each enzyme has well-defined substrate specificity. Catalysis by NPPs affects processes as diverse as cell proliferation and motility, angiogenesis, bone mineralization and digestion. In addition, there is emerging evidence for non-catalytic functions of NPPs in cell signaling. NPP-type ectophosphodiesterases are also implicated in the pathophysiology of cancer, insulin resistance and calcification diseases, and they hold great promise as easily accessible therapeutic targets.
Since the discovery of the first member ten years ago, the receptor-interacting protein (RIP) family kinases have emerged as essential sensors of cellular stress. The different members integrate both extracellular stress signals transmitted by various cell-surface receptors and signals emanating from intracellular stress. The cascades of events initiated by activated RIPs are complex. Not only are pro-survival, inflammatory and immune responses triggered by RIP kinases via the activation of transcription factors such as NF-kappa B and AP-1, but opposing, death-inducing programs can also be initiated by the RIP kinases. Hence, RIP kinases are crucial regulators of cell survival and cell death.
Two classes of short RNA molecule, small interfering RNA (siRNA) and microRNA (miRNA), have been identified as sequence-specific posttranscriptional regulators of gene expression. siRNA and miRNA are incorporated into related RNA-induced silencing complexes (RISCs), termed siRISC and miRISC, respectively. The current model argues that siRISC and miRISC are functionally interchangeable and target specific mRNAs for cleavage or translational repression, depending on the extent of sequence complementarity between the small RNA and its target. Emerging evidence indicates, however, that siRISC and miRISC are distinct complexes that regulate mRNA stability and translation. The assembly of RISCs can be traced from the biogenesis of the small RNA molecules and the recruitment of these RNAs by the RISC loading complex (RLC) to the transition of the RLC into the active RISC. Target recognition by the RISC can then take place through different interacting modes.
The lipid kinase phosphoinositide 3-kinase (PI3K) is activated in response to various extracellular signals including peptide growth factors such as insulin and insulin-like growth factors (IGFs). Phosphatidlylinositol (3,4,5) -trisphosphate [Ptdlns(3,4,5)P-3] generated by PI3K is central to the diverse responses elicited by insulin, including glucose homeostasis, proliferation, survival and cell growth. The actions of lipid phosphatases have been considered to be the main means of attenuating PI3K signalling, whereby the principal 3-phosphatase phosphatase and tensin homologue deleted on chromosome 10 (PTEN) - dephosphorylates Ptdlns(3,4,5)P3, reversing the action of PI3K. Recently, however, another pathway of regulation of PI3K has been identified in which activation of PI3K itself is prevented. This finding, together with earlier work, strongly suggests that a major form of negative feedback inhibition of PI3K results from activated growth signalling via mammalian target of rapamycin (mTOR) and the p70 S6 kinase (S6K) - a pathway that could have consequences for the development of type 2 diabetes and tuberous sclerosis complex.
Iron-sulfur (Fe-S) clusters (ISCs) are versatile, ancient co-factors of proteins that are involved in electron transport, enzyme catalysis and regulation of gene expression. The synthesis of ISCs and their insertion into apoproteins involves the function of complex cellular machineries. In eukaryotes, the mitochondrial ISC-assembly machinery is involved in the maturation of all cellular iron-sulfur proteins. A mitochondrial export machinery and a recently discovered cytosolic assembly system specifically participate in the maturation of cytosolic and nuclear iron-sulfur proteins. Of the similar to 20 assembly components, more than ten are encoded by essential genes, which indicates that the process is indispensable for life. Mutations in two of the assembly components lead to neurological diseases. The essential character of Fe-S-protein biogenesis in eukaryotes and its importance for human disease identifies this evolutionary ancient process as one of the most important biosynthetic pathways of life.
Glycine has important neurotransmitter functions at inhibitory and excitatory synapses in the vertebrate central nervous system. The effective synaptic concentrations of glycine are regulated by glycine transporters (GlyTs), which mediate its reuptake into nerve terminals and adjacent glial cells. GlyTs are members of the Na+/Cldependent transporter family, whose activities and subcellular distributions are regulated by phosphorylation and interactions with other proteins. The analysis of GlyT knockout mice has revealed distinct functions of individual GlyT subtypes in synaptic transmission and provided animal models for two hereditary human diseases, glycine encephalopathy and hyperekplexia. Selective GlyT inhibitors could be of therapeutic value in cognitive disorders, schizophrenia and pain.
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that join amino acids to tRNAs, thereby linking the genetic code to specific amino acids. Once considered a class of 'housekeeping' enzymes, ARSs are now known to participate in a wide variety of functions, including transcription, translation, splicing, inflammation, angiogenesis and apoptosis. Three nonenzymatic proteins ARS-interacting multi-functional proteins (AIMPs) associate with ARSs in a multi-synthetase complex of higher eukaryotes. Similarly to ARSs, AIMPs have novel functions unrelated to their support role in protein synthesis, acting as a cytokine to control angiogenesis, immune response and wound repair, and as a crucial regulator for cell proliferation and DNA repair. Evaluation of the functional roles of individual ARSs and AIMPs might help to elucidate why these proteins as a whole contribute such varied functions and interactions in complex systems.
Mediator is an essential component of the RNA polymerase II general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator was the first to be defined and is a high molecular mass complex composed of > 20 distinct subunits that performs multiple activities in transcription. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man.
Gene expression is regulated at multiple levels, and cells need to integrate and coordinate different layers of control to implement the information in the genome. Post-transcriptional levels of regulation such a's transcript turnover and translational control are an,integral part of gene expression and might rival the sophistication and importance of transcriptional control. Microarray-based methods are increasingly used to study not only transcription but also global patterns of transcript decay and translation rates in addition to comprehensively identify targets of RNA-binding proteins. Such large-scale analyses have recently provided supplementary and unique insights into gene expression programs. Integration of several different datasets will ultimately lead to a system-wide understanding of the varied and complex mechanisms for gene expression control.