Regulation Of Protein Localization Research Articles

Protein isoprenylation in biology and disease: general overview and perspectives from studies with genetically engineered animals

The posttranslational modification of proteins by lipids is a key mechanism in the regulation of protein localization and function. Isoprenylation is a process critical for the membrane association of a plethora of signalling proteins with fundamental roles in cell biology, including G proteins and nuclear lamins. Isoprenylation is irreversible but is frequently associated to reversible posttranslational modifications, such as palmitoylation or phosphorylation, that act like switches that modulate the dynamics of protein-membrane or protein-protein interactions and target the modified proteins to specific membrane compartments. The severe phenotype of animals deficient in the enzymes involved in isoprenylation and postprenylation processing highlights the significance of these processes. Moreover, alterations in the genes involved in the maturation of isoprenylated proteins have been found at the basis of some severe human diseases, like choroideremia or the premature ageing progeria syndromes. Given their critical role in the transformation potential of the Ras oncogenes, isoprenylation and postprenylation processing are targets for the development of inhibitors with antitumoral activity. The recent generation of animal models genetically engineered to target the enzymes involved in isoprenylation and associated modifications has unveiled unpredicted aspects of these modifications. Moreover, these models are proving of crucial importance for the elucidation of the mechanisms of disease, and the identification and validation of therapeutic targets. This review attempts to summarize general aspects of the posttranslational modification of proteins by isoprenylation, paying special attention to the evidences obtained from the use of genetically engineered animals and the avenues that these models open.

SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis

Sam68 (Src associated in mitosis; 68 kDa) is an RNA-binding protein and substrate of Src family kinases. It is thought to play a role in cell cycle progression. Overexpression of Sam68 in fibroblasts was reported to have two separable functions dependent on its ability to bind RNA--cell cycle arrest or the induction of apoptosis. Post-translational modification with SUMO (small ubiquitin-like modifier) is common to many transcription factors and can regulate protein localization, stability and function. Here we show Sam68 to be modified by SUMO, and demonstrate that the SUMO E3 ligase (PIAS1) (protein inhibitor of activated STAT1) can enhance Sam68 sumoylation. Lysine 96, the first lysine in the amino-terminal region of Sam68, was found to be the major SUMO acceptor site. Mutation of the SUMO acceptor lysine to arginine enhanced the ability of Sam68 to induce apoptosis but inhibited its ability to act as a transcriptional inhibitor of cyclin D1 expression. A SUMO-1 Sam68 fusion protein, on the other hand, inhibited the ability of Sam68 to induce apoptosis but was a strong repressor of cyclin D1 expression. Thus, SUMO may be an important regulator of Sam68 function in cell cycle progression.

Solution Structure of Human SUMO-3 C47S and Its Binding Surface for Ubc9,

Small ubiquitin-related modifier SUMO-3 is a member of a growing family of ubiquitin-like proteins (Ubls). So far, four isoforms of SUMO have been identified in humans. It is generally known that SUMO modification regulates protein localization and activity. Previous structure and function studies have been mainly focused on SUMO-1. The sequence of SUMO-3 is 46% identical with that of SUMO-1; nevertheless, functional heterogeneity has been found between the two homologues. Here we report the solution structure of SUMO-3 C47S (residues 14-92) featuring the beta-beta-alpha-beta-beta-alpha-beta ubiquitin fold. Structural comparison shows that SUMO-3 C47S resembles ubiquitin more than SUMO-1. On the helix-sheet interface, a strong hydrophobic interaction contributes to formation of the globular and compact fold. A Gly-Gly motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. In vivo, SUMO modification proceeds via a multistep pathway, and Ubc9 plays an indispensable role as the SUMO conjugating enzyme (E2) in this process. To develop a better understanding of SUMO-3 conjugation, the Ubc9 binding surface on SUMO-3 C47S has been detected by chemical shift perturbation using NMR spectroscopy. The binding site mainly resides on the hydrophilic side of the beta-sheet. Negatively charged and hydrophobic residues of this region are highly or moderately conserved among SUMO family members. Notably, the negatively charged surface of SUMO-3 C47S is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. This work indicates dissimilarities between SUMO-3 and SUMO-1 in tertiary structure and provides insight into the specific interactions of SUMO-3 with its modifying enzyme.

SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?

