Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.
J Mol Biol 2003 Nov 14
PMID:Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis. 1459 5

Insulin causes distinct cortical actin remodeling in muscle and fat cells, and interfering with actin dynamics halts glucose transporter 4 (GLUT4) translocation to the membrane. Phosphatidylinositol 3-kinase (PI3-K) and the small G protein Rac govern myocyte actin remodeling, whereas TC10 alpha contributes to adipocyte actin dynamics downstream of Cbl-associated protein (CAP) and Cbl, independently of PI3-K. Given the importance of insulin action in both cell types, it is paramount to determine whether signaling pathways and actin manifestations are cell type specific. We found CAP expression and insulin-mediated Cbl phosphorylation in differentiated myotubes but not in myoblasts. Unlike adipocytes, Cbl is phosphorylated on Y774 and Y731 in myotubes. TC10 alpha and beta-transcripts are amplified by RT-PCR in muscle cells, but the endogenous proteins are barely detectable using two unrelated antibodies. TC10 alpha transfected into myoblasts is activated by insulin despite the lack of CAP expression and Cbl phosphorylation. Moreover, dominant-negative TC10 alpha mutants do not prevent insulin-induced actin remodeling in either myoblasts or myotubes and do not interfere with insulin-mediated recruitment of c-myc epitope-tagged GLUT4 to the cell surface. In contrast to TC10 alpha, endogenous Rac is readily detectable in both muscle cells and adipocytes and binds GTP after insulin in a PI3-K-dependent manner. These data suggest that whereas individual components of the CAP to TC10 pathway are regulated by insulin, a functional TC10-dependent signaling pathway leading to actin remodeling and GLUT4 translocation may not operate in myocytes, as it does in adipocytes.
Mol Endocrinol 2004 Feb
PMID:Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulin-induced actin remodeling. 1461 6

HER2, a member of the human epidermal growth factor (EGF) receptor family, not only plays important roles in the progression of breast cancer tumorigenesis and metastasis, but may protect cancer cells from conventional cytotoxic therapies as well. In the current study, we evaluated the effect of targeting HER2 on radiosensitization of human breast cancer cells. Using six breast cancer cell lines with various levels of HER2 (BT474, SKBR3, MDA453, MCF7, ZR75B, and MDA468), we found that trastuzumab (Herceptin), a humanized monoclonal antibody that may inhibit breast cancer cell proliferation but does not induce apoptosis when used alone, enhanced radiation-induced apoptosis of the cells in a HER2 level-dependent manner. We furthered this study in MCF7 cells transfected for high levels of HER2 (MCF7HER2). Compared with parental or control vector-transfected MCF7 cells, MCF7HER2 cells showed increased phosphorylation of at least two important HER2 downstream molecules, protein kinase B/Akt and mitogen-activated protein kinase (MAPK), and increased resistance to radiotherapy, as shown by reduced induction of apoptosis and increased cell clonogenic survival after radiation. Exposure of the cells to trastuzumab down-regulated the levels of HER2 and reduced phosphorylation levels of Akt and MAPK in MCF7HER2 cells, and sensitized these cells to radiotherapy. When specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and MAPK kinase (MEK) pathways were used, we found that exposure of MCF7HER2 cells to the PI3-K inhibitor LY294002 inhibited Akt phosphorylation and radiosensitized the cells, whereas the radiosensitization effect by the MEK inhibitor PD98059 was relatively weaker, albeit the phosphorylation of MAPK was reduced by PD98059 treatment. Our results indicate that the PI3-K pathway might be the major pathway for trastuzumab-mediated radiosensitization of breast cancer cells.
Mol Cancer Ther 2003 Nov
PMID:Sensitization of breast cancer cells to radiation by trastuzumab. 1461 84

