Multiplexing the Identification of Microorganisms via Tandem Mass Tag Labeling Augmented by Interference Removal through a Novel Modification of the Expectation Maximization Algorithm.

Having fast, accurate, and broad spectrum methods for the identification of microorganisms is of paramount importance to public health, research, and safety. Bottom-up mass spectrometer-based proteomics has emerged as an effective tool for the accurate identification of microorganisms from microbial isolates. However, one major hurdle that limits the deployment of this tool for routine clinical diagnosis, and other areas of research such as culturomics, is the instrument time required for the mass spectrometer to analyze a single sample, which can take ∼1 h per sample, when using mass spectrometers that are presently used in most institutes. To address this issue, in this study, we employed, for the first time, tandem mass tags (TMTs) in multiplex identifications of microorganisms from multiple TMT-labeled samples in one MS/MS experiment. A difficulty encountered when using TMT labeling is the presence of interference in the measured intensities of TMT reporter ions. To correct for interference, we employed in the proposed method a modified version of the expectation maximization (EM) algorithm that redistributes the signal from ion interference back to the correct TMT-labeled samples. We have evaluated the sensitivity and specificity of the proposed method using 94 MS/MS experiments (covering a broad range of protein concentration ratios across TMT-labeled channels and experimental parameters), containing a total of 1931 true positive TMT-labeled channels and 317 true negative TMT-labeled channels. The results of the evaluation show that the proposed method has an identification sensitivity of 93-97% and a specificity of 100% at the species level. Furthermore, as a proof of concept, using an in-house-generated data set composed of some of the most common urinary tract pathogens, we demonstrated that by using the proposed method the mass spectrometer time required per sample, using a 1 h LC-MS/MS run, can be reduced to 10 and 6 min when samples are labeled with TMT-6 and TMT-10, respectively. The proposed method can also be used along with Orbitrap mass spectrometers that have faster MS/MS acquisition rates, like the recently released Orbitrap Astral mass spectrometer, to further reduce the mass spectrometer time required per sample.

Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

Point-of-Care HbA1c in Clinical Practice: Caveats and Considerations for Optimal Use.

Hemoglobin A1c (A1C) is widely used for the diagnosis and management of diabetes. Accurate measurement of A1C is necessary for optimal clinical value. Assay standardization has markedly improved the accuracy and consistency of A1C testing. Devices to measure A1C at point of care (POC) are commercially available, allowing rapid results when the patient is seen. In this review, we describe how standardization of A1C testing was achieved, leading to high-quality results in clinical laboratories. We address the use of POC A1C testing in clinical situations and summarize the advantages and disadvantages of POC A1C testing. We emphasize the importance of considering the limitations of these devices and following correct testing procedures to ensure that accurate A1C results are obtained for optimal care of patients.

Advanced Glycation Endproducts: A Marker of Long-term Exposure to Glycemia.

This article examines the importance of advanced glycation endproducts (AGEs) and summarizes the structure of AGEs, pathological changes associated with AGEs, the contribution of AGEs to metabolic memory, and the value of AGEs as a predictor of diabetic complications and cardiovascular disease in people with and without diabetes. As a practical focus, skin autofluorescence (SAF) is examined as an attractive approach for estimating AGE burden. The measurement of AGEs may be of significant value to specific individuals and groups, including Black and Hispanic/Latino Americans, as they appear to have higher concentrations of hemoglobin A1c (HbA1c) than would be predicted by other metrics of mean glycemia. We hypothesize that if the amount of glycation of HbA1c is greater than expected from measured glucose levels, and if AGEs are accumulating, then this accumulation of AGEs might account for the increased rate of complications of diabetes in populations with high rates of vascular disease and other complications. Thus, identifying and modifying the burden of AGEs based on measurement of AGEs by SAF may turn out to be a worthwhile metric to determine individuals who are at high risk for the complications of diabetes as well as others without diabetes at risk of vascular disease. We conclude that available evidence supports SAF as both a clinical measurement and as a means of evaluating interventions aimed at reducing the risks of vascular disease and diabetic complications.

Emerging Biomarkers in the Laboratory and in Practice: A Novel Approach to Diagnosing Heart Failure in Diabetes.