The small ubiquitin-related modifier SUMO posttranslationally modifies many proteins with roles in diverse processes including regulation of transcription, chromatin structure, and DNA repair. Similar to nonproteolytic roles of ubiquitin, SUMO modification regulates protein localization and activity. Some proteins can be modified by SUMO and ubiquitin, but with distinct functional consequences. It is possible that the effects of ubiquitination and SUMOylation are both largely due to binding of proteins bearing specific interaction domains. Both modifications are reversible, and in some cases dynamic cycles of modification may be required for activity. Studies of SUMO and ubiquitin in the nucleus are yielding new insights into regulation of gene expression, genome maintenance, and signal transduction.

Supramolecular complex formation in cell signaling and disease: an update on a recurrent theme in cell life and death.

Cells are constantly exposed to a multitudeof environmental cues. In order to respondappropriately to a given stimulus, integratedanalysis of the provided information isrequired. To this end, signal transductionpathways, the cellular communicationhighways responsible for linking externalcues via receptors to changes in cell cytosoland nucleus, must be cross-linked in anetwork. In the jargon of signaltransduction, this process is referred to as“cross-talk” between signaling pathways.Inherent to this process is the ability ofindividual elements to interact not onlywith components of the same pathway butalso those from different ones. At themolecular level, the importance of thisaspect to signaling is perhaps bestunderscored by the existence of so-calledscaffolding proteins, which, in the absenceof enzymatic activity, function by linkingmultiple proteins from the same or differentpathways. Hence, alterations in cellularbehavior may be viewed as the consequenceof a multitude of protein-protein, protein-lipid and protein-DNA interactionsassociated with signaling.Today, the secrets of signal transductionare being unraveled at a steadily increasingpace thanks to the availability of genomesequences from various species, includinghumans. Perhaps, one important insightfrom the analysis of such data is thatproteins are modular by nature. Althoughthe existence of specific protein moduleswas well accepted prior to the advent ofgenome sequencing, the sheer magnitudenow apparent was unexpected.Furthermore, given that genome size clearlydoes not reflect the functional complexityof an organism, some speculate that thischaracteristic may be more faithfullyencrypted in modularity of the resultingproteins (Lawler, 2003).Some years ago, the question was raisedas to why proteins are so large (Srere,1984). This enigma has now been solvedrather elegantly: protein domainsresponsible for enzymatic activity arecombined with modules responsible forcorrect spatial and temporal regulation ofprotein localization. Furthermore,functional analysis has revealed that thesetraits –activity and positional information–are crucial for correct protein function.Most intriguingly, many experiments cannow be designed more rationally thanks toguidance provided by computerizedanalysis of the wealth of informationavailable in different sequence databases.Currently, enormous resources are beinginvested in an attempt to combine genetics,molecular and cellular biology, medicine aswell as biochemistry to understand not only

Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5

The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 +/- 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(+I1A) or I3(-I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters.

Focus Issue: Protein Interactions Domains, the Second Set

Science's STKE continues to feature the structural motifs important in cellular signaling processes in a second focus issue on the theme of protein interaction domains. The first issue featured an overview published in Science by Pawson and Nash describing signaling domains and their function. STKE featured three Reviews. Lasko described proteins implicated in cellular signaling that have RNA binding motifs, thus linking posttranscriptional regulatory mechanisms to cellular signaling events. Zarrinpar et al . highlighted the domains that recognize proline-rich motifs complete with animations to allow STKE readers to appreciate the differences in binding mechanisms. Finally, Nourry et al . discussed the biological and medical relevance of proteins that contain PDZ domains, a motif that serves as a scaffold to organize multimolecular structures. The second issue in this series features two more interaction motifs: those that recognize phosphorylated tyrosine residues and those that form a β-propeller structure. In the Review by Schlessinger and Lemmon, the spotlight is on the Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains. Not only do they describe the structural features and differences in the binding characteristics of these motifs, but they also provide an overview of their roles in cellular signaling processes. SH2 and PTB domains play crucial roles in regulating protein localization, assembly, and activation and contribute to the regulation of many cellular processes in response to external signals. In the Perspective by Gettemans et al ., the spotlight is on two highly divergent classes of proteins, based on primary sequence, that show very similar overall structure. Gettemans et al . describe proteins with either WD-40 repeats or kelch repeats that adopt the β-propeller structure. These results are described in the context of yeast G protein-signaling cascades. By folding into a β propeller similar to that seen for the G protein β subunit, kelch-domain proteins may function as one subunit of the heterotrimeric G protein in place of the β subunit in particular signaling cascades. The results in yeast emphasize the importance of structure over sequence similarity and open a new avenue for exploring G protein partners and interactions. The final element to this second domains issue is the unveiling of a new section of the STKE site: Teaching Resources. In the Teaching Resources section, you will find various teaching and learning resources that can be used for academic educational purposes. This section will grow to become a collection of resources either developed during the publishing of STKE original articles or Connections Maps or resources contributed by the scientific community. Each resource developed by the STKE will have the scientific oversight of the scientist contributing the article or serving as the Pathway Authority and resources submitted by the community will be reviewed for scientific accuracy. In addition, many STKE articles, Connections Maps, and Teaching Resources will be indexed in the BioSciEdNet portal ( ), an index of peer-reviewed educational resources from the digital libraries at various scientific societies. The first item to be added to the STKE Teaching Resources is an animation created with Zarrinpar et al . that shows schematically how SH3 domains contribute to protein activation using the activation of Ras by the Grb2-SOS complex as an example. As always, the STKE editors welcome your comments, suggestions, and criticisms. The Teaching Resources section, as well as the ST on the Web and Events sections, has a special feedback form that allows users to nominate resources for inclusion in these various sections. Featured in This Focus Issue Reviews Perspectives Teaching Resources Related Resources at STKE Editorial Guides ST on the Web

