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PubMed2002

The experimental and clinical study on the effect of curcumin on cell cycle proteins and regulating proteins of apoptosis in acute myelogenous leukemia.

To investigate whether the Bcl-2 gene family is involved in modulating mechanism of apoptosis and change of cell cycle protein induced by curcumin in acute myeloid leukemia HL-60 cell line and primary acute myelogenous leukemic cells, the Bcl-2 family member Mcl-1, Bax and Bak and cell cycle proteins including P27kipl, P21wafl, cyclin D3 and pRbp- were selected and their expression detected by SABC immuno-histochemical stain method. The attitude of sub-G1 peak in DNA histogram was determined by FCM. The TUNEL positive cell percentage was identified by terminal deoxynucleotidyl transferase (TdT)-mediated Biotin dUNP end labeling technique. It was found that when HL-60 cells were treated with 25 mumol/L curcumin for 24 h, the expression level of Mcl-1 was down-regulated, but that of Bax and Bak up-regulated time-dependently. There was significant difference in the expression level of Mcl-1, Bax and Bak between the curcumin-treated groups and control group (P < 0.05-0.01). At the same time, curcumin had no effect on progress of cell cycle in primaty acute myelogenous leukemia at newly diagnosis, but could increase the peak of Sub-G1 (P < 0.05), and down-regulate the expression of Mcl-1 and up-regulate the expression of Bax and Bak with the difference being statistically significant. The expression of P27kipl, P21wafl and pRbp- were elevated and that of cyclin D3 decreased in the presence of curcumin. These findings suggested that the Bcl-2 gene family indeed participated in the regulatory process of apoptosis induced by curcumin in HL-60 cells and AML cells. Curcumin can induce apoptosis of primary acute myelogenous leukemic cells and disturb cell cycle progression of HL-60 cells. The mechanism appeared to be mediated by perturbing G0/G1 phases checkpoints which associated with up-regulation of P27kipl, P21wafl and pRbp- expression, and down-regulation of cyclin D3.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Cysteine string protein regulates G protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.

Adenosine Triphosphate↗
PubMed2004

[RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response].

RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins. By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction. Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins). The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies. In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways. They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation. They also take an essential part in inflammation. High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors. The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response

Animals↗
PubMed1988

Relationships between oestrogen receptor,epidermal growth factor receptor, ER-D5, and P24 oestrogen regulated protein in human breast cancer.

Proteins regulated by or related to the oestrogen receptor (ER) may prove to be more reliable indicators of prognosis and hormone sensitivity then expression of the receptor itself. It has been shown recently that expression of epidermal growth factor receptor (EGFR) is associated with a poor prognosis in breast cancer. In a series of 60 breast cancers, we have studied relationships between ER, ER-D5 oestrogen receptor related protein, P24 oestrogen regulated protein, and EGFR using an immunohistochemical technique employing monoclonal antibodies in each case. In addition, radioligand binding assays for ER and EGFR were carried out and tumour histological grade was determined. Seventy-one per cent and forty-three per cent of tumours stained for ER-D5 and P24, respectively, but there was no relationship between staining for these and ER or EGFR status. There was a significant correlation between staining for ER and EGFR, and the respective biochemical assays. Relating ER to EGFR, very few ER-positive cases expressed EGFR, but this relationship fell short of significance. The prognostic significance of expression of the epitopes recognized by the ERD5 and P24 antibodies must await assessment of clinical outcome.

Antibodies, Monoclonal↗
PubMed1994

Ethanol-responsive genes in neural cells include the 78-kilodalton glucose-regulated protein (GRP78) and 94-kilodalton glucose-regulated protein (GRP94) molecular chaperones.

