Publications by authors named "David B Sacks"

167 Publications

Calmodulin influences MAPK signaling by binding KSR1.

J Biol Chem 2021 Mar 22:100577. Epub 2021 Mar 22.

Department of Laboratory Medicine, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892, United States. Electronic address:

The mitogen activated protein kinase (MAPK) cascade is a fundamental signaling pathway that regulates cell fate decisions in response to external stimuli. Several scaffold proteins bind directly to kinase components of this pathway and regulate their activation by growth factors. One of the best studied MAPK scaffolds is kinase suppressor of Ras1 (KSR1), which is induced by epidermal growth factor (EGF) to translocate to the plasma membrane where it activates ERK. While Ca has been shown to modulate MAPK signaling, the molecular mechanisms by which this occurs are incompletely understood. Here we tested the hypothesis that Ca alters MAPK activity at least in part via KSR1. Using several approaches, including fusion proteins, immunoprecipitation, confocal microscopy and a cell-permeable chemical inhibitor, we investigated the functional interaction between KSR1 and calmodulin. In vitro analysis with pure proteins reveals that calmodulin binds directly to KSR1. Moreover, endogenous calmodulin and KSR1 co-immunoprecipitate from mammalian cell lysates. Importantly, Ca is required for the association between calmodulin and KSR1, both in vitro and in cells. The cell-permeable calmodulin antagonist CGS9343B significantly reduced activation of ERK by EGF in mouse embryo fibroblasts that overexpress KSR1, but not in control cells. Moreover, CGS9343B impaired the ability of EGF to induce KSR1 translocation to the plasma membrane and to stimulate formation of KSR1-ERK and KSR1-pERK complexes in cells. Collectively, our data identify a previously unrecognized mechanism by which the scaffold protein KSR1 couples Ca and calmodulin signaling to the MAPK cascade.
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http://dx.doi.org/10.1016/j.jbc.2021.100577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079274PMC
March 2021

B-Raf autoinhibition in the presence and absence of 14-3-3.

Structure 2021 Mar 8. Epub 2021 Mar 8.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer ImmunoMetabolism, National Cancer Institute, Frederick, MD 21702, USA; Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address:

Raf-activating mutations are frequent in cancer. In the basal state, B-Raf is autoinhibited by its upstream Ras-binding domain (RBD) and cysteine-rich domain (RBD-CRD) interacting with its kinase domain (KD) and the 14-3-3 dimer. Our comprehensive molecular dynamics simulations explore two autoinhibition scenarios in the presence and absence of the 14-3-3 dimer. When present, the 14-3-3 interaction with B-Raf stabilizes the RBD-CRD-KD interaction, interfering with the KD dimerization. Raf's pSer365 removal fails to induce large disruption. RBD-CRD release promotes KD fluctuations and reorientation for dimerization, consistent with experimental data. In the absence of 14-3-3, our sampled B-Raf conformations suggest that RBD-CRD can block the KD dimerization surface. Our results suggest a B-Raf activation mechanism, whereby one KD monomer is donated by 14-3-3-free B-Raf KD and the other by 14-3-3-bound KD. This mechanism can lead to homo- and heterodimers. These autoinhibition scenarios can transform autoinhibited B-Raf monomers into active B-Raf dimers.
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http://dx.doi.org/10.1016/j.str.2021.02.005DOI Listing
March 2021

IQGAP1 Is a Scaffold of the Core Proteins of the Hippo Pathway and Negatively Regulates the Pro-Apoptotic Signal Mediated by This Pathway.

Cells 2021 Feb 23;10(2). Epub 2021 Feb 23.

Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.

The Hippo pathway regulates a complex signalling network which mediates several biological functions including cell proliferation, organ size and apoptosis. Several scaffold proteins regulate the crosstalk of the members of the pathway with other signalling pathways and play an important role in the diverse output controlled by this pathway. In this study we have identified the scaffold protein IQGAP1 as a novel interactor of the core kinases of the Hippo pathway, MST2 and LATS1. Our results indicate that IQGAP1 scaffolds MST2 and LATS1 supresses their kinase activity and YAP1-dependent transcription. Additionally, we show that IQGAP1 is a negative regulator of the non-canonical pro-apoptotic pathway and may enable the crosstalk between this pathway and the ERK and AKT signalling modules. Our data also show that bile acids regulate the IQGAP1-MST2-LATS1 signalling module in hepatocellular carcinoma cells, which could be necessary for the inhibition of MST2-dependent apoptosis and hepatocyte transformation.
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http://dx.doi.org/10.3390/cells10020478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926663PMC
February 2021

Pannexin 1 binds β-catenin to modulate melanoma cell growth and metabolism.