The androgen receptor (AR) is an androgen-inducible transcription factor characterized by a modular primary structure, with each module representing a distinct functional unit. After its interaction with androgens, the cytoplasmic AR is activated and translocated to the nucleus where it binds to target genes at the androgen responsive element(s) and recruits coregulators to form a multiprotein complex that interacts with transcriptional mediators and the basal transcription machinery to regulate gene transcription. Androgens play an essential role in the morphogenesis and physiology of the normal prostate. The etiology of benign prostatic hyperplasia (BPH) and prostatic neoplasia, which can progress to adenocarcinoma, is androgen-dependent, and reduction/obliteration of androgen action in the prostate has been the therapy of choice for BPH and prostate cancer. After androgen withdrawal and antiandrogen treatment, the androgen responsive prostate cancer cells cease to proliferate and undergo apoptosis, causing tumor regression. However, relapses are seen invariably, when tumors emerge as androgen-independent and apoptosis-resistant. Gene amplification and amino acid substitutions in the AR are detected at a high frequency in recurrent tumors. These changes confer growth advantage to the tumor cells due to either hypersensitivity of AR to low, castrate-level androgens or a realignment of the receptor conformation, leading to altered ligand specificity that enables antiandrogens, adrenal androgens and non-androgen steroids act agonistically to increase AR activity. Persistence of signaling by the wild-type AR in therapy-resistant tumors is due to the increased receptor activity caused by cross talk of AR with multiple intracellular signaling cascades, especially the growth factor activated MAP kinase/ERK and PI3 kinase/Akt pathways. Ablation of AR function using antisense oligodeoxynucleotides, ribozymes or small interference RNAs (RNAi) holds promise as future approaches to the successful treatment of hormone-refractory, apoptosis-resistant prostate tumors.
Mol Cell Biochem 2003 Nov
PMID:The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer. 1461 59

Long-term estrogen deprivation causes hypersensitivity of MCF-7 cells to the mitogenic effect of estradiol (E2) which is associated with activation of mitogen-activated protein kinase (MAPK). However, several lines of evidence indicate that MAPK activation is not the exclusive mechanism for E2 hypersensitivity and multiple signal pathways might be involved. The current study explores the possible role of the PI3 kinase (PI3K) pathway in development of E2 hypersensitivity. Basal PI3K activity in long-term estrogen deprived MCF-7 cells (LTED) was elevated as evidenced by increased phosphorylation of three downstream effectors, Akt, p70 S6 kinase, and eukaryotic initiation factor-4E binding protein (4E-BP1), which was blocked by the specific inhibitor of PI3K, LY294002. Dual blockade of both MAPK and PI3K completely reversed E2 hypersensitivity of LTED cells. Enhancement in aromatase activity is another phenomenon accompanied with E2 hypersensitivity. In aromatase over-expressing MCF-7 cells, aromatase activity was reduced by inhibitors of MAPK and PI3K suggesting the involvement of protein phosphorylation in the regulation of aromatase activity. Our data suggest that in addition to the MAP kinase pathway, activation of the PI3 kinase pathway is involved in E2 hypersensitivity, which develops during adaptation of MCF-7 cells to the low estrogen environment.
J Steroid Biochem Mol Biol 2003 Sep
PMID:Adaptive hypersensitivity following long-term estrogen deprivation: involvement of multiple signal pathways. 1462 20

On cell maturation following culture in medium containing 26 mM potassium (high K+; HK), a change to medium containing 5 mM potassium (low K+; LK) rapidly induces apoptosis in rat cerebellar granule neurons. Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) have survival-promoting effects on the neurons via PI3-K. However, it remains unclear how they prevent the apoptosis in the pathway downstream of phosphatidylinositol-3 kinase (PI3-K). Recently, we have reported that PI3-K-ASK1 pathway is involved in signal-transduction to p38 MAPK (p38)-c-Jun pathway. Here we found that IGF-1 had a greater survival-promoting effect than BDNF, and activated PI3-K to a higher level and maintained the level for a longer time. BDNF and IGF-1 suppressed the activation of p38 and c-Jun, but not of c-Jun N-terminal kinase (JNK), caused by lowering the potassium concentration. The inhibitory effects of IGF-1 were much greater than those of BDNF. In addition, LY294002, a specific inhibitor of PI3-K, cancelled the inhibitory effects of BDNF and IGF-1. These results suggest that the greater inhibitory effects of IGF-1 than BDNF, on activation of p38 and c-Jun and apoptosis, are caused by the higher level of PI3-K activation during LK-induced apoptosis of cultured cerebellar granule neurons.
Brain Res Mol Brain Res 2003 Nov 26
PMID:Comparison of inhibitory effects of brain-derived neurotrophic factor and insulin-like growth factor on low potassium-induced apoptosis and activation of p38 MAPK and c-Jun in cultured cerebellar granule neurons. 1462 85