Cell shape, division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development.

In the last decade, the use of cytological techniques, together with the analysis of complete genomes, has dramatically advanced our understanding of bacterial development. Work on several well-developed model systems such as Bacillus subtilis, Caulobacter crescentus, Myxococcus xanthus and Streptomyces spp., has provided us with an in-depth understanding of processes such as sporulation, multicellular behaviour and the bacterial cell cycle. At the same time, these studies have revolutionized our view of the bacterial cell and shown it to be a highly complex entity with spatial and temporal organization. The recent American Society for Microbiology (ASM) conference on prokaryotic development demonstrated that several laboratories have now started to connect data obtained through functional genomic analysis with subcellular organization, thereby generating three-dimensional regulatory networks. This meeting report highlights new findings in the field, such as regulation of protein localization during sporulation and the cell cycle, control of cell-cell interaction and the initiation of cell division.

Intracellular compartmentalization of leukotriene synthesis: unexpected nuclear secrets

Leukotrienes are important lipid mediators implicated in the regulation of various cellular processes and in disease states as well as homeostasis. Regulation of leukotriene biosynthesis is therefore of considerable interest. Although the levels of expression and catalytic activity of leukotriene-forming proteins have long been recognized as important determinants of leukotriene biosynthesis, it has recently become apparent that their intracellular compartmentalization also affects the integrated output of this biosynthetic pathway. In this minireview, we focus on the unexpected discovery that the nucleus is the key intracellular site for leukotriene biosynthesis and discuss the mechanisms that regulate protein localization and the potential implications of these findings.

Signaling and subcellular targeting by membrane-binding domains.

Protein kinase C homology-1 and -2, FYVE, and pleckstrin homology domains are ubiquitous in eukaryotic signal transduction and membrane-trafficking proteins. These domains regulate subcellular localization and protein function by binding to lipid ligands embedded in cell membranes. Structural and biochemical analysis of these domains has shown that their molecular mechanisms of membrane binding depend on a combination of specific and nonspecific interactions with membrane lipids. In vivo studies of green fluorescent protein fusions have highlighted the key roles of these domains in regulating protein localization to plasma and internal membranes in cells.

SUMO/sentrin: protein modifiers regulating important cellular functions

Regulation of protein functions can be achieved by posttranslational protein modifications. One of the most studied modifications has been conjugation to ubiquitin, which mainly targets substrate proteins for degradation by the 26 S proteasome. Recently, SUMO/sentrin, a ubiquitin-like protein has been characterized. This evolutionary conserved protein is conjugated to specific proteins in a way similar, but not identical, to ubiquitin and seems also to be involved in the regulation of protein localization or function. An increasing number of SUMO/sentrin substrates are currently described. We focus here on three major substrates of modification by SUMO: RanGAP1, PML, and IkappaBalpha proteins. These different examples illustrate how SUMO conjugation may be involved in the control of the level of critical proteins within the cell or in the modulation of subcellular localization and nucleocytoplasmic trafficking.

Regulation of endosome sorting by a specific PP2A isoform.

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869-5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205-216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.

Structural Compartments within Neurons: Developmentally Regulated Organization of Microfilament Isoform mRNA and Protein

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarilyin vivo,has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Ourin vivoandin vitrostudies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and β-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.