Advances in Enzyme Regulation

George Weber

Catherine E. Forrest Weber

Lucio Cocco

Acknowledgements

致谢

Akt/PKB is a key master regulator of a wide range of physiological functions including metabolism, proliferation, survival, growth, angiogenesis and migration and invasion. The Akt protein kinase family comprises three highly related isoforms encoded by different genes. The initial observation that the Akt isoforms share upstream activators as well as several downstream effectors, together with the high sequence homology suggested that their functions were mostly redundant. By contrast, an increasing body of evidence has recently uncovered the concept of Akt isoform signaling specificity, supported by distinct phenotypes displayed by animal strains genetically modified for each of the three genes, as well as by the identification of isoform-specific substrates and association with discrete subcellular locations. Given that Akt is regarded as a promising therapeutic target in a number of pathologies, it is essential to dissect the relative contributions of each isoform, as well as the degree of compensation in pathophysiological function. Here we summarize our view of how Akt selectivity is achieved in the context of subcellular localization, isoform-specific substrate phosphorylation and context-dependent functions in normal and pathophysiological settings.

Nadir M. Maraldi

Giovanna Lattanzi

Vittoria Cenni

Alberto Bavelloni

Sandra Marmiroli

Francesco A. Manzoli

Laminopathies and A-type lamin-associated signalling pathways

核纤层病与A型核纤层相关信号通路

Sanguk Yun

Ha-Young Byun

Young-Seok Oh

Yong Ryoul Yang

Sung Ho Ryu

Pann-Ghill Suh

Adv Enzyme Regul

Protein kinase C-α negatively regulates EGF-induced PLC-ɛ activity through direct phosphorylation

蛋白激酶C-α通过直接磷酸化负调控EGF诱导的PLC-ɛ活性

The need of large-scale chromatin organization in the nucleus has become more and more appreciated. The higher order nuclear organization ultimately regulate a plethora of biological processes including transcription, DNA replication, and DNA repair. In this context, it is of critical importance to understand the mechanisms that allow higher order nuclear organization. Scaffold Attachment Factor A (SAF-A/hnRNPU), which was originally identified as the component of nuclear matrix, has emerged as an important regulator of higher order nuclear organization. It is shown that SAF-A/hnRNPU binds to tandem repeats (TRs) and scaffold/matrix attachment regions (S/MAR) in a sequence-non-specific, but structure-specific manner (e.g. DNA curvature). Recent studies showed that SAF-A interacts with chromatin-associated RNAs (caRNAs) to regulate interphase chromatin structures in a transcription-dependent manner. It is proposed that SAF-A/hnRNPU and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes chromatin in a large scale. The common structural features of S/MAR and pericentromeric (periCEN) TR promotes SAF-A-mediated association with each other. Collectively a model is presented wherein SAF-A/hnRNPU and periCEN TR are the key players in large-scale nuclear organization that supports general transcription.

Kishore S. Malyavantham

Sambit Bhattacharya

Ronald Berezney

The architecture of functional neighborhoods within the mammalian cell nucleus

哺乳动物细胞核内功能邻域的结构

Inositols are natural compounds involved in many biological pathways. Among them myo-inositol (myo-Ins) is the most important and widespread, and it is detectable ubiquitously in almost all biological systems. It is a very ancient molecule, defined as a prebiotic compound. d-Chiro-inositol (d-chiro-Ins), a stereoisomer of myo-Ins, is another molecule of great interest. The endogenous production of these two stereoisomers fluctuates depending on the specific tissue needs. In such a context, their transport system is another crucial component. One of the most significant activities exerted by myo-Ins and d-chiro-Ins concerns glycemia regulation. Both stereoisomers exert an insulin-mimetic activity and are also effective against insulin resistance. They also play other pivotal physiological roles, such as promoting ovulation and fertility. Besides their metabolic activity, myo-Ins and d-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Moreover, inositols in the form of inositol phosphates have the function of essential second messengers. Based on their physiological role and the pathological implications of the altered myo-Ins and d-chiro-Ins ratios, inositol therapy should be designed with two different aims: on one hand, restoring their physiological ratio; on the other hand, altering this ratio in a controlled way to achieve specific effects.

Marco M. Tsui

John D. York

Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear processes

肌醇磷酸和肌醇焦磷酸在发育、细胞信号传导和核过程中的作用

Nit2/ω-amidase catalyzes the hydrolysis of α-ketoglutaramate (KGM, the α-keto acid analogue of glutamine) to α-ketoglutarate and ammonia. The enzyme also catalyzes the amide hydrolysis of monoamides of 4- and 5-C-dicarboxylates, including α-ketosuccinamate (KSM, the α-keto acid analogue of asparagine) and succinamate (SM). Here we describe an inexpensive procedure for high-yield expression of human Nit2 (hNit2) in Escherichia coli and purification of the expressed protein. This work includes: 1) the design of a genetic construct (pQE-Nit22) obtained from the previously described construct (pQE-Nit2) by replacing rare codons within an 81 bp-long DNA fragment "preferred" by E. coli near the translation initiation site; 2) methods for producing and maintaining the pQE-Nit22 construct; 3) purification of recombinant hNit2; and 4) activity measurements of the purified enzyme with KGM and SM. Important features of the hNit2 gene within the pQE-Nit22 construct are: 1) optimized codon composition, 2) the presence of an N-terminus His6 tag immediately after the initiating codon ATG (Met) that permits efficient purification of the end-product on a Ni-NTA-agarose column. We anticipate that the availability of high yield hNit2/ω-amidase will be helpful in elucidating the normal and pathological roles of this enzyme and in the design of specific inhibitors.

