Wild-type Overexpression Research Articles

A reevaluation of the role of the heterotrimeric G protein in coupling light responses in Arabidopsis.

Previous studies implicated the involvement of a heterotrimeric G protein in red (R) and far-red (FR) light signal transduction, but these studies utilized pharmacological or gain-of-function approaches and, therefore, are indirect tests. Here, we reexamine the role of the single canonical heterotrimeric G protein in R and FR control of hypocotyl growth using a loss-of-function approach. Single- and double-null mutants for the GPA1, AGB1 genes encoding the alpha and beta subunit of the heterotrimeric G protein, respectively, have wild-type sensitivity to R and FR. Ectopic overexpression of wild type and a constitutive active form of the alpha subunit and of the wild-type beta subunit had no effect that can be unequivocally attributed to altered R and FR responsiveness. These results preclude a direct role for the heterotrimeric G complex in R and FR transduction in Arabidopsis leading to growth control in the hypocotyl.

AGGRESOME FORMATION IN NEUROPATHY MODELS BASED ON PERIPHERAL MYELIN PROTEIN 22 MUTATIONS

Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degradation lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been observed with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and alphaB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also observed in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degradation are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.

Aggresome Formation in Neuropathy Models Based on Peripheral Myelin Protein 22 Mutations

Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degradation lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been observed with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and αB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also observed in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degradation are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.

Activation of Constitutive Nitric-oxide Synthase Activity Is an Early Signaling Event Induced by Ionizing Radiation

Ionizing radiation at clinical dose levels activates both pro- and anti-proliferative signal transduction pathways, the balance of which determines cell fate. The initiating and amplifying mechanisms involved in the activation are poorly understood. We demonstrate that one mechanism involves stimulation of constitutive nitric-oxide synthase (NOS) activity. NOS activity of Chinese hamster ovary cells was measured by the arginine --> citrulline conversion assay. Irradiation stimulated a transient activation of NOS with maximal activity at 5 min of post-irradiation. Western blot analysis and genetic manipulation by overexpression of wild type or dominant negative NOS mutant identify the radiation-induced isoform as NOS-1. Further evidence that NOS-1 is activated by radiation was the demonstration of radiation-induced cGMP formation in cells transiently transfected with the NO-dependent soluble guanylate cyclase. Protein Tyr nitration, a footprint of peroxynitrite formation, followed radiation exposure and was inhibited by expression of a dominant negative NOS-1 mutant. Radiation-induced ERK1/2 kinase activity, a cytoprotective response to radiation, was also blocked by inhibiting NOS activity. These experiments establish NO-dependent signal transduction pathways as being radio-responsive. Given the lipophilic and relatively stable properties of NO, these results also suggest a possible mechanism by which ionization events in one cell may activate signaling processes in adjacent cells.

CAMP Response Element-binding Protein Content Is a Molecular Determinant of Smooth Muscle Cell Proliferation and Migration

We hypothesized that cAMP response element-binding protein (CREB) could function as a molecular determinant of smooth muscle cell fate. In arterial sections from the systemic and pulmonary circulation, CREB content was high in proliferation-resistant medial subpopulations of smooth muscle cells and low in proliferation-prone regions. In vessels from neonatal calves exposed to chronic hypoxia, CREB content was depleted and smooth muscle cell (SMC) proliferation was accelerated. Induction of quiescence by serum deprivation in culture led to increased CREB content. Highly proliferative SMC in culture were observed to have low CREB content. Exposure to proliferative stimuli such as hypoxia or platelet-derived growth factor decreased SMC CREB content. Assessment of CREB gene transcription by nuclear run-on analysis and transcription from a CREB promoter-luciferase construct indicate that CREB levels in SMC are in part controlled at the level of transcription. Overexpression of wild type or constitutively active CREB in primary cultures of SMC arrested cell cycle progression. Additionally, expression of constitutively active CREB decreased both proliferation and chemokinesis. Consistent with these functional properties, active CREB decreased the expression of multiple cell cycle regulatory genes, as well as genes encoding growth factors, growth factor receptors, and cytokines. Our data suggest a unique mode of cellular phenotype determination at the level of the nuclear content of CREB.

Caspase-mediated Cleavage of the Ca2+/Calmodulin-dependent Protein Kinase-like Kinase Facilitates Neuronal Apoptosis

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.