The Biomarkers for the Diagnosis of Heart Failure in Diabetes webinar was hosted by Diabetes Technology Society on September 20, 2023, with the objective to review current evidence and management practices of biomarker screening for heart failure in people with diabetes. The webinar discussed (1) the four stages of heart failure, (2) diabetes and heart failure, (3) natriuretic peptide and troponin for diagnosing heart failure in diabetes, (4) emerging composite and investigational biomarkers for diagnosing heart failure, and (5) prevention of heart failure progression. Experts in heart failure from the fields of clinical chemistry, cardiology, and diabetology presented data about the importance of screening for heart failure as an often-unnoticed complication of people with type 1 and type 2 diabetes.

Dynamic Elevation of Aromatic Amino Acids in Hepatitis C Virus-Induced Cirrhosis After a Standard Meal.

INTRODUCTION: Perturbations in aromatic (AAAs) and branched-chain amino acids (BCAAs) are seen in decompensated liver disease. The aim of this study was to evaluate the dynamic, postprandial relationship between hepatitis C virus-induced liver disease and amino acid concentrations in patients with compensated liver disease. METHODS: Patients infected with hepatitis C virus underwent a baseline liver biopsy to determine Ishak Fibrosis Score and evaluate the liver transcriptome. Patients ate a standard meal and underwent peripheral vein sampling at defined intervals. Quantitative analysis of amino acids was performed using liquid chromatography-tandem mass spectrometry. RESULTS: At baseline, there was no difference in AAA and BCAA concentrations between patients with cirrhosis and non-cirrhotic patients. After a standard meal, AAAs, but not BCAAs, were elevated in patients with cirrhosis compared with non-cirrhotic patients at every time point. The HepQuant SHUNT fraction was significantly higher in patients with cirrhosis and positively correlated with AAA concentration at all time points, but not BCAA. Analysis of the hepatic transcriptome demonstrated greater downregulation of the AAA degradation pathways than the BCAA degradation pathways. DISCUSSION: At baseline, cirrhotic patients with compensated liver disease have adequate reserve liver function to metabolize AAAs and BCAAs. When faced with a metabolic stressor, such as a standard meal, patients with cirrhosis are less able to metabolize the increased load of AAAs. This impairment correlates with portosystemic shunting. Further evaluation of AAA levels in compensated liver disease might further the understanding of the liver-muscle axis and the role it may play in the development of sarcopenia in liver disease.

A Longitudinal Study of Plasma Glycated Albumin across Pregnancy and Associations with Maternal Characteristics and Cardiometabolic Biomarkers.

BACKGROUND: Glycated albumin (GA) has recently been proposed as a screening marker for diabetes among non-pregnant individuals. However, data on GA during pregnancy are sparse and lacking among women of diverse race/ethnicity. We investigated longitudinal concentrations of GA among multiracial pregnant women in the National Institute of Child Health and Human Development (NICHD) Fetal Growth Studies-Singletons. METHODS: We quantified GA and cardiometabolic biomarkers using longitudinal plasma samples collected at 10 to 14, 15 to 26 (fasting), 23 to 31, and 33 to 39 gestational weeks from 214 pregnant women without gestational diabetes. We examined the distribution of GA across pregnancy and its association with participants' characteristics including race/ethnicity, pre-pregnancy body mass index (ppBMI), and selected cardiometabolic biomarkers. GA trajectories were estimated using a latent class approach. RESULTS: Medians (interquartile range) of GA concentrations were 12.1% (10.6%-13.4%), 12.5% (10.7%-13.8%), 12.4% (10.9%-13.5%), and 11.5% (10.4%-12.5%) at 10 to 14, 15 to 26, 23 to 31, and 33 to 39 weeks, respectively. There were no significant differences in the pattern among different race/ethnic groups (P > 0.53). A minority of women exhibited a GA trajectory characterized by a high concentration of GA at 15 to 26 weeks. GA concentrations were inversely related to ppBMI and plasma low-density lipoprotein and triglyceride concentrations, but were not significantly related to hemoglobin A1c, fasting insulin, or glucose over pregnancy. CONCLUSIONS: In this study of individuals who were normoglycemic before pregnancy, plasma GA concentrations stayed relatively constant over pregnancy, decreasing only in late pregnancy. GA concentrations were inversely related to ppBMI and suboptimal lipid profiles, but did not appear to be a sensitive marker for glucose metabolism in pregnancy.

The effectiveness of blood glucose and blood ketone measurement in identifying significant acidosis in diabetic ketoacidosis patients.

