Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity

Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.

Parag Patwardhan and Marilyn D. Resh*

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065

Molecular and Cellular Biology, September 2010, p. 4094-4107, Vol. 30, No. 17
0270-7306/10/$12.00+0     doi:10.1128/MCB.00246-10

Antidote for leishmaniasis

New research has identified several organic compounds that could inhibit the growth ofLeishmania parasites, known to cause leishmaniasis in humans¹. Guided by a computer-aided drug design system, the research has shown that the organic compounds block the activity of an enzyme that is key to the survival of these parasites in the host.

The study may help the development of drugs against leishmaniasis, which claims around 500,000 to 1 million lives every year across the globe. Existing therapies for leishmaniasis are toxic to humans and have also resulted in the emergence of drug-resistant parasitic strains.

The researchers studied a potential drug development target trypanothione reductase (TryR), an enzyme that helps the metabolic activities of the parasite for its survival in the host. TryR has ealier been successful in drug design studies on Trypanosoma cruzi, a parasite that causes Chagas disease.

The researchers chose to target TryR of Leishmania infantum and tested the inhibitory activities of three types of trycyclic organic compounds. They found that all the compounds bind to the active site of the enzyme through the formation of hydrogen bonds. The modelled binding modes provide an insight into the interactions of these compounds with the enzyme, and thus could be used for the design and synthesis of specific inhibitors.

"Because TryR is unique in the Leishmania parasite and is not found in the mammalian host, these TryR-targeted compounds may be exploited to yield safe, affordable drugs," says lead researcher Vikash Kumar Dubey.

• References

  1. Kannan, S. et al. Molecular docking studies of selected tricyclic and quinone derivatives on trypanothione reductase of_Leishmania infantum_. J. Comput. Chem. 31, 2463–2475 (2010)

http://www.nature.com/nindia/2010/100728/full/nindia.2010.100.html

doi:10.1038/nindia.2010.100; Published online 28 July 2010

Bacteria-borne antidote for cancer

http://www.nature.com/nindia/2010/100802/full/nindia.2010.105.html

doi:10.1038/nindia.2010.105; Published online 2 August 2010

Researchers have isolated a new organic compound that halts the migration of cancer cells. They found the compound in a species of Streptomyces, a type of bacteria known to yield antibiotics¹.

The compound inhibited the migration of cultured human and mouse cancer cells by blocking the activity of cysteine protease, an enzyme whose level increases in cancer cells. This suggests that the compound could be a potential drug for treating cancer.

To hunt down effective inhibitors of this enzyme, the researchers screened severalStreptomyces species by exposing them to high-protein-growth media.

They found one strain of Streptomyces species — NCIM 2081 — with protease inhibitory effect. They isolated the active compound CPI-2081 — a mixture of two novel pentapeptides — from the bacteria. The compound's inhibitory effect against migration of cancer cells was tested using human breast and skin cancer cells and mouse skin cancer cells.

The compound successfully inhibited migration of human breast and mouse skin cancer cells at concentrations lower than the cytotoxic dose.

The study is significant since the compound could be developed as a complementary therapy to existing anticancer drugs, which have harmful side effects due to non-specific action on non-cancerous host tissues. The researchers say spread of a tumour (metastasis) can be restricted by applying CPI-2081, followed by antitumour drugs.

The authors of this study are from: National Chemical Laboratory & National Centre for Cell Science, Pune, India; Yale University School of Medicine, New Haven, Connecticut, USA; Memorial Sloan Kettering Cancer Center (MSKCC), New York, USA and CEA Grenoble, LEMOH/INAC/SPrAM, Grenoble, France.

• References

  1. Singh, P. J. et al. Isolation, structure, and functional elucidation of a modified pentapeptide, cysteine protease inhibitor (CPI-2081) from Streptomyces species 2081 that exhibit inhibitory effect on cancer cell migration. J. Med. Chem. 53, 5121-5128 (2010) | Article | PubMed

Guardian of genome: Protein helps prevent damaged DNA in yeast

http://www.eurekalert.org/pub_releases/2010-08/cu-ot073010.php

Like a scout that runs ahead to spot signs of damage, a protein in yeast safeguards the yeast cell’s genome during replication.

Researchers from Cornell University’s Weill Institute for Cell and Molecular Biology have discovered how a protein called Mec1 plays the role of “guardian of the genome”, explained Marcus Smolka, assistant professor of molecular biology and genetics. The findings, “DNA Damage Signalling Recruits the Rtt 107-Six4 Scaffolds via Dpb11 to Mediate Replication Stress Response,” published in Jornal Molecualar Cell, July 30,2010.