Previously we found that ethanol increases expression of the constitutive 70-kDa heat shock protein (Hsc70) in NG108-15 neuroblastoma x glioma cells. We suggested that known ethanol actions on cellular protein trafficking may relate to Hsc70 induction because Hsc70 functions as a molecular chaperone. Here we use a subtractive hybridization protocol to isolate ethanol-responsive genes (EtRGs). Northern blot hybridization verified ethanol-induced increases in mRNA abundance for five cDNA clones isolated from ethanol-treated NG108-15 neuroblastoma x glioma cells. DNA sequence analysis identified one EtRG as 94-kDa glucose-regulated protein (GRP94), a member of the "glucose-responsive" subgroup of stress proteins. Other identified EtRGs included an insulin-induced growth-response protein gene and an intracisternal A-type particle gene. Sequence analysis of the remaining two EtRGs showed no homology in DNA sequence databases. All EtRGs showed wide tissue expression, except SL64, which was not detected in Northern blot analyses of adult mouse or rat tissues. Ethanol also increased mRNA abundance for 78-kDa glucose-regulated protein (GRP78), a molecular chaperone known to function in glycoprotein trafficking and usually coordinately regulated with GRP94. However, ethanol induced GRP94 more than GRP78, a pattern distinct from those of other inducers of these genes. All EtRGs, including GRP94 and GRP78, showed similar ethanol concentration-dependent increases in mRNA abundance. In contrast, thapsigargin and other inducers of glucose-responsive proteins increased GRP94 and GRP78 mRNA levels without altering expression of other EtRGs. Our studies demonstrate that several molecular chaperones constitute a subset of EtRGs. Ethanol appears to regulate these EtRGs by a unique mechanism, rather than one shared by classical inducers of stress proteins.

Carrier Proteins↗
PubMed1979

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer.

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.

Animals↗
PubMed1979

The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis.

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32 degrees C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61+/-3, 46+/-3, 27+/-4 and 18+/-4% lower than in the cells pretreated without cycloheximide respectively (means+/-s.e.m., n=4-6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36+/-0.5 to 36.5+/-1.0ng/10(6) cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2-4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.

Animals↗
PubMed2002

Immunohistochemical demonstration of apoptosis-regulated proteins, Bcl-2 and Bax, in resected non-small-cell lung cancers.

OBJECTIVE: Bcl-2 and Bax proteins regulate apoptosis independently, cooperatively, or both in vitro. The purpose of this study was to clarify the association between their expression with spontaneous apoptosis and various clinicopathologic features in patients with non-small-cell lung cancer (NSCLC). METHODS: Bcl-2 protein, Bax protein, and spontaneous apoptosis were evaluated retrospectively in 70 resected specimens from NSCLC patients. Immunohistochemical (IHC) tests were used to assess the expression of Bcl-2 and Bax in the samples. The apoptotic index (AI) was also measured in these samples by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. We then evaluated the clinicopathologic features of all 70 samples and their relationships with malignancy. RESULTS: Bcl-2 overexpression was significantly associated with male sex and squamous cell carcinoma (SCC). Bax overexpression was not associated with any clinicopathologic features. AI was significantly associated with SCC and negative nodal metastasis. No clear associations were found among Bcl-2 expression, Bax expression, and AI. Bcl-2/Bax ratios were not associated significantly with AI. Bcl-2, Bax, Bcl-2/Bax ratio, and the grading of AI did not have prognostic values. CONCLUSION: Bcl-2 overexpression was significantly associated with SCC. Spontaneous apoptosis was significantly associated with SCC and with negative nodal metastasis. Apoptosis-regulated proteins, Bcl-2 and Bax, and AI lack significant associations with each other and prognostic values in patients with resected NSCLC.

Aged↗
PubMed1998

A role for Saccharomyces cerevisiae fatty acid activation protein 4 in regulating protein N-myristoylation during entry into stationary phase.

Saccharomyces cerevisiae contains four known acyl-CoA synthetases (fatty acid activation proteins, Faaps). Faa1p and Faa4p activate exogenously derived fatty acids. Acyl-CoA metabolism plays a critical role in regulating protein N-myristoylation by the essential enzyme, myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). In this report, we have examined whether Faa1p and Faa4p have distinct roles in affecting protein N-myristoylation as cells transition from growth in rich media to a growth-arrested state during nutrient deprivation (stationary phase). The colony-forming potential of 10 isogenic strains was defined as a function of time spent in stationary phase. These strains contained either a wild type or mutant NMT1 allele, and wild type or null alleles of each FAA. Only the combination of the Nmt mutant (nmt451Dp; reduced affinity for myristoyl-CoA) and loss of Faa4p produced a dramatic loss of colony-forming units (CFU). The progressive millionfold reduction in CFU was associated with a deficiency in protein N-myristoylation that first appeared during logarithmic growth, worsened through the post-diauxic phase, and became extreme in stationary phase. Northern and Western blot analyses plus N-myristoyltransferase assays showed that Nmt is normally present only during the log and diauxic/post-diauxic periods, indicating that N-myristoylproteins present in stationary phase are "inherited" from these earlier phases. Moreover, FAA4 is the only FAA induced during the critical diauxic/early post-diauxic transition. Although substitution of nmt1-451D for NMT1 results in deficiencies in protein N-myristoylation, these deficiencies are modest and limited by compensatory responses that include augmented expression of nmt1-451D and precocious induction of FAA4 in log phase. Loss of Faa4p from nmt1-451D cells severely compromises their capacity to adequately myristoylate Nmt substrates prior to entry into stationary phase since none of the other Faaps are able to functionally compensate for its absence. To identify Nmt1p substrates that may affect maintenance of proliferative potential during stationary phase, we searched the yeast genome for known and putative N-myristoylproteins. Of the 64 genes found, 48 were successfully deleted in NMT1 cells. Removal of any one of the following nine substrates produced a loss of CFU similar to that observed in nmt1-451Dfaa4Delta cells: Arf1p, Arf2p, Sip2p, Van1p, Ptc2p, YBL049W (homology to Snf7p), YJR114W, YKR007W, and YMR077C. These proteins provide opportunities to further define the molecular mechanisms that regulate survival during stationary phase.