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.
Mol Cell Biol 2003 Dec
PMID:Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A. 1464 48

In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.
Mol Cell Biol 2004 Feb
PMID:Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation. 1472 69

Keratinocyte growth factor (KGF or FGF-7) stimulates alveolar type II cell proliferation, but little is known about the signaling pathways involved. We investigated the role of the ERK (p42/44 mitogen activated protein [MAP] kinase) and phosphatidylinositol 3-OH kinase (PI3 kinase) pathways on alveolar type II cell proliferation and differentiation. Rat type II cells were cultured on tissue culture plastic and Matrigel in the presence or absence of KGF and specific chemical inhibitors PD98059, LY294002, and rapamycin at various concentrations. Proliferation was measured by thymidine incorporation and DNA quantitation, and differentiation was measured by expression of surfactant protein A and alkaline phosphatase. We demonstrate that KGF activates distal effectors of the PI3 kinase pathway, PKB/Akt, and p70S6 kinase, as well as p42/44 MAP kinase proteins. Inhibition of these pathways with PD98059, LY294002, or rapamycin inhibited type II cell proliferation but had no significant effect on differentiation. KGF did not activate the c-Jun kinase or p38 MAP kinase pathways. We conclude that the p42/44 MAP kinase and PI3 kinase pathways are important in regulating alveolar type II cell proliferation in response to KGF.
Am J Respir Cell Mol Biol 2004 Jun
PMID:Keratinocyte growth factor stimulates alveolar type II cell proliferation through the extracellular signal-regulated kinase and phosphatidylinositol 3-OH kinase pathways. 1474 97

We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure of bovine lung microvascular cells (BLMVEC) to vanadate increased the release of biologically active NO in endothelium/smooth muscle cocultures, as measured by the accumulation of its surrogate marker, cGMP. This release was sensitive to NO synthase (NOS) inhibition and was greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal and ionomycin-induced NO production, they failed to inhibit vanadate-induced NO release. Moreover, in the absence of calcium/calmodulin, cell lysates from vanadate-treated cells exhibited greater NOS activity compared with control cells. Vanadate activates the phosphoinositide3-kinase (PI3-K)/Akt pathway, which is known to increase endothelial NOS (eNOS) activity by direct phosphorylation of Ser-1179. Treatment of BLMVEC with vanadate resulted in phosphorylation of both Akt and endothelial NOS. In addition, wortmannin, a PI3-K inhibitor, blocked both the vanadate-induced phosphorylation of eNOS and the increase in cGMP accumulation. Similarly, adenovirus-mediated gene transfer of an activation deficient form of Akt (AA-Akt) blocked the release of NO brought about by vanadate. To further investigate the mechanism of action of vanadate, eNOS was immunoprecipitated and its association with proteins that alter eNOS activity was tested. Immunoblots demonstrated that the eNOS-caveolin interaction remained unaffected by vanadate, whereas vanadate promoted recruitment of the 90-kDa heat shock protein (hsp90) to eNOS. We conclude that vanadate causes NO release via a mechanism that involves Akt-induced eNOS phosphorylation and increased binding of the activator protein hsp90 to eNOS.
Mol Pharmacol 2004 Feb
PMID:Vanadate is a potent activator of endothelial nitric-oxide synthase: evidence for the role of the serine/threonine kinase Akt and the 90-kDa heat shock protein. 1474 83


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