J Biol Chem 2021 Feb 26:100478. Epub 2021 Feb 26.

Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada, N6A 5C1; Department of Oncology, Division of Experimental Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada, N6A 5C1. Electronic address:

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of β-catenin, a key transcription factor in the Wnt pathway. At the protein level, β-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of β-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with β-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and β-catenin interact as a potential new component of the Wnt signaling pathway.
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http://dx.doi.org/10.1016/j.jbc.2021.100478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027267PMC
February 2021

Call for Action: Journals Need to Insist on Full Reporting of the Analytical Characteristics of Biomarkers.

Lab Med 2021 01;52(1):7-9

Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland.

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http://dx.doi.org/10.1093/labmed/lmaa097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781521PMC
January 2021

IQGAP1 binds AMPK and is required for maximum AMPK activation.

J Biol Chem 2020 Nov 15. Epub 2020 Nov 15.

National Institutes of Health, United States.

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
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http://dx.doi.org/10.1074/jbc.RA120.016193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948462PMC
November 2020

Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.

J Biol Chem 2020 12 21;295(52):18105-18121. Epub 2020 Oct 21.

Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA. Electronic address:

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.
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http://dx.doi.org/10.1074/jbc.RA120.015891DOI Listing
December 2020

Continuous Glucose Monitors and Automated Insulin Dosing Systems in the Hospital Consensus Guideline.

J Diabetes Sci Technol 2020 11 28;14(6):1035-1064. Epub 2020 Sep 28.

Mills-Peninsula Medical Center, San Mateo, CA, USA.

This article is the work product of the Continuous Glucose Monitor and Automated Insulin Dosing Systems in the Hospital Consensus Guideline Panel, which was organized by Diabetes Technology Society and met virtually on April 23, 2020. The guideline panel consisted of 24 international experts in the use of continuous glucose monitors (CGMs) and automated insulin dosing (AID) systems representing adult endocrinology, pediatric endocrinology, obstetrics and gynecology, advanced practice nursing, diabetes care and education, clinical chemistry, bioengineering, and product liability law. The panelists reviewed the medical literature pertaining to five topics: (1) continuation of home CGMs after hospitalization, (2) initiation of CGMs in the hospital, (3) continuation of AID systems in the hospital, (4) logistics and hands-on care of hospitalized patients using CGMs and AID systems, and (5) data management of CGMs and AID systems in the hospital. The panelists then developed three types of recommendations for each topic, including clinical practice (to use the technology optimally), research (to improve the safety and effectiveness of the technology), and hospital policies (to build an environment for facilitating use of these devices) for each of the five topics. The panelists voted on 78 proposed recommendations. Based on the panel vote, 77 recommendations were classified as either strong or mild. One recommendation failed to reach consensus. Additional research is needed on CGMs and AID systems in the hospital setting regarding device accuracy, practices for deployment, data management, and achievable outcomes. This guideline is intended to support these technologies for the management of hospitalized patients with diabetes.
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http://dx.doi.org/10.1177/1932296820954163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645140PMC
November 2020

Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration.

J Exp Med 2021 Jan;218(1)

Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL.

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.
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http://dx.doi.org/10.1084/jem.20192378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953625PMC
January 2021

Improved Detection of Abnormal Glucose Tolerance in Africans: The Value of Combining Hemoglobin A With Glycated Albumin.

Diabetes Care 2020 10 14;43(10):2607-2613. Epub 2020 Aug 14.

Section on Ethnicity and Health, Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD

Objective: In African-born Blacks living in America, we determined by BMI category ) prevalence of abnormal glucose tolerance (Abnl-GT) and ) diagnostic value and reproducibility of hemoglobin A (HbA), fructosamine, and glycated albumin (GA).

