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Biomedical subjects

David B Sacks

Publications and source records attributed to David B Sacks.

At least 19 recordsLinked to original sources
PubMed2006

Targeting of calcium:calmodulin signals to the cytoskeleton by IQGAP1.

Mast cells reorganize their actin cytoskeleton in response to cytosolic calcium signals while in parallel secreting histamine and other inflammatory mediators. The effect of calcium on actin is mediated in large part through calmodulin. EGFP-tagged calmodulin is concentrated in the actin-rich cortex of RBL-2H3 mast cells. Transfection with small interfering RNA directed against the actin and calmodulin-binding protein IQGAP1 dramatically reduced expression of the latter protein and reduced or eliminated the concentration of calmodulin at the actin-rich cortex. Both actin reorganization and secretion were enhanced in IQGAP1 knockdown cells. Our results suggest a model in which calmodulin is targeted to and sequestered at the actin cytoskeleton by IQGAP1. Upon cell stimulation and the subsequent [Ca2+]i increase, it is immediately available to activate local downstream targets.

Animals↗
PubMed2006

Functional interactions between calmodulin and estrogen receptor-alpha.

The effects of estrogen in breast epithelial cells are mediated primarily via estrogen receptor alpha and estrogen receptor beta (ERalpha and ERbeta). Accumulating evidence implicates a role for Ca2+ and calmodulin in breast carcinoma, as well as in the function of ERalpha. Calmodulin contributes to the regulation of both ERalpha degradation and ERalpha-mediated transcriptional activation. Recent data will be summarized in this review. Models to explain the molecular mechanisms by which calmodulin modulates ERalpha function are proposed.

Animals↗
PubMed2006

Proteomic analysis of ischemia-reperfusion injury upon human liver transplantation reveals the protective role of IQGAP1.

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.

Actins↗
PubMed2006

IQGAP1 in cellular signaling: bridging the GAP.

IQGAP1 was identified in 1994 as a widely expressed IQ domain-containing protein with a region containing sequence similarity to the Ras GTPase-activating proteins. IQGAP1 has roles in many different aspects of cell physiology and interacts with numerous proteins. It modulates the actin cytoskeleton through Rac1 and Cdc42, and cell-cell adhesion through E-cadherin and beta-catenin. It also regulates the mitogen activated protein kinase pathway, which influences cell proliferation and differentiation. Evidence suggests that IQGAP1 is a scaffold protein that links components of signaling cascades. Here, we evaluate recent data that identify the participation of IQGAP1 in signaling networks and we illustrate how this influences diverse cellular functions. These findings suggest that IQGAP1 integrates signaling pathways and coordinates several fundamental cellular activities.

Animals↗
PubMed2006

Identification of a conserved negative regulatory sequence that influences the leukemogenic activity of NOTCH1.

NOTCH1 is a large type I transmembrane receptor that regulates normal T-cell development via a signaling pathway that relies on regulated proteolysis. Ligand binding induces proteolytic cleavages in NOTCH1 that release its intracellular domain (ICN1), which translocates to the nucleus and activates target genes by forming a short-lived nuclear complex with two other proteins, the DNA-binding factor CSL and a Mastermind-like (MAML) coactivator. Recent work has shown that human T-ALL is frequently associated with C-terminal NOTCH1 truncations, which uniformly remove sequences lying between residues 2524 and 2556. This region includes the highly conserved sequence WSSSSP (S4), which based on its amino acid content appeared to be a likely site for regulatory serine phosphorylation events. We show here that the mutation of the S4 sequence leads to hypophosphorylation of ICN1; increased NOTCH1 signaling; and the stabilization of complexes containing ICN1, CSL, and MAML1. Consistent with these in vitro studies, mutation of the WSSSSP sequence converts nonleukemogenic weak gain-of-function NOTCH1 alleles into alleles that cause aggressive T-ALLs in a murine bone marrow transplant model. These studies indicate that S4 is an important negative regulatory sequence and that the deletion of S4 likely contributes to the development of human T-ALL.

Amino Acid Sequence↗
PubMed2005

The receptor protein-tyrosine phosphatase PTPmu interacts with IQGAP1.

The receptor protein-tyrosine phosphatase PTPmu is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTPmu contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTPmu contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTPmu make it an attractive molecule to transduce signals upon cell-cell contact. PTPmu has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTPmu signaling pathway utilized to regulate these events. To aid in the further characterization of PTPmu signaling pathways, we used a series of GST-PTPmu fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTPmu-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTPmu-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTPmu, which include the juxtamembrane domain and 35 residues of the first phosphatase domain, mediate the binding to IQGAP1. Furthermore, we demonstrate that constitutively active Cdc42, and to a lesser extent Rac1, enhances the interaction of PTPmu and IQGAP1. These data indicate PTPmu may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTPmu signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTPmu-mediated neurite outgrowth.

Amino Acid Motifs↗
PubMed2005

E6AP and calmodulin reciprocally regulate estrogen receptor stability.

Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.

Animals↗
PubMed2005

Self-association of IQGAP1: characterization and functional sequelae.

The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763-863. In support of this observation, a peptide comprising residues 763-863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.

Binding Sites↗
PubMed2005

IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner.

In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cepsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.

Amino Acid Sequence↗
PubMed2005

The transcriptional activity of estrogen receptor-alpha is dependent on Ca2+/calmodulin.