Rb and TP53 Pathway Alterations in Sporadic and NF1-Related Malignant Peripheral Nerve Sheath Tumors

Karyotypic complexities associated with frequent loss or rearrangement of a number of chromosome arms, deletions, and mutations affecting the TP53 region, and molecular alterations of the INK4A gene have been reported in sporadic and/or neurofibromatosis type I (NF1)-related malignant peripheral nerve sheath tumors (MPNSTs). However, no investigations addressing possible different pathogenetic pathways in sporadic and NF1-associated MPNSTs have been reported. This lack is unexpected because, despite similar morphologic and immunophenotypic features, NF1-related cases are, by definition, associated with NF1 gene defects. Thus, we investigated the occurrence of TP53 and p16INK4A gene deregulation and the presence of microsatellite alterations at markers located at 17p, 17q, 9p21, 22q, 11q, 1p, or 2q loci in MPNSTs and neurofibromas either related (14 cases) or unrelated (14 cases) to NF1. Our results indicate that, in MPNSTs, p16INK4A inactivation almost equally affects both groups. However, TP53 mutations and loss of heterozygosity involving the TP53 locus (43% versus 9%), and p53 wild type overexpression, related or not to mdm2 overexpression (71% versus 25%), seem to mainly be restricted to sporadic MPNSTs. In NF1-associated MPNSTs, our microsatellite results are consistent with the occurrence of somatic inactivation by loss of heterozygosity of the second NF1 allele.

P53 associates with and targets Delta Np63 into a protein degradation pathway.

A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27-29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or Delta N-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of Delta Np63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain Delta Np63 proteins (p40 and Delta Np63 alpha). The association between p53 and Delta Np63 supports a previously unrecognized role for p53 in regulation of Delta Np63 stability. The ability of p53 to mediate Delta Np63 degradation may balance the capacity of Delta Np63 to accelerate tumorigenesis or to induce epithelial proliferation.

Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity

In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.

L-type Ca 2+ channel-mediated Zn 2+ toxicity and modulation by ZnT-1 in PC12 cells

In view of evidence that Zn 2+ neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn 2+ neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 μM ZnCl 2 for 15 min under depolarizing conditions resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca 2+ channel antagonist, nimodipine, and increased by the L-type Ca 2+ channel opener, S(−)-Bay K 8644. At lower insult levels (200 μM Zn 2++Bay K 8644), Zn 2+-induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH 2F (Z-VAD); at higher insult levels (1000 μM+Bay K 8644), cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane transporter, ZnT-1, modulates Zn 2+ neurotoxicity, we generated stable PC12 cell lines overexpressing wild type or dominant negative forms of rat ZnT-1 (rZnT-1). Clones T9 and T23 overexpressing wild type rZnT-1 exhibited enhanced Zn 2+ efflux and reduced vulnerability to Zn 2+-induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.

REGULATION OF TNF-α EXPRESSION IN NORMAL MACROPHAGES: THE ROLE OF C/EBPβ

C/EBPβ is present in monocytes and macrophages, binds to the proximal region of the TNF-α promoter, and contributes to its regulation. This study was performed to characterize the ability of C/EBPβ to regulate the TNF-α gene in myelomonocytic cells and primary macrophages. In transient transfection assays, overexpression of wild type C/EBPβ resulted in a 3–4-fold activation of a 120 base pair TNF-α promoter-reporter construct, while overexpression of a dominant negative (DN) C/EBPβ inhibited LPS-induced activation. In vitro monocyte-differentiated macrophages, infected with an adenoviral vector expressing the DN C/EBPβ (AdDNC/EBPβ) or the control Adβgal, expressed their transgenes weakly, however expression was greatly enhanced in the presence of PMA. Infection with AdDNC/EBPβ resulted in 60% suppression of LPS induced TNFα secretion compared to Adβgal infection (P<0.001) in PMA-treated macrophages. Northern blot analysis demonstrated approximately a 40% reduction of the TNF-α mRNA in the presence of the DN C/EBPβ, suggesting that the effect of the DN C/EBPβ was at the transcriptional level. In contrast, AdDNC/EBPβ infection did not result in inhibition of LPS-induced TNF-α secretion in the absence of PMA. Further, DN versions of both C/EBPβ and c-Jun, but not NF-κB p65, suppressed PMA-induced TNF-α secretion in macrophages. These observations demonstrate that, C/EBPβ and c-Jun contribute to the regulation of the TNF-α gene in normal macrophages following treatment with PMA.