BACKGROUND: Patients with diabetic ketoacidosis (DKA), a potentially fatal complication of type 1 diabetes, have hyperglycemia, ketonemia and metabolic acidosis. Blood glucose and blood ketone results are often used to triage patients with suspected DKA. This study aimed to establish how effective blood glucose and blood ketone (beta-hydroxybutyrate, BOHB) measurements are in identifying patients with significant acidosis and sought to validate existing diagnostic BOHB thresholds. METHODS: Initial Emergency Department results on 161 presumptive DKA episodes in 95 patients (42 F, 53 M, age range 14-89 years) containing a complete dataset of D (glucose), K (BOHB) and A (Bicarbonate [HCO3] and pH) results. RESULTS: Blood glucose correlated poorly with BOHB (r = 0.28 p = 0.0003), pH (r= -0.25, p = 0.002) and HCO3 (r= -0.17, p = 0.04). BOHB, though better, was still limited in predicting pH (r = -0.44, p < 0.0001) and HCO3 (r = -0.49, p < 0.0001). A HCO3 of 18mmol/L equated to a BOHB concentration of 4.3mmol/L, whilst a HCO3 of 15mmol/L equated to a BOHB of 4.7mmol/L. Of the 133 of 161 events with HCO3 < 18mmol/L, 22 were not hyperglycemic (> 13.9mmol/L, n = 8), ketonemic (≤ 3mmol/L, n = 9) or either (n = 5). CONCLUSIONS: The commonly employed BOHB diagnostic cutoff of 3mmol/L could not be verified. Since acid-base status was poorly predicted by both glucose and BOHB, this highlights that, regardless of their results, pH and/or HCO3 should also be tested in any patient suspected of DKA.

Fructosamine is Not a Reliable Test for the Detection of Hyperglycemia: Insight from the Africans in America Study.

Objective: To improve detection of abnormal glucose tolerance (Abnl-GT), attention has moved beyond the oral glucose tolerance test (OGTT), to non-fasting markers of glycemia, specifically, HbA1c, fructosamine (FA) and glycated albumin (GA). Emerging data suggest that in African descent populations, the combination of HbA1c and GA is superior to the combination of HbA1c and FA. However, the diagnosis of Abnl-GT is usually based on tests which are performed only once. As reproducibility of Abnl-GT diagnosis by HbA1c, fructosamine (FA) and glycated albumin (GA) is unknown, reproducibility of Abnl-GT diagnosis by HbA1c, FA and GA were assessed in 209 African-born Blacks living in America. Methods: At Visits 1 and 2 (9 ± 4 days apart), samples were obtained for HbA1c, FA and GA levels. Glucose tolerance status was determined at Visit 1 by OGTT. Reproducibility was based on the К-statistic and paired t-tests. Thresholds for the diagnosis of Abnl-GT by FA and GA which corresponded to an HbA1c of 5.7% were 235umol/L and 14.6%, respectively. Results: Abnl-GT occurred in 38% (80/209). Diagnostic reproducibility was excellent for HbA1c (К≥0.86) and GA (К≥0.89), but only moderate for FA (К=0.59). Neither HbA1c nor GA levels varied between visits (both P≥0.3). In contrast, FA was significantly lower at Visit 2 than Visit 1(P<0.01). Conclusion: As HbA1c and GA provided similar diagnostic results on different days and FA did not, HbA1C and GA are superior to FA in both clinical care settings and epidemiologic studies.

Ca2+ signaling and the Hippo pathway: Intersections in cellular regulation.

The Hippo signaling pathway is a master regulator of organ size and tissue homeostasis. Hippo integrates a broad range of cellular signals to regulate numerous processes, such as cell proliferation, differentiation, migration and mechanosensation. Ca2+ is a fundamental second messenger that modulates signaling cascades involved in diverse cellular functions, some of which are also regulated by the Hippo pathway. Studies published over the last five years indicate that Ca2+ can influence core Hippo pathway components. Nevertheless, comprehensive understanding of the crosstalk between Ca2+ signaling and the Hippo pathway, and possible mechanisms through which Ca2+ regulates Hippo, remain to be elucidated. In this review, we summarize the multiple intersections between Ca2+ and the Hippo pathway and address the biological consequences.

Executive Summary: Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus.