Research Design And Methods: Participants ( = 416; male, 66%; BMI 27.7 ± 4.5 kg/m [mean ± SD]) had an oral glucose tolerance test with HbA, GA, and fructosamine assayed. These glycemic markers were repeated 11 ± 7 days later. Abnl-GT diagnosis required 0 h ≥5.6 mmol/L (≥100 mg/dL) and/or 2 h ≥7.8 mmol/L (≥140 mg/dL). Thresholds for HbA, GA, and fructosamine were the values at the 75th percentile for the population (39 mmol/mol [5.7%], 14.2%, and 234 μmol/L, respectively).

Results: Abnl-GT prevalence in the nonobese was 34% versus 42% in the obese ( = 0.124). Reproducibility was excellent for HbA and GA (both κ ≥ 0.8), but moderate for fructosamine (κ = 0.6). Focusing on HbA and GA in the nonobese, we found as single tests the sensitivities of HbA and GA were 36% versus 37% ( = 0.529). Combining HbA and GA, sensitivity increased to 58% because GA identified 37% of Africans with Abnl-GT not detected by HbA ( value for both tests vs. HbA alone was <0.001). For the obese, sensitivities for HbA, GA, and the combined tests were 60%, 27%, and 67%, respectively. Combined test sensitivity did not differ from HbA alone ( = 0.25) because GA detected only 10% of obese Africans with Abnl-GT not detected by HbA.

Conclusions: Adding GA to HbA improves detection of Abnl-GT in nonobese Africans.
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http://dx.doi.org/10.2337/dc20-1119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510044PMC
October 2020

IQGAP1 causes choroidal neovascularization by sustaining VEGFR2-mediated Rac1 activation.

Angiogenesis 2020 11 11;23(4):685-698. Epub 2020 Aug 11.

The John A Moran Eye Center, University of Utah, 65 Mario Capecchi Drive, Salt Lake City, UT, 84132, USA.

Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermate Iqgap1, Iqgap1 mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration, and tube formation. Our findings indicate that CEC IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.
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http://dx.doi.org/10.1007/s10456-020-09740-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530064PMC
November 2020

Evaluation of Three Commercial Automated Assays for the Detection of Anti-SARS-CoV-2 Antibodies.

Clin Chem 2020 10;66(10):1351-1353

Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD.

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http://dx.doi.org/10.1093/clinchem/hvaa193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454464PMC
October 2020

The YSI 2300 Analyzer Replacement Meeting Report.

J Diabetes Sci Technol 2020 05 16;14(3):679-686. Epub 2020 Mar 16.

Diabetes Research Institute, Mills-Peninsula Medical Center, San Mateo, CA, USA.

This is a summary report of the most important aspects discussed during the YSI 2300 Analyzer Replacement Meeting. The aim is to provide the interested reader with an overview of the complex topic and propose solutions for the current issue. This solution should not only be adequate for the United States or Europe markets but also for all other countries. The meeting addendum presents three outcomes of the meeting.
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http://dx.doi.org/10.1177/1932296820911471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576944PMC
May 2020

Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42.

J Biol Chem 2020 04 24;295(15):4822-4835. Epub 2020 Feb 24.

Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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http://dx.doi.org/10.1074/jbc.RA119.011491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152761PMC
April 2020

Diagnosis of Gestational Diabetes Mellitus Will Be Flawed until We Can Measure Glucose.

Clin Chem 2020 02;66(2):265-267

Department of Laboratory Medicine, NIH Clinical Center, Bethesda, MD.

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http://dx.doi.org/10.1093/clinchem/hvz027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055661PMC
February 2020

Ca-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1.

Biochemistry 2019 12 26;58(49):4903-4911. Epub 2019 Nov 26.

Center for Cancer Research, National Cancer Institute , National Institutes of Health , Frederick , Maryland 20892 , United States.

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca as a key modulator of the CaM-IQGAP1 interactions.
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http://dx.doi.org/10.1021/acs.biochem.9b00854DOI Listing
December 2019

Insulin suppresses transcriptional activity of yes-associated protein in insulin target cells.

Mol Biol Cell 2020 01 6;31(2):131-141. Epub 2019 Nov 6.

Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD 20892.

