Der3p/Hrd1p Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Lumenal and Integral Membrane Proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3–1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61–2 allele. This is accompanied by the stabilization of the Sec61–2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61–2 strain at the permissive temperature of 25°C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61–2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.

Epstein–Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors

Epstein–Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-κB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.

STIM and Orai proteins: players in sexual differences in hypertension-associated vascular dysfunction?

Sex-associated differences in hypertension have been observed repeatedly in epidemiological studies; however, the mechanisms conferring vascular protection to females are not totally elucidated. Sex-related differences in intracellular Ca(2+) handling or, more specifically, in mechanisms that regulate Ca(2+) entry into vascular smooth muscle cells have been identified as players in sex-related differences in hypertension-associated vascular dysfunction. Recently, new signalling components that regulate Ca(2+) influx, in conditions of intracellular store depletion, were identified: STIM1 (stromal interaction molecule 1), which works as an intracellular Ca(2+) sensor; and Orai1, which is a component of the CRAC (Ca(2+) release-activated Ca(2+)) channels. Together, these proteins reconstitute store-operated Ca(2+) channel function. Disturbances in STIM1/Orai1 signalling have been implicated in pathophysiological conditions, including hypertension. In the present article, we analyse evidence for sex-related differences in Ca(2+) handling and propose a new hypothesis where sex-related differences in STIM/Orai signalling may contribute to hypertension-associated vascular differences between male and female subjects.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

Compartmentalization in membrane rafts defines a pool of N-cadherin associated with catenins and not engaged in cell-cell junctions in melanoma cells.

Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

Arenavirus budding resulting from viral-protein-associated cell membrane curvature

Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations.

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Expressions- und Funktionsanalysen von "Tetraspan Vesicle Membrane Proteins" in Caenorhabditis elegans

Tetraspan vesicle membrane proteins (TVPs) sind ubiquitäre Komponenten von Transportvesikeln. Bei den Säugetieren unterscheidet man drei Familien, die Physine, Gyrine und SCAMPs (secretory carrier-associated membrane proteins). Ihre Funktion ist weitgehend unbekannt, es wird jedoch vermutet, dass sie eine Rolle bei der Vesikelbildung und der Vesikelrezirkulierung spielen. In Caenorhabditis elegans existiert von jeder Familie jeweils nur ein einziges Polypeptid: für die Physine Synaptophysin (SPH-1), für die Gyrine Synaptogyrin (SNG-1) und für die SCAMPs SCAMP (SCM-1). Ziel der Arbeit war es die Verteilung der C. elegans TVPs zu untersuchen und ihre Funktion unter besonderer Berücksichtigung der vesikelvermittelten synaptischen Kopplung zu bestimmen. Wenn die C. elegans TVPs in humanen Epithelzellen synthetisiert werden, lokalisieren sie in zytoplasmatischen Vesikeln. In Kotransfektionsexperimenten wurde gezeigt, dass sie größtenteils in den gleichen Strukturen enthalten sind. In C. elegans synthetisierte TVP-Reporterkonstrukte können in unterschiedlichen Geweben nachgewiesen werden. Dabei ist SNG-1 fast ausschließlich in Neuronen zu finden. SPH-1 und SCM-1 hingegen weisen komplexe und teilweise überlappende Verteilungsmuster auf. Während für SPH-1 eine starke Fluoreszenz im Pharynx, auf der apikalen Seite der Darmzellen oberhalb des sog. terminal webs und in adluminalen Regionen von exkretorischen Geweben gefunden wurde, war SCM-1 stark in der Muskulatur und den Coelomozyten vertreten. Die Expression von SCM-1 in Pharynx und Darm war deutlich schwächer. Die C. elegans TVPs werden früh in der Entwicklung ab der Gastrulation (SPH-1 und SCM-1) bzw. ab der Neurulation im sog. Komma-Stadium (SNG-1) produziert. Um die Funktion der TVPs in C. elegans zu untersuchen, wurden TVP-Mutanten analysiert. Durch Kombination aller drei TVP-Gen-Mutanten wurden TVP-Dreifachmutanten generiert. Diese wiesen keinen offensichtlichen Defekt im Bewegungsmuster auf, entwickelten sich normal und bildeten ein normales Nervensystem aus. Auch auf unterschiedliche chemische und physikalische Reize in sensorischen Tests reagierten die TVP-Dreifachmutanten in gleicher Weise wie Wildtyptiere. Ebenso zeigen die TVP-Dreifachmutanten elektrophysiologisch unter normalen Bedingungen keine anormalen Reaktionsmuster. In ultrastrukturellen Untersuchungen wurde lediglich eine signifikant erhöhte Anzahl Clathrin-ummantelter Vesikel in cholinergen Synapsen gefunden. Erst unter Stressbedingungen, hervorgerufen durch den GABA-Antagonisten Pentylentetrazol (PTZ), wiesen sowohl die TVP-Dreifach- als auch die TVP-Einzelmutanten eine deutlich erhöhte Krampfbereitschaft auf. Zusammengenommen zeigen die Analysen, dass TVPs zwar für grundlegende neuronale Prozesse nicht notwendig sind, dass sie aber auf der anderen Seite vermutlich an alternativen redundanten Wegen der Neurotransmitterfreisetzung beteiligt sind.