Acyl Coenzyme A↗
PubMed2000

GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins.

GTPase-activating proteins (GAPs) regulate heterotrimeric G proteins by increasing the rates at which their subunits hydrolyze bound GTP and thus return to the inactive state. G protein GAPs act allosterically on G subunits, in contrast to GAPs for the Ras-like monomeric GTP-binding proteins. Although they do not contribute directly to the chemistry of GTP hydrolysis, G protein GAPs can accelerate hydrolysis >2000-fold. G protein GAPs include both effector proteins (phospholipase C-¿, p115RhoGEF) and a growing family of regulators of G protein signaling (RGS proteins) that are found throughout the animal and fungal kingdoms. GAP activity can sharpen the termination of a signal upon removal of stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network. GAPs are regulated by various controls of their cellular concentrations, by complex interactions with G¿ or with G¿5 through an endogenous G-like domain, and by interaction with multiple other proteins.

Amino Acid Sequence↗
PubMed1999

Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly, substrates of all these SDKs so far identified are molecular chaperones or adapters capable of binding to enzymes and key molecules involved in signal transduction, maintaining tertiary structure of bioactive molecules, or maintaining cellular homeostasis in response to environmental stress. Thus, the essential role of Sph and DMS is to activate molecular chaperones, thereby providing a link to the mechanism by which SDK activity regulates cellular homeostasis and signal transduction.

3T3 Cells↗
PubMed2000

Endogenous regulators of G protein signaling proteins regulate presynaptic inhibition at rat hippocampal synapses.

Presynaptic inhibition mediated by G protein-coupled receptors (GPCRs) can develop and decay in a few seconds. This time course is too rapid to be accounted for by the intrinsic GTPase activity of Galpha subunits alone. Here, we test the hypothesis that endogenous regulators of G protein signaling (RGS proteins) are required for rapid, brief presynaptic inhibition. Endogenous G protein alpha subunits were uncoupled from GPCRs by treating cultures with pertussis toxin (PTX). Adenoviral expression of mutant PTX-insensitive (PTX-i) Galpha(i1-3) or Galpha(o) subunits rescued adenosine-induced presynaptic inhibition in cultured hippocampal neurons. Expression of double mutant Galpha(i1) or Galpha(o) subunits that were both PTX-insensitive and unable to bind RGS proteins (PTX/RGS-i) also rescued presynaptic inhibition. Presynaptic inhibition mediated by PTX/RGS-i subunits decayed much more slowly after agonist removal than that mediated by PTX-i subunits or native G proteins. The onset of presynaptic inhibition mediated by PTX/RGS-i Galpha(o) was also slower than that mediated by PTX-i Galpha(o). In contrast, the onset of presynaptic inhibition mediated by PTX/RGS-i Galpha(i1) was similar to that mediated by PTX-i Galpha(i1). These results suggest that endogenous RGS proteins regulate the time course of G protein signaling in mammalian central nervous system presynaptic terminals.

Adenoviridae↗
PubMed1979

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. I. Effect of calcium-dependent regulator protein on the calcium sensitivity of microtubule assembly.