BACKGROUND: Numerous laboratory tests are used in the diagnosis and management of patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. An expert committee compiled evidence-based recommendations for laboratory analysis in patients with diabetes. The overall quality of the evidence and the strength of the recommendations were evaluated. The draft consensus recommendations were evaluated by invited reviewers and presented for public comment. Suggestions were incorporated as deemed appropriate by the authors (see Acknowledgments in the full version of the guideline). The guidelines were reviewed by the Evidence Based Laboratory Medicine Committee and the Board of Directors of the American Association of Clinical Chemistry and by the Professional Practice Committee of the American Diabetes Association. CONTENT: Diabetes can be diagnosed by demonstrating increased concentrations of glucose in venous plasma or increased hemoglobin A1c (Hb A1c) in the blood. Glycemic control is monitored by the patients measuring their own blood glucose with meters and/or with continuous interstitial glucose monitoring devices and also by laboratory analysis of Hb A1c. The potential roles of noninvasive glucose monitoring; genetic testing; and measurement of ketones, autoantibodies, urine albumin, insulin, proinsulin, and C-peptide are addressed. SUMMARY: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are found to have minimal clinical value at the present time, and measurement of them is not recommended.

Executive Summary: Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus.

BACKGROUND: Numerous laboratory tests are used in the diagnosis and management of patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. An expert committee compiled evidence-based recommendations for laboratory analysis in patients with diabetes. The overall quality of the evidence and the strength of the recommendations were evaluated. The draft consensus recommendations were evaluated by invited reviewers and presented for public comment. Suggestions were incorporated as deemed appropriate by the authors (see Acknowledgments in the full version of the guideline). The guidelines were reviewed by the Evidence Based Laboratory Medicine Committee and the Board of Directors of the American Association for Clinical Chemistry and by the Professional Practice Committee of the American Diabetes Association. CONTENT: Diabetes can be diagnosed by demonstrating increased concentrations of glucose in venous plasma or increased hemoglobin A1c (HbA1c) in the blood. Glycemic control is monitored by the patients measuring their own blood glucose with meters and/or with continuous interstitial glucose monitoring devices and also by laboratory analysis of HbA1c. The potential roles of noninvasive glucose monitoring; genetic testing; and measurement of ketones, autoantibodies, urine albumin, insulin, proinsulin, and C-peptide are addressed. SUMMARY: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are found to have minimal clinical value at the present time, and measurement of them is not recommended.

Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus.

BACKGROUND: Numerous laboratory tests are used in the diagnosis and management of diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. APPROACH: An expert committee compiled evidence-based recommendations for laboratory analysis in screening, diagnosis, or monitoring of diabetes. The overall quality of the evidence and the strength of the recommendations were evaluated. The draft consensus recommendations were evaluated by invited reviewers and presented for public comment. Suggestions were incorporated as deemed appropriate by the authors (see Acknowledgments). The guidelines were reviewed by the Evidence Based Laboratory Medicine Committee and the Board of Directors of the American Association for Clinical Chemistry and by the Professional Practice Committee of the American Diabetes Association. CONTENT: Diabetes can be diagnosed by demonstrating increased concentrations of glucose in venous plasma or increased hemoglobin A1c (HbA1c) in the blood. Glycemic control is monitored by the people with diabetes measuring their own blood glucose with meters and/or with continuous interstitial glucose monitoring (CGM) devices and also by laboratory analysis of HbA1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of ketones, autoantibodies, urine albumin, insulin, proinsulin, and C-peptide are addressed. SUMMARY: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are found to have minimal clinical value at the present time, and measurement of them is not recommended.

Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus.

BACKGROUND: Numerous laboratory tests are used in the diagnosis and management of diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. APPROACH: An expert committee compiled evidence-based recommendations for laboratory analysis in screening, diagnosis, or monitoring of diabetes. The overall quality of the evidence and the strength of the recommendations were evaluated. The draft consensus recommendations were evaluated by invited reviewers and presented for public comment. Suggestions were incorporated as deemed appropriate by the authors (see Acknowledgments). The guidelines were reviewed by the Evidence Based Laboratory Medicine Committee and the Board of Directors of the American Association of Clinical Chemistry and by the Professional Practice Committee of the American Diabetes Association. CONTENT: Diabetes can be diagnosed by demonstrating increased concentrations of glucose in venous plasma or increased hemoglobin A1c (Hb A1c) in the blood. Glycemic control is monitored by the people with diabetes measuring their own blood glucose with meters and/or with continuous interstitial glucose monitoring (CGM) devices and also by laboratory analysis of Hb A1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of ketones, autoantibodies, urine albumin, insulin, proinsulin, and C-peptide are addressed. SUMMARY: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are found to have minimal clinical value at the present time, and measurement of them is not recommended.

Biomarkers for the Diagnosis of Heart Failure in People with Diabetes: A Consensus Report from Diabetes Technology Society.