A biomimetic model membrane system on oxidic surfaces designed for the investigation of cytochrome c oxidase and other membrane proteins by fluorescence and waveguide spectroscopy

Die transmembrane Potenzialdifferenz Δφm ist direkt mit der katalytischen Aktivität der Cytochrom c Oxidase (CcO) verknüpft. Die CcO ist das terminale Enzym (Komplex IV) in der Atmungskette der Mitochondrien. Das Enzym katalysiert die Reduktion von O2 zu 2 H2O. Dabei werden Elektronen vom natürlichen Substrat Cytochrom c zur CcO übertragen. Der Eleltronentransfer innerhalb der CcO ist an die Protonentranslokation über die Membran gekoppelt. Folglich bildet sich über der inneren Membrane der Mitochondrien eine Differenz in der Protonenkonzentration. Zusätzlich wird eine Potenzialdifferenz Δφm generiert.rnrnDas Transmembranpotenzial Δφm kann mit Hilfe der Fluoreszenzspektroskopie unter Einsatz eines potenzialemfindlichen Farbstoffs gemessen werden. Um quantitative Aussagen aus solchen Untersuchungen ableiten zu können, müssen zuvor Kalibrierungsmessungen am Membransystem durchgeführt werden.rnrnIn dieser Arbeit werden Kalibrierungsmessungen von Δφm in einer Modellmembrane mit inkorporiertem CcO vorgestellt. Dazu wurde ein biomimetisches Membransystem, die Proteinverankerte Doppelschicht (protein-tethered Bilayer Lipid Membrane, ptBLM), auf einem transparenten, leitfähigem Substrat (Indiumzinnoxid, ITO) entwickelt. ITO ermöglicht den simultanen Einsatz von elektrochemischen und Fluoreszenz- oder optischen wellenleiterspektroskopischen Methoden. Das Δφm in der ptBLM wurde durch extern angelegte, definierte elektrische Spannungen induziert. rnrnEine dünne Hydrogelschicht wurde als "soft cushion" für die ptBLM auf ITO eingesetzt. Das Polymernetzwerk enthält die NTA Funktionsgruppen zur orientierten Immobilisierung der CcO auf der Oberfläche der Hydrogels mit Hilfe der Ni-NTA Technik. Die ptBLM wurde nach der Immobilisierung der CcO mittels in-situ Dialyse gebildet. Elektrochemische Impedanzmessungen zeigten einen hohen elektrischen Widerstand (≈ 1 MΩ) der ptBLM. Optische Wellenleiterspektren (SPR / OWS) zeigten eine erhöhte Anisotropie des Systems nach der Bildung der Doppellipidschicht. Cyklovoltammetriemessungen von reduziertem Cytochrom c bestätigten die Aktivität der CcO in der Hydrogel-gestützten ptBLM. Das Membranpotenzial in der Hydrogel-gestützten ptBLM, induziert durch definierte elektrische Spannungen, wurde mit Hilfe der ratiometrischen Fluoreszenzspektroskopie gemessen. Referenzmessungen mit einer einfach verankerten Dopplellipidschicht (tBLM) lieferten einen Umrechnungsfaktor zwischen dem ratiometrischen Parameter Rn und dem Membranpotenzial (0,05 / 100 mV). Die Nachweisgrenze für das Membranpotenzial in einer Hydrogel-gestützten ptBLM lag bei ≈ 80 mV. Diese Daten dienen als gute Grundlage für künftige Untersuchungen des selbstgenerierten Δφm der CcO in einer ptBLM.

Kinetoplastid-specific pATOM36 mediates membrane insertion of a subset of mitochondrial outer membrane proteins

In trypanosomes, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported into the mitochondrion. The recently characterized multisubunit ATOM complex mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial matrix proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein insertase acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis furthermore shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the kDNA as it forms a physical connection between the kDNA and the basal body of the flagellum throughout the cell cycle. Thus, the presence of pATOM36 in the TAC provides an exciting link between mitochondrial protein import and kDNA inheritance.

A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-ErbB signaling to actin stress fiber disassembly

The avian erythroblastosis viral oncogene (v-erbB) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-erbB in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-erbB-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc–Grb2–Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.