Previous Studies have shown that cells lacking Mec1 accumulate damaged DNA and become more sensitive to agents that interfere with replication. The researchers report that the Mec1 protein monitors and repairs the machinery responsible for replicating the DNA. At times, when DNA becomes damaged, the replication machinery can actually detach from the DNA, but Mec1 coordinates the repair of the machinery and the DNA itself, allowing it to restart and continue replicating.

“Mec1 organize the cell’s response against things that jeopardize the integrity of the genome,” Smolka said.

During replication process, Mec1 accumulates at trouble spots such as lesions in the DNA or other blocks to replication. Mec1 is know as a kinase, a type of enzyme that modifies other proteins by adding a phosphate group to them, phosphorylation, which then leads to functional change in the protein. Mec1 adds a phosphate group to a protein known as Six4, which then triggers Six4 to anchor to the replication machinery. Six4 then can employ a variety of tools to repair DNA and the replication machinery.

The findings are important because researchers have discovered  counterparts, orthologues to Mec1 & related proteins with similar biological pathways in humans. Also, mutations to the human genes that produce Mec1 and related proteins can lead to cancer predisposition and neurological disorders.At the same time, cancer cells employ their own similar replication repair system.

Recently, other researchers discovered that the human version of Mec1 called ATR, phosphorylates a protein that is the human counterpart to Six4. The next step, Smolka said, will be to see if after phosphorylation the human Six4 also anchors to the replication machinery to repair any damaged machinery or DNA.

Cdk1-Dependent Regulation of the Mitotic Inhibitor Wee1

Cell, Volume 122, Issue 3, 12 August 2005, Pages 407-420

Stacy L. Harvey, Alyson Charlet, Wilhelm Haas, Steven P. Gygi and Douglas R. Kellogg

Summary

• The Wee1 kinase phosphorylates and inhibits cyclin-dependent kinase 1 (Cdk1), thereby delaying entry into mitosis until appropriate conditions have been met.
  
• An understanding of the mechanisms that regulate Wee1 should provide new insight into how cells make the decision to enter mitosis. We report here that Swe1, the budding-yeast homolog of Wee1, is directly regulated by Cdk1.
• Phosphorylation of Swe1 by Cdk1 activates Swe1 and is required for formation of a stable Swe1-Cdk1 complex that maintains Cdk1 in the inhibited state. Dephosphorylation of Cdk1 leads to further phosphorylation of Swe1 and release of Cdk1. Thus, Cdk1 both positively and negatively regulates its own inhibitor. Regulation of the Swe1-Cdk1 complex is likely to play a critical role in controlling the transition into mitosis.

Disease-Associated Prion Protein Oligomers Inhibit the 26S Proteasome

• The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein.
• We report that disease-associated prion protein specifically inhibits the proteolytic subunits of the 26S proteasome.
  
• Using reporter substrates, fluorogenic peptides, and an activity probe for the subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines.
• By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative sheet conformation inhibits the 26S proteasome at stoichiometric concentrations.
  
• Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice.
  
• Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein. new proteins. that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. The overall system of ubiquitination and proteasomal degradation is known as the

• Proteasomes are large protein complexes inside all eukaryotes and archaea, as well as in some bacteria.

1. In eukaryotes, they are located in the nucleus and the cytoplasm.^[1] The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases.
   
2. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into amino acids and used in synthesizing^[2]
3. Proteins are tagged for degradation by a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein.^[2]
4. In structure, the proteasome is a large barrel-like complex containing a "core" of four stacked rings around a central pore.
   
5. Each ring is composed of seven individual proteins. The inner two rings are made of seven β subunits that contain the six protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded.
   
6. The outer two rings each contain seven α subunits whose function is to maintain a "gate" through which proteins enter the barrel. These α subunits are controlled by binding to "cap" structures or regulatory particlesubiquitin-proteasome system.
7. The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress.
   
8. The importance of proteolytic degradation inside cells and the role of ubiquitin in proteolytic pathways was acknowledged in the award of the 2004 Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose.^[3]

• amyloidogenic proteins:-The various protein deposits of brain amyloidosis share common ultrastructural, biophysical, and histological properties. These amyloidogenic deposits can be composed of distinct proteins, which are conceptually associated with different diseases.
  

1. Amyloidogenic proteins are typically soluble monomeric precursors, which undergo remarkable conformation changes associated with the polymerization into 8- to 10-nm wide fibrils, which culminate in the formation of amyloid aggregates.
2. Some amyloidogenic inclusions are extracellular, such as senile plaques of Alzheimer’s disease, which are composed of amyloid beta (Abeta) peptides.
3. Intracytoplasmic amyloid aggregates, such as neurofibrillary tangles in Alzheimer’s disease and Lewy bodies in Parkinson’s disease, are composed of the proteins tau and alpha-synuclein, respectively. These proteins are directly linked to the etiology of spectrum of neurodegenerative diseases, coining the terms "tauopathies" and "synucleinopathies."

