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PubMed2000

Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIb beta 3 and collagen receptors.

Mice deficient in the hematopoietic cell-specific adapter protein SLP-76 demonstrate a failure of T cell development and fetal hemorrhage. Although SLP-76-deficient platelets manifest defective collagen receptor signaling, this alone may not explain the observed bleeding diathesis. Because alpha IIb beta 3, the platelet fibrinogen receptor, is required for normal hemostasis, we explored a potential role for SLP-76 in alpha IIb beta 3 signaling. Interaction of soluble or immobilized fibrinogen with normal human or murine platelets triggers rapid tyrosine phosphorylation of SLP-76. Moreover, platelet adhesion to fibrinogen stimulates actin rearrangements, filopodial and lamellipodial extension, and localization of tyrosine phosphorylated proteins to the cell periphery. In contrast, SLP-76-deficient murine platelets bind fibrinogen normally, but spread poorly and exhibit reduced levels of phosphotyrosine. The in vivo bleeding diathesis as well as the defects in platelet responses to fibrinogen and collagen are reversed by retroviral transduction of SLP-76 into bone marrow derived from SLP-76-deficient mice. These studies establish that SLP-76 functions downstream of alpha IIb beta 3 and collagen receptors in platelets. Furthermore, expression of SLP-76 in hematopoietic cells, including platelets, plays a necessary role in hemostasis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes.

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL. Surprisingly, antibodies to beta1 or beta 3 integrins were also unable to stimulate MAP kinase activation, suggesting that although beta 1 integrins are capable of stimulating MAP kinase activation in other cells, they cannot do so in CTL. In CTL, phosphorylation of proline-rich tyrosine kinase 2 downstream of integrin stimulation did not result in recruitment of the adaptor protein Grb2. Additionally, we examined the role of MAP kinases in regulating integrin-mediated adhesion. Anti-CD3-triggered adhesion to fibronectin was largely insensitive to the MAP kinase kinase inhibitor PD98059. Triggered cell-spreading on fibronectin was inhibited by PD98059 but not by U0126. In summary, ligation of beta 3 integrin by antibodies or fibronectin or of beta1 integrin by monoclonal antibodies fails to activate ERK MAP kinases, but integrin ligation synergizes with T cell receptor stimulation upstream of MAP kinases.

Animals↗
PubMed1997

Postprandial stimulation of muscle protein synthesis is independent of changes in insulin.

Protein synthesis in skeletal muscle is markedly stimulated (approximately 180% of control rate) within 3 h of oral feeding in mice subjected to an overnight fast (18 h). The stimulation of protein synthesis is the result of a faster rate of translation initiation; however, neither the mediators (i.e., hormones or nutrients) nor the mechanisms responsible for the effect of feeding are well understood. Results of the present study revealed that the amount of eukaryotic initiation factor 4E (eIF-4E) present in the phosphorylated form (i.e., 70%) was not changed after overnight starvation or a subsequent 3-h refeeding period compared with muscles from freely fed mice. In contrast, the phosphorylation state of the eIF-4E binding protein 1 (4E-BP1) was changed with nutritional state. Starvation increased the proportion of the unphosphorylated form of 4E-BP1, whereas feeding promoted a shift to the more highly phosphorylated forms of the protein. Moreover, starvation increased the amount of 4E-BP1 recovered by almost threefold, indicative of an increase in the eIF-4E.4E-BP1 complex. The increased association of 4E-BP1 with eIF-4E was completely reversed within 3 h of feeding. Starvation and refeeding also altered the amount of eIF-4G that coimmunoprecipitated with eIF-4E. However, in contrast to the results obtained for 4E-BP1, starvation decreased the amount of eIF-4G recovered in the eIF-4E immunoprecipitate, suggesting that starvation causes a decrease in the formation of the active eIF-4F complex. The alterations in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4G with eIF-4E observed in control mice in response to starvation and refeeding were also observed in diabetic mice exhibiting characteristics of type I or type II diabetes subjected to the same conditions, suggesting that insulin alone does not mediate the observed changes. Thus the integrated feeding response represents an important area of investigation for understanding the regulation of translation initiation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Patterns of meiotic recombination in human fetal oocytes.