Yes-associated protein (YAP), the main transcriptional coactivator of the Hippo pathway, integrates multiple inputs from different signaling cascades. Evidence implicates YAP in the control of cellular nutrient and energy status, but the underlying mechanisms are not fully elucidated. Here we show that insulin modulates YAP transcriptional activity in classic insulin target cells, namely HepG2 and C2C12. Insulin increases YAP phosphorylation and significantly decreases YAP abundance in HepG2 cell nuclei. Proximity ligation assay analysis revealed a marked reduction in the interaction of YAP with TEA domain (TEAD) transcription factors in the nuclei of insulin-exposed cells. Consistent with these findings, insulin impaired both YAP/TEAD-mediated transcription and transcription of YAP target genes in HepG2 and C2C12 cells. Serum starvation abrogated the effect of insulin on YAP phosphorylation and YAP transcription. Both the expression of two gluconeogenesis genes, G6PC and PCK1, and the inhibitory effect of insulin on these genes were attenuated in YAP-deficient HepG2 cells. Our results identify insulin as a previously undescribed suppressor of YAP activity in insulin target cells and provide insight into cross-talk between the insulin and Hippo pathways.
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http://dx.doi.org/10.1091/mbc.E19-04-0205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960410PMC
January 2020

Inadequate Reporting of Analytical Characteristics of Biomarkers Used in Clinical Research: A Threat to Interpretation and Replication of Study Findings.

Clin Chem 2019 12 31;65(12):1554-1562. Epub 2019 Oct 31.

Department of Laboratory Medicine, Clinical Center, NIH, Bethesda, MD;

Background: Analytical characteristics of methods to measure biomarkers determine how well the methods measure what they claim to measure. Transparent reporting of analytical characteristics allows readers to assess the validity and generalizability of clinical studies in which biomarkers are used. Our aims were to assess the reporting of analytical characteristics of biomarkers used in clinical research and to evaluate the extent of reported characterization procedures for assay precision.

Methods: We searched 5 medical journals (, , , , and ) over a 10-year period for the term "biomarker" in the full-text field. We included studies in which biomarkers were used for inclusion/exclusion of study participants, for patient classification, or as a study outcome. We tabulated the frequencies of reporting of 11 key analytical characteristics (such as analytical accuracy of test results) in the included studies.

Results: A total of 544 studies and 1299 biomarker uses met the inclusion criteria. No information on analytical characteristics was reported for 67% of the biomarkers. For 65 biomarkers (3%), ≥4 characteristics were reported (range, 4-8). The manufacturer of the measurement procedure could not be determined for 688 (53%) of the 1299 biomarkers. The extent of assessments of assay imprecision, when reported, did not meet expectations for clinical use of biomarkers.

Conclusions: Reporting of the analytical performance of biomarker measurements is variable and often absent from published clinical studies. We suggest that readers need fuller reporting of analytical characteristics to interpret study results, assess generalizability of conclusions, and compare results among clinical studies.
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http://dx.doi.org/10.1373/clinchem.2019.309575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055667PMC
December 2019

Point-of-Care Hemoglobin A1c.

JAMA 2019 Oct;322(14):1404-1405

Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland.

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http://dx.doi.org/10.1001/jama.2019.14063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065945PMC
October 2019

Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis.

J Exp Med 2019 11 8;216(11):2582-2601. Epub 2019 Aug 8.

Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL

Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires the coordination of membrane movements and cytoskeletal interactions, including, prominently, recruitment of the lateral border recycling compartment (LBRC). The scaffold protein IQGAP1 was recently identified in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we show for the first time that IQGAP1 in the endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.
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http://dx.doi.org/10.1084/jem.20190008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829592PMC
November 2019

Endogenous IQGAP1 and IQGAP3 do not functionally interact with Ras.

Sci Rep 2019 07 30;9(1):11057. Epub 2019 Jul 30.

From the Department of Laboratory Medicine, National Institutes of Health, 10 Center Drive, Bethesda, Maryland, 20892, USA.

The Ras family of small GTPases modulates numerous essential processes. Activating Ras mutations result in hyper-activation of selected signaling cascades, which leads to human diseases. The high frequency of Ras mutations in human malignant neoplasms has led to Ras being a desirable chemotherapeutic target. The IQGAP family of scaffold proteins binds to and regulates multiple signaling molecules, including the Rho family GTPases Rac1 and Cdc42. There are conflicting data in the published literature regarding interactions between IQGAP and Ras proteins. Initial reports showed no binding, but subsequent studies claim associations of IQGAP1 and IQGAP3 with K-Ras and H-Ras, respectively. Therefore, we set out to resolve this controversy. Here we demonstrate that neither endogenous IQGAP1 nor endogenous IQGAP3 binds to the major Ras isoforms, namely H-, K-, and N-Ras. Importantly, Ras activation by epidermal growth factor is not altered when IQGAP1 or IQGAP3 proteins are depleted from cells. These data strongly suggest that IQGAP proteins are not functional interactors of H-, K-, or N-Ras and challenge the rationale for targeting the interaction of Ras with IQGAP for the development of therapeutic agents.
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http://dx.doi.org/10.1038/s41598-019-46677-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667474PMC
July 2019

The National Glycohemoglobin Standardization Program: Over 20 Years of Improving Hemoglobin A Measurement.