Further Evidence for BRCA1 Communication with the Inactive X Chromosome

Cell, Volume 128, Issue 5, 9 March 2007, Pages 991-1002
Daniel P. Silver, Stoil D. Dimitrov, Jean Feunteun, Rebecca Gelman, Ronny Drapkin, Shihua D. Lu, Elena Shestakova, Soundarapandian Velmurugan, Nicholas DeNunzio, Serban Dragomir, Jessica Mar, Xiaoling Liu, Sven Rottenberg, Jos Jonkers, Shridar Ganesan and David M. Livingston

Summary

, a breast and ovarian cancer-suppressor gene, exerts tumor-suppressing functions that appear to be associated, at least in part, with its DNA repair, checkpoint, and mitotic regulatory activities. Earlier work from our laboratory also suggested an ability of BRCA1 to communicate with the inactive X chromosome (Xi) in female somatic cells (). (this issue of ) have challenged this conclusion. Here we discuss recently published data from our laboratory and others and present new results that, together, provide further support for a role of BRCA1 in the regulation of XIST concentration on Xi in somatic cells.

Mutations in the ERGolgi Intermediate Compartment Protein ERGIC-53 Cause Combined Deficiency of Coagulation Factors V and VIII

Cell, Volume 93, Issue 1, 3 April 1998, Pages 61-70
William C Nichols, Uri Seligsohn, Ariella Zivelin, Valeri H Terry, Colette E Hertel, Matthew A Wheatley, Micheline J Moussalli, Hans-Peter Hauri, Nicola Ciavarella, Randal J Kaufman and David Ginsburg

Summary

Combined deficiency of factors V and VIII is an autosomal recessive bleeding disorder resulting from alterations in an unknown gene on chromosome 18q, distinct from the factor V and factor VIII genes. ERGIC-53, a component of the ERGolgi intermediate compartment, was mapped to a YAC and BAC contig containing the critical region for the combined factors V and VIII deficiency gene. DNA sequence analysis identified two different mutations, accounting for all affected individuals in nine families studied. Immunofluorescence and Western analysis of immortalized lymphocytes from patients homozygous for either of the two mutations demonstrate complete lack of expression of the mutated gene in these cells. These findings suggest that ERGIC-53 may function as a molecular chaperone for the transport from ER to Golgi of a specific subset of secreted proteins, including coagulation factors V and VIII.

Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase

Cell, Volume 121, Issue 5, 3 June 2005, Pages 725-737
Françoise Wyers, Mathieu Rougemaille, Gwenaël Badis, Jean-Claude Rousselle, Marie-Elisabeth Dufour, Jocelyne Boulay, Béatrice Régnault, Frédéric Devaux, Abdelkader Namane, Bertrand Séraphin, Domenico Libri and Alain Jacquier

Summary

Since detection of an RNA molecule is the major criterion to define transcriptional activity, the fraction of the genome that is expressed is generally considered to parallel the complexity of the transcriptome. We show here that several supposedly silent intergenic regions in the genome of are actually transcribed by RNA polymerase II, suggesting that the expressed fraction of the genome is higher than anticipated. Surprisingly, however, RNAs originating from these regions are rapidly degraded by the combined action of the exosome and a new poly(A) polymerase activity that is defined by the Trf4 protein and one of two RNA binding proteins, Air1p or Air2p. We show that such a polyadenylation-assisted degradation mechanism is also responsible for the degradation of several Pol I and Pol III transcripts. Our data strongly support the existence of a posttranscriptional quality control mechanism limiting inappropriate expression of genetic information.

Nuclear Retention of Unspliced mRNAs in Yeast Is Mediated by Perinuclear Mlp1

Cell, Volume 116, Issue 1, 9 January 2004, Pages 63-73
Vincent Galy, Olivier Gadal, Micheline Fromont-Racine, Alper Romano, Alain Jacquier and Ulf Nehrbass

Summary

The molecular mechanism underlying the retention of intron-containing mRNAs in the nucleus is not understood. Here, we show that retention of intron-containing mRNAs in yeast is mediated by perinuclearly located Mlp1. Deletion of impairs retention while having no effect on mRNA splicing. The Mlp1-dependent leakage of intron-containing RNAs is increased in presence of , a splicing mutant. When overall pre-mRNA levels are increased by deletion of a nuclear exosome component, deletion augments leakage of only the intron-containing portion of mRNAs. Our data suggest, moreover, that Mlp1-dependent retention is mediated via the 5 splice site. Intriguingly, we found Mlp-proteins to be present only on sections of the NE adjacent to chromatin. We propose that at this confined site the perinuclear Mlp1 implements a quality control step prior to export, physically retaining faulty pre-mRNAs.