Abnormal patterns of meiotic recombination (i.e., crossing-over) are believed to increase the risk of chromosome nondisjunction in human oocytes. To date, information on recombination has been obtained using indirect, genetic methods. Here we use an immunocytological approach, based on detection of foci of a DNA mismatch-repair protein, MLH1, on synaptonemal complexes at prophase I of meiosis, to provide the first direct estimate of the frequency of meiotic recombination in human oocytes. At pachytene, the stage of maximum homologous chromosome pairing, we found a mean of 70.3 foci (i.e., crossovers) per oocyte, with considerable intercell variability (range 48-102 foci). This mean equates to a genetic-map length of 3,515 cM. The numbers and positions of foci were determined for chromosomes 21, 18, 13, and X. These chromosomes yielded means of 1.23 foci (61.5 cM), 2.36 foci (118 cM), 2.5 foci (125 cM), and 3.22 foci (161 cM), respectively. The foci were almost invariably located interstitially and were only occasionally located close to chromosome ends. These data confirm the large difference, in recombination frequency, between human oocytes and spermatocytes and demonstrate a clear intersex variation in distribution of crossovers. In a few cells, chromosomes 21 and 18 did not have any foci (i.e., were presumptively noncrossover); however, configurations that lacked foci were not observed for chromosomes 13 and X. For the latter two chromosome pairs, the only instances of absence of foci were observed in abnormal cells that showed chromosome-pairing errors affecting these chromosomes. We speculate that these abnormal fetal oocytes may be the source of the nonrecombinant chromosomes 13 and X suggested, by genetic studies, to be associated with maternally derived chromosome nondisjunction.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells.

The ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts. While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process. We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes. Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli. Using an epithelial cell infection model, C. pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria. Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. These results suggest that activation of specific cell signalling pathways is an essential strategy used by C. pneumoniae to invade epithelial cells.

Actins↗
PubMed2003

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

Adaptor Protein Complex 2↗
PubMed1999

Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1.

Wnt proteins transduce their signals through dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3beta (GSK), leading to the accumulation of cytosolic beta-catenin and activation of TCF/LEF-1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF-1, we investigated the roles of Axin and Frat1 in Wnt-mediated activation of LEF-1 in mammalian cells. We found that Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. We also found that Dvl, Axin and GSK can form a ternary complex bridged by Axin, and that Frat1 can be recruited into this complex probably by Dvl. The observation that the Dvl-binding domain of either Frat1 or Axin was able to inhibit Wnt-1-induced LEF-1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt-1 appeared to promote the disintegration of the Frat1-Dvl-GSK-Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1-mediated dissociation of GSK from Axin.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways.

Insulin-stimulated glucose uptake in adipose tissue and striated muscle is critical for reducing post-prandial blood glucose concentrations and the dysregulation of this process is one hallmark of Type II (non-insulin-dependent) diabetes mellitus. It has been well established that the insulin-stimulated redistribution of the insulin responsive glucose transporter, GLUT-4, from intracellular storage sites to the plasma membrane depends on the production of phosphoinositide 3,4,5 trisphosphate by the Class IA Phosphatidylinositol 3' kinase. Recent discoveries however, have shown the presence of a second insulin signalling pathway leading to GLUT-4 translocation, a pathway dependent on insulin receptor signalling emanating from caveolae or lipid rafts at the plasma membrane. This pathway begins with the phosphorylation of the adaptor protein Cbl by the insulin receptor, and results in the activation of a small GTP binding protein, TC10, a member of the Rho family. TC10 is able to modulate actin structure in 3T3L1 adipocytes, and its overexpression inhibits insulin-stimulated GLUT-4 translocation, an inhibition completely dependent on localization of TC10 to the caveolae or lipid rafts. The spatial compartmentalization of insulin signalling from caveolae or lipid rafts provides a novel signalling pathway that functions in concert with general signalling mechanisms in the control of actin dynamics regulating insulin-dependent GLUT-4 translocation.

Animals↗
PubMed2005

mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events.

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Is it time to re-classify Crohn's disease?