Clin Chem 2019 07 5;65(7):839-848. Epub 2018 Dec 5.

Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD.

Background: Measurement of hemoglobin A1c (HbA) in the blood is integral to and essential for the treatment of patients with diabetes mellitus. HbA reflects the mean blood glucose concentration over the preceding 8 to 12 weeks. Although the clinical value of HbA was initially limited by large differences in results among various methods, the investment of considerable effort to implement standardization has brought about a marked improvement in analysis.

Content: The focus of this review is on the substantial progress that has been achieved in enhancing the accuracy and, therefore, the clinical value of HbA assays.

Summary: The interactions between the National Glycohemoglobin Standardization Program and manufacturers of HbA methods have been instrumental in standardizing HbA. Proficiency testing using whole blood has allowed accuracy-based assessment of methods in individual clinical laboratories that has made an important contribution to improving the HbA measurement in patient samples. These initiatives, supported by the efforts of the IFCC network, have led to a continuing enhancement of HbA methods.Many of the factors that previously influenced HbA results independently of blood glucose have been eliminated from most modern methods. These include carbamylation, labile intermediates, and common hemoglobin variants. Nevertheless, some factors (e.g., race and aging) may alter HbA interpretation, but whether these differences have clinical implications remains contentious. HbA has a fundamental role in the diagnosis and management of diabetes. Ongoing improvements in HbA measurement and quality will further enhance the clinical value of this analyte.
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http://dx.doi.org/10.1373/clinchem.2018.296962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693326PMC
July 2019

Glucose Management Indicator (GMI): A New Term for Estimating A1C From Continuous Glucose Monitoring.

Diabetes Care 2018 11 17;41(11):2275-2280. Epub 2018 Sep 17.

American Diabetes Association, Arlington, VA.

While A1C is well established as an important risk marker for diabetes complications, with the increasing use of continuous glucose monitoring (CGM) to help facilitate safe and effective diabetes management, it is important to understand how CGM metrics, such as mean glucose, and A1C correlate. Estimated A1C (eA1C) is a measure converting the mean glucose from CGM or self-monitored blood glucose readings, using a formula derived from glucose readings from a population of individuals, into an estimate of a simultaneously measured laboratory A1C. Many patients and clinicians find the eA1C to be a helpful educational tool, but others are often confused or even frustrated if the eA1C and laboratory-measured A1C do not agree. In the U.S., the Food and Drug Administration determined that the nomenclature of eA1C needed to change. This led the authors to work toward a multipart solution to facilitate the retention of such a metric, which includes renaming the eA1C the glucose management indicator (GMI) and generating a new formula for converting CGM-derived mean glucose to GMI based on recent clinical trials using the most accurate CGM systems available. The final aspect of ensuring a smooth transition from the old eA1C to the new GMI is providing new CGM analyses and explanations to further understand how to interpret GMI and use it most effectively in clinical practice. This Perspective will address why a new name for eA1C was needed, why GMI was selected as the new name, how GMI is calculated, and how to understand and explain GMI if one chooses to use GMI as a tool in diabetes education or management.
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http://dx.doi.org/10.2337/dc18-1581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196826PMC
November 2018

IQGAP1 binds the Axl receptor kinase and inhibits its signaling.

Biochem J 2018 10 9;475(19):3073-3086. Epub 2018 Oct 9.

Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD 20892, U.S.A.