Transcriptional Regulation of the Cyclooxygenase-2 Gene in Activated Mast Cells

Activation of mast cells by aggregation of their IgE receptors induces rapid and transient synthesis of cyclooxygenase-2 (COX-2). In this study we investigated (i) the cis-acting response elements and transcription factors active at the COX-2 promoter and (ii) the signal transduction pathways mediating COX-2 induction following aggregation of mast cell IgE receptors. Transient transfection assays with COX-2 promoter/luciferase constructs suggest that a consensus cyclic AMP response element is essential for induced COX-2 expression. Cotransfection studies with plasmids expressing c-Jun, dominant negative Ras, dominant negative c-Jun NH(2)-terminal kinase, and dominant negative MEKK1 demonstrate that activation of the Ras/MEKK1/c-Jun NH(2)-terminal kinase/c-Jun pathway is required for COX-2 promoter-mediated luciferase expression. Attenuation of COX-2 promoter activity by dominant negative constructs for Raf-1, ERK1, and ERK2 suggests that the Ras/Raf-1/extracellular signal-regulated kinase pathway is also necessary for COX-2 induction. Although mutating the two NF-IL6 sites individually did not affect COX-2 promoter activity, mutating both NF-IL6 sites substantially inhibits COX-2 promoter activity. Moreover, overexpression of wild type CCAAT/enhancer-binding protein-beta (C/EBPbeta) augments COX-2 promoter activity in activated mast cells and cotransfection of a dominant negative C/EBPbeta construct completely blocks COX-2 promoter/luciferase expression. Our data suggest that in activated mast cells, a Ras/MEKK1/c-Jun NH(2)-terminal kinase signal transduction pathway activating c-Jun, a Ras/Raf-1/extracellular signal-regulated kinase pathway, and activated C/EBPbeta facilitate COX-2 induction via the cyclic AMP response element and NF-IL6 sites of the COX-2 promoter.

Both p38αMAPK and JNK/SAPK Pathways Are Important for Induction of Nitric-oxide Synthase by Interleukin-1β in Rat Glomerular Mesangial Cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.

The Mitogen-activated Protein (MAP) Kinase p38 and Its Upstream Activator MAP Kinase Kinase 6 Are Involved in the Activation of Signal Transducer and Activator of Transcription by Hyperosmolarity

Environmental stress (e.g. aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the "stress-responsive" mitogen-activated protein kinases and nuclear factor kappaB. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6.

Gα13 Stimulates Rho-dependent Activation of the Cyclooxygenase-2 Promoter

Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells. Here, we examine the regulation of COX-2 promoter activity by alpha subunits of heterotrimeric G proteins in NIH 3T3 cells. Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for alpha subunits of G-proteins, we show that overexpression of wild type and constitutively active Galpha(13) and Galpha(q) induced transcription from the COX-2 promoter. The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active Galpha(13)(Q226L). We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter. Co-expression of Clostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either Galpha(13)Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin. We conclude that Galpha(13) signals through a Rho-dependent pathway leading to activation of the COX-2 promoter. This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by Galpha(13)/Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.

Etk/Bmx, a PH-domain containing tyrosine kinase, protects prostate cancer cells from apoptosis induced by photodynamic therapy or thapsigargin.

Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability confirmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A specific inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an efficient effector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.

Cloning, characterisation, and functional expression of the Mus musculus SKD1 gene in yeast demonstrates that the mouse SKD1 and the yeast VPS4 genes are orthologues and involved in intracellular protein trafficking.

The mouse SKD1 protein displays a high degree of sequence identity (62%) to the yeast Vps4 protein, which is involved in the transport of proteins out of a prevacuolar/endosomal compartment. We isolated the mouse SKD1 locus and found that the SKD1 gene is split into 11 exons covering a region of 29kb of the genome. Interestingly, the exon/intron structure reflects to a certain degree the proposed domain structure of the protein, since the 5' located coiled-coil region and the AAA domain are flanked by introns. Analysis of the promoter region, which revealed features common for 'housekeeping genes', is consistent with previous results of a mouse multi-tissue Northern blot, confirming that SKD1 is a ubiquitously expressed gene. Expression of the full-length SKD1 cDNA in a vps4 disrupted yeast strain suppressed the temperature-sensitive growth defect of the vps4 mutant strain. Overexpression of wild type and expression of mutant Vps4 and SKD1 proteins, harbouring single amino acid exchanges in their AAA domains, induced a dominant-negative vacuolar protein sorting defect in wild type yeast cells, indicating that mouse SKD1 protein and yeast Vps4p fulfil similar functions.