From the original anatomic classification of Crohn's disease in 1975, there have been three subsequent published classifications. The most recent, the Vienna Classification arose from a 1998 World Congress of Gastroenterology Working Party that attempted to prospectively design a simple and objective Crohn's disease phenotypic classification that encompassed components of age at onset, anatomic location and disease behavior. Subsequent application of the Vienna Classification to clinical practice has demonstrated that the Crohn's disease phenotype changes markedly over time, with nearly 80% of inflammatory disease ultimately evolving into a stricturing or penetrating pattern of behavior, and 15% undergoing a change in anatomic location. Furthermore, in controlled studies, the ability of inflammatory bowel disease experts to similarly identify the disease phenotypes, using the Vienna classification, ranges from poor to fair. Taken together, these failings markedly limit the utilization of the Vienna classification of Crohn's disease in clinical trails or disease management. Recent advances in determining the genetic linkages associated with Crohn's disease will undoubtedly lead to a revised Crohn's disease classification that will combine genotype with phenotype. Nevertheless, before this new classification can become a reality we will need to reconcile a number of key elements, including; (1) localization and grouping of all inflammatory bowel disease(IBD) gene mutations, (2) discovering the function of the IBD genes, (3) understanding the impact of the environment on phenotypic expression, and, (4) linking to well-defined phenotypic databases that will permit accurate prediction of disease natural history and therapeutic response. Indeed, the consistency of the phenotypic data available may be the greatest single challenge in unraveling the complexities of inflammatory bowel disease and developing a new classification of Crohn's disease.

Crohn Disease↗
PubMed2003

Sequence-based discovery of a synthetic peptide inhibitor of caspase 6.

We recently demonstrated that tumor necrosis factor alpha activates caspase 6, which in turn cleaves transcription factor AP-2 alpha. We mapped the cleavage site at 19 amino acids from the N-terminus at the sequence aspartate-argenine-histidine-aspartate (DRHD). Mutating aspartic acid at position 19 abrogated the cleavage site. From these observations, we hypothesized that the DRHD peptide could act as a caspase 6 inhibitor. To test this hypothesis, the peptide zAsp(Ome)-Arg-His-Asp(Ome)-fluoromethyl ketone (zDRHDfmk) was synthesized. Here we show that zDRHDfmk inhibits TNFalpha-induced caspase 6 activity and apoptosis in breast cancer cells. When compared to other caspase inhibitors, zDRHDfmk inhibited caspase 6 activity more effectively than the general caspase inhibitor zVal-Ala-Lys(Ome)-fluoromethy ketone (zVADfmk) or the caspase 6 inhibitor zVal-Glu-Ile-Asp-(Ome)-fluoromethyl ketone (zVEIDfmk). However, it was less effective in inhibiting TNFalpha-induced apoptosis than zVADfmk or zVEIDfmk, presumably because caspase 6 is only one of at least three effector caspases, the others being caspase 3 and 7, that are active during caspase-dependent apoptosis. The discovery of this sequence-based caspase 6 inhibitor provides a new tool for studying caspase 6. More importantly, it could be used, in combination with other agents, as a drug to inhibit apoptosis in neurodegenrative diseases such as Alzheimer's, Parkinson and amyotrophic lateral sclerosis.

Adaptor Protein Complex 2↗
PubMed2002

Nephrocystin-conserved domains involved in targeting to epithelial cell-cell junctions, interaction with filamins, and establishing cell polarity.

Nephrocystin is the protein product of the gene mutated in juvenile nephronophthisis, an autosomal recessive cystic kidney disease afflicting children and young adults. Because the normal cellular function of nephrocystin is largely unknown, the molecular defects underlying disease pathogenesis remain obscure. Analysis of nephrocystin amino acid sequences from human and other species revealed three distinct conserved domains including Src homology 3 and coil-coil domains in the N-terminal region, as well as a large highly conserved C-terminal region bearing no obvious homology to other proteins and hence referred to as the "nephrocystin homology domain" (NHD). The objective of this study was to gain insight into nephrocystin function by defining functional properties of the conserved domains. We analyzed a series of nephrocystin deletion mutants expressed in Madin-Darby canine kidney and COS-7 cells. This analysis revealed previously unrecognized functional attributes of the NHD, including abilities to promote both self-association and epithelial cell-cell junctional targeting. We further observed that Madin-Darby canine kidney cell lines stably expressing a nephrocystin mutant with a deletion of the Src homology 3 domain have reduced ability to establish tight junctions as measured by transepithelial electrical resistance. Finally, from a two-hybrid screen and coimmunoprecipitation studies we identified members of the filamin family of actin-binding proteins as having the capacity to interact with the NHD. These findings support a functional role for nephrocystin as a docking protein involved in organizing a protein complex to regulate the actin cytoskeleton at sites of epithelial cell-cell adhesion and further suggest that these properties are important for establishing epithelial cell polarity.

Actins↗
PubMed2002

Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line.