Axl is a tyrosine kinase receptor that is important for hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival. Binding of growth arrest-specific protein 6 (Gas6) activates Axl signaling, but the mechanism of inactivation of the Axl receptor is poorly understood. In the present study, we show that IQGAP1 modulates Axl signaling. IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules. Our analysis revealed a direct interaction between the IQ domain of IQGAP1 and Axl. Analysis by both immunoprecipitation and proximity ligation assays demonstrated an association between Axl and IQGAP1 in cells and this interaction was decreased by Gas6. Unexpectedly, reducing IQGAP1 levels in cells significantly enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, IQGAP1 regulates the interaction of Axl with the epidermal growth factor receptor. Our data identify IQGAP1 as a previously undescribed suppressor of Axl and provide insight into regulation of Axl function.
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http://dx.doi.org/10.1042/BCJ20180594DOI Listing
October 2018

Calmodulin (CaM) Activates PI3Kα by Targeting the "Soft" CaM-Binding Motifs in Both the nSH2 and cSH2 Domains of p85α.

J Phys Chem B 2018 12 8;122(49):11137-11146. Epub 2018 Aug 8.

Cancer and Inflammation Program, Leidos Biomedical Research, Inc. , Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute , Frederick , Maryland 21702 , United States.

PI3Kα is a key lipid kinase in the PI3K/Akt pathway. Its frequent oncogenic mutations make it a primary drug target. Calmodulin (CaM) activates PI3Kα independently of extracellular signals, indicating a significant role in oncogenic PI3Kα activation. Here, we reveal the atomic-scale structures of CaM in complexes with the nSH2 and cSH2 domains of the regulatory p85α subunit of PI3Kα, and illustrate how CaM activates PI3Kα by targeting the "soft 1-5-10" CaM-binding motifs in both nSH2 and cSH2 domains. Experiment observed CaM binding cSH2 first, followed by nSH2 binding hours later. CaM typically prefers binding helical peptides. Here we observe that, unlike in cSH2, the CaM-binding motif in nSH2 populates a mixed β-sheet/α-helix/random coil structure. The population shift from a β-sheet toward CaM's favored α-helical conformation explains why the nSH2 domain needs a longer time for CaM binding in the experiments. The "soft" CaM-binding motifs in both nSH2 and cSH2 domains establish strong CaM-PI3Kα interactions, collectively facilitating PI3Kα activation. This work uncovers the structural basis for CaM-driven PI3Kα activation.
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http://dx.doi.org/10.1021/acs.jpcb.8b05982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422767PMC
December 2018

Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry.

J Am Soc Mass Spectrom 2018 Aug 5;29(8):1721-1737. Epub 2018 Jun 5.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20894, USA.

Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html . Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1986-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061032PMC
August 2018

Establishment of Community-Based Reference Intervals for Fructosamine, Glycated Albumin, and 1,5-Anhydroglucitol.

Clin Chem 2018 05 7;64(5):843-850. Epub 2018 Feb 7.

Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN.

Background: There is growing interest in fructosamine, glycated albumin, and 1,5-anhydroglucitol (1,5-AG) as alternative measures of hyperglycemia, particularly for use in settings where traditional measures (glucose and HbA1c) are problematic or where intermediate (2-4 weeks) glycemic control is of interest. However, reference intervals for these alternative biomarkers are not established.

Methods: We measured fructosamine, glycated albumin, and 1,5-AG in a community-based sample of US black and white adults who participated in the Atherosclerosis Risk in Communities (ARIC) Study. We calculated reference intervals, evaluated demographic differences, and derived cutoffs aligned with current diagnostic cutpoints for HbA1c and fasting glucose.

Results: In a healthy reference population of 1799 individuals (mean age, 55 years; 51% women; 15% black), the 2.5 and 97.5 percentiles, respectively, were 194.8 and 258.0 μmol/L for fructosamine, 10.7% and 15.1% for glycated albumin, and 8.4 and 28.7 μg/mL for 1,5-AG. Distributions differed by race, sex, and body mass index. Equivalent concentrations of fructosamine and glycated albumin corresponding to an HbA1c of 6.5% (96.5 percentile) were 270.2 μmol/L and 15.6%, respectively. Equivalent concentrations of fructosamine and glycated albumin corresponding to a fasting glucose of 126 mg/dL (93.9 percentile) were 261.7 μmol/L and 15.0%, respectively.

Conclusions: The reference intervals for these biomarkers should inform their clinical use. Diagnostic cutpoint equivalents for fructosamine and glycated albumin could be useful to identify persons with hyperglycemia in settings where fasting glucose or HbA1c are not available or where the interpretation of these traditional measures is problematic.
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http://dx.doi.org/10.1373/clinchem.2017.285742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924648PMC
May 2018