Estrogen binds to estrogen receptors in cells, thereby activating the receptors and eliciting biological effects. One of the best characterized effects of estrogen receptor-alpha (ERalpha) is transcriptional activation that regulates selected target genes in the nucleus. Work from several laboratories has documented a Ca2+-dependent interaction between ERalpha and calmodulin. In addition, we previously showed that antagonism of calmodulin function in cells prevented estradiol from inducing ERalpha transcriptional activity, suggesting that association of ERalpha with calmodulin participates in ERalpha function. In this study we adopted a multifaceted approach to directly address this hypothesis. The calmodulin binding domain on ERalpha was identified and several mutant ERalpha constructs unable to bind calmodulin were generated. Elimination of calmodulin binding prevented estradiol from stimulating ERalpha transcriptional activation. Essentially identical results were obtained when intracellular Ca2+ was chelated with the cell-permeable chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM). Moreover, CaM1234, a calmodulin mutant that is unable to bind Ca2+, functioned as a dominant negative construct. Transfection of cells with CaM1234 reduced estradiol-stimulated ERalpha transcriptional activity. These data indicate that binding to calmodulin is required for normal transcriptional function of ERalpha.

Animals↗
PubMed2005

IQGAP1 is a scaffold for mitogen-activated protein kinase signaling.

IQGAP1 modulates many cellular functions such as cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds extracellular signal-regulated kinase (ERK) 2 and regulates growth factor-stimulated ERK activity. Here we show that MEK, the molecule immediately upstream of ERK in the Ras/mitogen-activated protein (MAP) kinase signaling cascade, also interacts directly with IQGAP1. Both MEK1 and MEK2 bound IQGAP1 in vitro and coimmunoprecipitated with IQGAP1. The addition of ERK2 enhanced by fourfold the in vitro interaction of MEK2 with IQGAP1 without altering binding of MEK1. Similarly, ERK1 promoted MEK binding to IQGAP1, but either MEK protein altered the association between IQGAP1 and ERK. Epidermal growth factor (EGF) differentially regulated binding, enhancing MEK1 interaction while reducing MEK2 binding to IQGAP1. In addition, both knockdown and overexpression of IQGAP1 reduced EGF-stimulated activation of MEK and ERK. Analyses with selective IQGAP1 mutant constructs indicated that MEK binding is crucial for IQGAP1 to modulate EGF activation of ERK. Our data strongly suggest that IQGAP1 functions as a molecular scaffold in the Ras/MAP kinase pathway.

Epidermal Growth Factor↗
PubMed2004

IQGAP1 binds ERK2 and modulates its activity.

IQGAP1 binds several proteins including actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. The interaction with these targets enables IQGAP1 to participate in many cellular functions varying from regulation of the cytoskeleton to gene transcription. Here we show that extracellular signal-regulated kinase (ERK) 2 binds to IQGAP1. In vitro analysis with purified proteins demonstrated a direct interaction between ERK2 and IQGAP1. Moreover, binding occurred in cells as endogenous ERK2 co-immunoprecipitated with IQGAP1 from human breast epithelial cell lysates. The association between ERK2 and IQGAP1 was independent of epidermal growth factor. The in vivo interaction has functional significance. Manipulation of intracellular IQGAP1 levels significantly reduced growth factor-stimulated ERK1 and ERK2 activity. Similarly, stimulation of ERK1 and ERK2 activity by insulin-like growth factor I was reduced when IQGAP1 levels were changed. In contrast, overexpression of an IQGAP1 construct lacking the ERK2 binding region did not interfere with activation of ERK1 and ERK2 by epidermal growth factor. Our data disclose a previously unidentified communication between IQGAP1 and the ERK pathway and imply that IQGAP1 modulates the Ras/mitogen-activated protein kinase signaling cascade.

Binding Sites↗
PubMed2004

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis.

The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [(32)P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC. Phosphorylation of CaM by purified CK2 in vitro reduces the V(max) of immunopurified eNOS by a factor of 2 but has no effect on the K(A) for CaM or calcium. Additionally, [(32)P]orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM ("phosphonull" S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the "phosphomimetic" Ser-81 to aspartate (S81D) mutant is unchanged. Further studies using Escherichia coli-expressed and phenyl-Sepharose-purified CaM mutants revealed that the S81A mutation abrogates in vitro CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved. Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS. Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue. We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses.

Animals↗
PubMed2003

Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells.

Ca(2+) and calmodulin (CaM) play a critical role in proliferation and viability of a wide variety of cells, including prostate cancer cells. We examined two prostate cancer cell lines, androgen-sensitive LNCaP and androgen-independent PC-3. Proliferation of LNCaP cells was six to eight times more sensitive to the inhibitory effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) than were PC-3 cells. Because LNCaP cell proliferation is sensitive to stimulation by androgen, we assessed the physical and functional interaction between androgen receptor (AR) and CaM. We observed tight binding of AR to CaM when LNCaP cell extracts were subjected to CaM-affinity column chromatography. AR binding to CaM was Ca(2+)-dependent and was inhibited by pretreatment of the cell extracts with W-7. Using immunofluorescence staining and confocal microscopy, we demonstrated colocalization of AR and CaM in the nucleus of LNCaP cells. Furthermore, the functional relevance of AR-CaM interactions in intact cells was revealed by the observation that W-7 was as effective as Casodex, an antiandrogen, in blocking AR-regulated expression of prostate-specific antigen in LNCaP cells. AR seems to interact with CaM directly because purified human AR could bind to CaM-agarose, and CaM could be detected in AR-immunoprecipitate prepared from purified soluble proteins. These studies provide direct evidence for physical and functional interaction between AR and CaM and suggest the potential usefulness of CaM antagonists in blocking AR activity in prostate cancer.

Calmodulin↗