The interpodocyte slit diaphragm is an essential structure for maintaining the functional glomerular filtration barrier. The slit diaphragm is proposed to consist of an interacting meshwork of nephrin molecules. Earlier studies with tagged proteins have suggested that the intracellular part of nephrin interacts with CD2-associated protein (CD2AP). This study was addressed to show by coimmunoprecipitation and pulldown assays an interaction of endogenously expressed nephrin and CD2AP in the kidney-derived mouse epithelial M-1 cell line, to provide evidence of the domain(s) of CD2AP involved in the interaction, and to show the localization of the respective proteins by immunoelectron microscopy in kidney cortex. In addition, the localization of CD2AP, podocin, alpha-actinin 4, and nephrin was studied in human kidney glomeruli and in M-1 cells by immunofluorescence microscopy. The results indicate an endogenous interaction between nephrin and CD2AP in M-1 cells and suggest that this interaction is mediated by the third Src homology 3 (SH3) domain of CD2AP. We also show by immunoelectron microscopy that nephrin and CD2AP are detected at the slit diaphragm area, supporting their interaction in the glomeruli in vivo. In addition, nephrin was found to partially colocalize with CD2AP and podocin in double immunofluorescence microscopy, confirming the close proximity of these proteins and proposing that these proteins may belong to nephrin-associated protein complex in glomeruli. The existence of nephrin, CD2AP, podocin, and alpha-actinin 4 enables further characterization of their relationship in M-1 cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Disruption of Trrap causes early embryonic lethality and defects in cell cycle progression.

The transactivation/transformation-domain associated protein (TRRAP) belongs to the Ataxia-telangiectasia mutated (ATM) super-family and has been identified as a cofactor for c-MYC-mediated oncogenic transformation. TRRAP and its yeast homolog (Tra1p) are components of histone acetyltransferase (HAT) complexes, SAGA (refs. 2,4,5), PCAF (ref. 3) and NuA4 (ref. 6), which are important for the regulation of transcription and cell cycle progression and also have a role in cell viability. Yet the biological function of this molecule and how it controls proliferation are still unclear. Here we show that null mutation of Trrap in mice results in peri-implantation lethality due to a blocked proliferation of blastocysts. We use an inducible Cre-loxP system to show that loss of Trrap blocks cell proliferation because of aberrant mitotic exit accompanied by cytokinesis failure and endoreduplication. Trrap-deficient cells fail to sustain mitotic arrest despite chromosome missegregation and disrupted spindles, and display compromised cdk1 activity. Trrap is therefore essential for early development and required for the mitotic checkpoint and normal cell cycle progression.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network.

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.

Animals↗
PubMed1996

Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein.

Lfc and Lsc are two recently identified oncoproteins that contain a Dbl homology domain in tandem with a pleckstrin homology domain and thus share sequence similarity with a number of other growth regulatory proteins including Dbl, Tiam-1, and Lbc. We show here that Lfc and Lsc, like their closest relative Lbc, are highly specific guanine nucleotide exchange factors (GEFs) for Rho, causing a >10-fold stimulation of [3H]GDP dissociation from Rho and a marked stimulation of GDP-[35S]GTPgammas (guanosine 5'-O-(3-thiotriphosphate) exchange. All three proteins (Lbc, Lfc, and Lsc) are able to act catalytically in stimulating the guanine nucleotide exchange activity, such that a single molecule of each of these oncoproteins can activate a number of molecules of Rho. Neither Lfc nor Lsc shows any ability to stimulate GDP dissociation from other related GTP-binding proteins such as Rac, Cdc42, or Ras. Thus Lbc, Lfc, and Lsc appear to represent a subgroup of Dbl-related proteins that function as highly specific GEFs toward Rho and can be distinguished from Dbl, Ost, and Dbs which are less specific and show GEF activity toward both Rho and Cdc42. Consistent with these results, Lbc, Lfc, and Lsc each form tight complexes with the guanine nucleotide-depleted form of Rho and bind weakly to the GDP- and GTPgammaS-bound states. None of these oncoproteins are able to form complexes with Cdc42 or Ras. However, Lfc (but not Lbc nor Lsc) can bind to Rac, and this binding occurs equally well when Rac is nucleotide-depleted or is in the GDP- or GTPgammaS-bound state. These findings raise the possibility that in addition to acting directly as a GEF for Rho, Lfc may play other roles that influence the signaling activities of Rac and/or coordinate the activities of the Rac and Rho proteins.

3T3 Cells↗
PubMed1995

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.

Adaptor Proteins, Signal Transducing↗
PubMed2005

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

Adaptor Proteins, Signal Transducing↗