Kay Huebner

Joshua C. Saldivar

Jin Sun

Hidetaka Shibata

Teresa Druck

Hits, Fhits and Nits: Beyond enzymatic function

Hits、Fhits 和 Nits：超越酶学功能

In summary, recently developed technologies have begun to draw back the curtain of mystery that obscures some of the basic mechanisms of DNA replication at multiple levels. Studies using extended DNA and chromatin fiber techniques have proven valuable for identifying the location of origins of replication at specific genomic sites and determining their temporal order of replication, for identifying and quantifying sites of DNA damage and localizing chromatin proteins in relation to sites of DNA replication. The future potential of these methods include further discoveries in functional genomics and contributions to the elucidation of the histone code. Such studies could prove very valuable in studies of the mechanisms of cancer development, aging, and other processes of disordered genomic functioning.

David G. Kaufman

Stephanie M. Cohen

Paul D. Chastain

Temporal and functional analysis of DNA replicated in early S phase

早期S期DNA复制的时序和功能分析

The RTKs are one of the most important families mediating transmembrane signaling and they participate and are instrumental in regulating a broad range of physiological activities. Indeed, tyrosine kinases in general, and the processes that they control and/or stimulate, provide a rich source of drug targets, particularly in growth related disorders such as cancer (Zwick et al., 2002; Krause and Van Etten, 2005). However, there remain many questions regarding their activation and downstream signaling and the application of proteomic analyses promises to answer many of them. There have been relatively few detailed studies of this type to date and it will require considerably more of them to better define the pathways with respect to both the major and minor PTMs that, along with the protein-protein interactions, are the means to direct the flow of the signals generated. It will take such approaches to define the specificity that characterize the individual families, even appreciating that to some degree all are capable of activating many, if not all, of the principal pathways. It will also be necessary to understand, in the highly complex networks of intracellular phosphorylation (that contain thousands of sites of modification and clearly have not yet been fully determined in any paradigm), exactly which kinases modify which substrates, and to work out the inter-relationships with other modifications such as O-GlcNAcylation and acetylation. Only then will it be possible to determine which modifications are physiologically significant and which are simply background. Along theway, these studies should continue to provide potential drug targets and perhaps improve the current lackluster biomarker discovery track record.

Jordane Biarc

Robert J. Chalkley

A.L. Burlingame

Ralph A. Bradshaw

Receptor tyrosine kinase signaling – a proteomic perspective

受体酪氨酸激酶信号传导——蛋白质组学视角

Since we first identified the PLC-β isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-β subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-β subtype exist. Most cells express multiple subtypes of PLC-β in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-β subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-β interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-β subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-β subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-β subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-β subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-β in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-β subtypes. It has been observed for a long time that the expression of certain PLC-β subtype fluctuates during diverse physiological conditions. For example, the expression of PLC-β1 is selectively increased during myoblast and adipocyte differentiation (Faenza et al., 2004; O'Carroll et al., 2009). Likewise, PLC-β2 is highly up-regulated during breast cancer progression and plays a critical role in cell migration and mitosis (Bertagnolo et al., 2007). Although PLC-β3 is selectively down-regulated in neuroendocrine tumors, the expression of PLC-β1 is increased in small cell lung carcinoma (Stalberg et al., 2003; Strassheim et al., 2000). In our hypothetical model, it is most likely that up- and down regulation of certain PLC-β subtypes are due to their selective coupling with specific GPCR-mediated signaling, implicated in these pathophysiologic conditions. Therefore, better understanding of selective coupling between PLC-β subtypes, PDZ domain proteins, and GPCRs will shed light on new prognosis and therapy of diverse diseases, and provide potential targets for drug development.

Jung Kuk Kim

Seyoung Lim

Jinho Kim

Sanguk Kim

Jae Ho Kim

Subtype-specific roles of phospholipase C-β via differential interactions with PDZ domain proteins

磷脂酶C-β通过与PDZ域蛋白的不同相互作用发挥亚型特异性作用

Ins and Ins phospholipids are present in and are made by most Archaea and all eukaryotes. Relatively few bacteria possess Ins phospholipids: and only one major grouping, the Actinobacteria, is known to have evolved multiple functions for Ins derivatives. The Ins phospholipids of all organisms, whether they have diradylglycerol or ceramide backbones, seem to use the same Ins1P headgroup stereochemistry, so they are probably made by evolutionarily conserved pathways. It seems likely that an early member of the Archaea made the first phospholipid with an Ins1P headgroup -maybe three billionyears ago – and that amuchlater archaeal descendentwas the ancestral contributor that brought these molecules into the common ancestor of all eukaryotes – maybe two billionyears ago (Michell, 2007, 2008). It will only be possible to infer the likely details of these processes when we have learned much more about the Ins lipid biochemistry of modern archaeons. All eukaryotes make substantial amounts of PtdIns, both as a ‘bulk’ membrane phospholipid and as the precursor of seven phosphorylated derivatives of PtdIns (the polyphosphoinositides; PPIn) and of the ‘GPI anchors’ of cell surface ectoproteins. PtdIns(4,5)P2 – with its many functions – and its precursor PtdIns4P are found in all in eukaryotes. So are PtdIns3P and PtdIns(3,5)P2, which have ubiquitous roles in the regulation of membrane trafficking events. However, synthesis of and signalling by PtdIns(3,4,5) P 3 appears to be confined to a later-evolved group of eukaryotes.

Robert H. Michell

Inositol and its derivatives: Their evolution and functions

肌醇及其衍生物：它们的演变与功能

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