SH2-B Is Required for Nerve Growth Factor-induced Neuronal Differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.

The N terminus of the Qcr7 protein of the cytochrome bc1 complex is not essential for import into mitochondria in Saccharomyces cerevisiae but is essential for assembly of the complex.

Subunit 7 of the yeast cytochrome bc1 complex is encoded by the nuclear QCR7 gene and is essential for respiration. This protein does not contain a cleavable N-terminal mitochondrial targeting sequence, and it is not understood how the Qcr7 protein is imported into mitochondria and assembled into the complex. To test the role of the N terminus of the Qcr7 protein in mitochondrial import, assembly of the complex, and proton translocation, we inactivated the endogenous QCR7 gene and expressed mutated qcr7 genes capable of synthesizing proteins truncated by 7, 10, 14, and 20 residues (Qcr7p-delta7, Qcr7p-delta10, Qcr7p-delta14, and Qcr7p-delta20, respectively) from the N terminus. In addition, we studied two mutants containing Qcr7 proteins with point mutations in addition to a delta7 truncation, Qcr7p-delta7(D13V) and Qcr7p-delta7(R10K). All the mutant proteins with the exception of Qcr7p-delta10 were present in the mitochondria at 30 degrees C, although most at lower steady-state levels than the Qcr7p from the strain overexpressing wild type QCR7. The absence of the Qcr7p-delta10 may be the result of an unstable protein or a decrease in the efficiency of mitochondrial import due to its compromised amphipathic alpha-helix and the presence of a negative charge exposed at the N terminus. Cytochrome c reductase activities and the amounts of ATP synthesized were comparable with the wild type in the strain expressing Qcr7p-delta7. The strain expressing Qcr7p-delta7(R10K) had an identical phenotype to the one containing the Qcr7p-delta7, whereas strains expressing the Qcr7p-delta10, Qcr7p-delta14, Qcr7p-delta20, and Qcr7p-delta7(D13V) were all respiration-deficient. Examination of the steady-state levels of complex III subunits showed that core protein 2, cytochrome c1, the iron-sulfur protein, and the 11-kDa subunit are reduced in respiration-deficient mutant strains. Results from deletion analyses indicate that the N-terminal 20 residues (after Met-1) of the Qcr7 protein are not essential for import into mitochondria and that the N-terminal seven residues (after Met-1) are not involved in proton translocation. The results of this work show, however, that the N terminus of the Qcr7 protein is essential for the biosynthesis of ubiquinol-cytochrome c reductase.

Protein-tyrosine Phosphatase 1B Is a Negative Regulator of Insulin- and Insulin-like Growth Factor-I-stimulated Signaling

To understand the physiological role of protein-tyrosine phosphatase 1B (PTPase 1B) in insulin and insulin-like growth factor-I (IGF-I) signaling, we established clonal cell lines overexpressing wild type or inactive mutant (C215S) PTPase 1B in cells overexpressing insulin (Hirc) or IGF-I (CIGFR) receptors. PTPase 1B overexpression in transfected cells was verified by immunoblot analysis with a monoclonal PTPase 1B antibody. Subfractionation of parental cells demonstrated that greater than 90% of PTPase activity was localized in the Triton X-100-soluble particulate (P1) cell fraction. PTPase activity in the P1 fraction of cells overexpressing wild type PTPase 1B was 3-6-fold higher than parental cells but was unaltered in all fractions from C215S PTPase 1B-containing cells. The overexpression of wild type and C215S PTPase 1B had no effects on intrinsic receptor kinase activity, growth rate, or general cell morphology. The effects of PTPase 1B overexpression on cellular protein tyrosine phosphorylation were examined by anti-phosphotyrosine immunoblot analysis. No differences were apparent under basal conditions, but hormone-stimulated receptor autophosphorylation and/or insulin receptor substrate tyrosine phosphorylation were inhibited in cells overexpressing wild type PTPase 1B and increased in cells expressing mutant PTPase 1B, in comparison with parental cells. Metabolic signaling, assessed by ligand-stimulated [14C]glucose incorporation into glycogen, was also inhibited in cells overexpressing active PTPase 1B and enhanced in cells containing C215S PTPase 1B. These data strongly suggest that PTPase 1B acts as a negative regulator of insulin and IGF-I signaling.