We examined the effect of porcine brain Ca2+-dependent regulator (CDR) protein on microtubule (MT) assembly from microtubular proteins isolated from porcine brain by temperature-dependent cycles of assembly-disassembly. CDR exhibited a potent inhibitory effect on MT assembly in the presence of Ca2+, whereas it had little or no effect on the extent of MT assembly in the absence of Ca2+. The increase in KCl concentration greatly potentiated the Ca2+-dependent inhibitory effect of CDR. The effect of CDR was reversible in a Ca2+ concentration-dependent manner, and the extent of inhibition by CDR at a fixed concentration of free Ca2+ was roughly proportional to the concentration of CDR. Moreover, the Ca2+ concentration required for the half-maximal inhibition of MT assembly from a fixed concentration of purified microtubular proteins (PMP) decreased with increasing CDR concentration. On the basis of these results, together with data on the Ca2+-dependent association of CDR and tubulin (J. Biochem., accompanying paper), we propose the following model; Ca2+ + CDR in equilibrium Ca2+-CDR Ca2+-CDR + tubulin in equilibrium Ca2+-CDR-tubulin (nonpolymerizable).

Animals↗
PubMed2000

The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptor-effector coupling.

The M(3) muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to G(q) and G(12) proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M(3) mAChR-PLD coupling in comparison to M(3) mAChR-PLC coupling, we expressed various Galpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for G(q)- or G(12)-type G proteins. PLD stimulation by the M(3) mAChR was enhanced by the overexpression of Galpha(12) and Galpha(13), whereas the overexpression of Galpha(q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha(12) and Galpha(13), blunted the M(3) mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha(q)- but not Galpha(12)-type G proteins, suppressed the M(3) mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M(3) mAChR in HEK-293 cells apparently signals to PLD via G(12)- but not G(q)-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.

Carbachol↗
PubMed2001

A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans.

Efg1p in the human fungal pathogen Candida albicans is a member of the conserved APSES class of proteins regulating morphogenetic processes in fungi. We have analyzed the importance for hyphal morphogenesis of a putative phosphorylation site for protein kinase A (PKA), threonine-206, within an Efg1p domain highly conserved among APSES proteins. Alanine substitution of T206, but not of the adjacent T207 and T208 residues, led to a block of hypha formation on solid and in liquid media, while a T206E exchange caused hyperfilamentation. The extent of the morphogenetic defect caused by the T206A mutation depended on hypha-induction conditions. Extragenous suppression of mutations in signaling components, including tpk2 and cek1 mutations, was achieved by wild-type- and T206E-, but not by the T206A-variant-encoding allele of EFG1. All muteins tested were produced at equal levels and at high production levels supported pseudohyphal formation. The results are consistent with a role of Efg1p as a central downstream component of a PKA-signaling pathway including Tpk2p or other PKA isoforms. Threonine-206 of Efg1p is essential as a putative phosphorylation target to promote hyphal induction by a subset of environmental cues.

Alanine↗
PubMed2006

Dexamethasone-induced Ras protein 1 negatively regulates protein kinase C delta: implications for adenylyl cyclase 2 signaling.

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) delta, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gbetagamma signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Galpha(s)-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCdelta autophosphorylation. This effect was selective for the delta isoform because Dexras1 did not alter autophosphorylation of PKCalpha or PKCepsilon. Dexras1 disruption of PKCdelta autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [gamma-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCdelta coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCdelta; however, the ability of Dexras1 to interfere with PKCdelta autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCdelta at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCdelta activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gbetagamma- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.

Adenylyl Cyclases↗
PubMed2004

Selective estrogen receptor modulator regulated proteins in endometrial cancer cells.

Tamoxifen is the primary hormonal therapy for breast cancer and is also used as a breast cancer chemopreventative agent. A major problem with tamoxifen therapy is undesirable endometrial proliferation. To identify proteins associated with the growth stimulatory effects of tamoxifen in an ER-positive model, the present study profiled total cellular and secreted proteins regulated by estradiol and selective estrogen receptor modifiers (SERMs) in the Ishikawa endometrial adenocarcinoma cell line using two-dimensional gel electrophoresis. Following 24 h incubation with 10(-8) M estradiol, 10(-7) M 4-hydroxytamoxifen, or 10(-7) M EM-652 (Acolbifene), nine proteins exhibited significant increase in expression. The proteins identified were heat shock protein 90-alpha, and -beta, heterogeneous nuclear ribonucleoprotein F, RNA polymerase II-mediating protein, cytoskeletal keratin 8, cytoskeletal keratin 18, ubiquitin-conjugating enzyme E2-18 kDa and nucleoside diphosphate kinase B. These protein profiles may serve as novel indices of SERM response and may also provide insight into novel mechanisms of SERM-mediated growth.

Biomarkers↗