Diabetes Technology Society assembled a panel of clinician experts in diabetology, cardiology, clinical chemistry, nephrology, and primary care to review the current evidence on biomarker screening of people with diabetes (PWD) for heart failure (HF), who are, by definition, at risk for HF (Stage A HF). This consensus report reviews features of HF in PWD from the perspectives of 1) epidemiology, 2) classification of stages, 3) pathophysiology, 4) biomarkers for diagnosing, 5) biomarker assays, 6) diagnostic accuracy of biomarkers, 7) benefits of biomarker screening, 8) consensus recommendations for biomarker screening, 9) stratification of Stage B HF, 10) echocardiographic screening, 11) management of Stage A and Stage B HF, and 12) future directions. The Diabetes Technology Society panel recommends 1) biomarker screening with one of two circulating natriuretic peptides (B-type natriuretic peptide or N-terminal prohormone of B-type natriuretic peptide), 2) beginning screening five years following diagnosis of type 1 diabetes (T1D) and at the diagnosis of type 2 diabetes (T2D), 3) beginning routine screening no earlier than at age 30 years for T1D (irrespective of age of diagnosis) and at any age for T2D, 4) screening annually, and 5) testing any time of day. The panel also recommends that an abnormal biomarker test defines asymptomatic preclinical HF (Stage B HF). This diagnosis requires follow-up using transthoracic echocardiography for classification into one of four subcategories of Stage B HF, corresponding to risk of progression to symptomatic clinical HF (Stage C HF). These recommendations will allow identification and management of Stage A and Stage B HF in PWD to prevent progression to Stage C HF or advanced HF (Stage D HF).

The IQGAP scaffolds: Critical nodes bridging receptor activation to cellular signaling.

The scaffold protein IQGAP1 assembles multiprotein signaling complexes to influence biological functions. Cell surface receptors, particularly receptor tyrosine kinases and G-protein coupled receptors, are common IQGAP1 binding partners. Interactions with IQGAP1 modulate receptor expression, activation, and/or trafficking. Moreover, IQGAP1 couples extracellular stimuli to intracellular outcomes via scaffolding of signaling proteins downstream of activated receptors, including mitogen-activated protein kinases, constituents of the phosphatidylinositol 3-kinase pathway, small GTPases, and ß-arrestins. Reciprocally, some receptors influence IQGAP1 expression, subcellular localization, binding properties, and post-translational modifications. Importantly, the receptor:IQGAP1 crosstalk has pathological implications ranging from diabetes and macular degeneration to carcinogenesis. Here, we describe the interactions of IQGAP1 with receptors, summarize how they modulate signaling, and discuss their contribution to pathology. We also address the emerging functions in receptor signaling of IQGAP2 and IQGAP3, the other human IQGAP proteins. Overall, this review emphasizes the fundamental roles of IQGAPs in coupling activated receptors to cellular homeostasis.

Establishing Pragmatic Analytical Performance Specifications for Blood Beta-Hydroxybutyrate Testing.

BACKGROUND: Currently, no authoritative guidelines exist recommending the analytical performance specification (APS) of blood beta-hydroxybutyrate (BOHB) testing in order to meet the clinical needs of patients. This study has applied existing diabetic ketoacidosis (DKA) BOHB diagnostic thresholds and the recommended rates of fall in BOHB concentrations during DKA treatment to establish pragmatic APSs for BOHB testing. METHODS: Required analytical performance was based on 2 clinical requirements: (a) to reliably distinguish between non-adjacent DKA BOHB diagnostic categories of <0.6, 0.6 to 1.5, 1.6 to 2.9, and ≥3 mmol/L, and (b) to be assured that a measured 0.5 mmol/L reduction in BOHB indicates the true concentration is at least falling (meaning >0 mmol/L decline). RESULTS: An analytical coefficient of variation (CV) of <21.5% could reliably distinguish all non-adjacent diagnostic categories with >99% certainty, assuming zero bias. In contrast, within-day CVs of 4.9%, 7.0%, and 9.1% at 3 mmol/L BOHB were required to assure truly falling ketone concentrations with 99% (optimal), 95% (desirable), and 90% (minimal) probability, respectively. These CVs are larger at lower BOHB concentrations and smaller at higher concentrations. CONCLUSIONS: Reliable tracking of changes in BOHB during DKA treatment largely drives the requirement for analytical performance. These data can be used to guide minimal, desirable, and optimal performance targets for BOHB meters and laboratory assays.

IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins.

The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr1510. Separately, Src homology 2 (SH2) domains mediate protein-protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr1510 of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr1510 of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr1510 of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling.

Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species.

RAS-ERK (extracellular signal-regulated kinase) pathway signals are modulated by scaffold proteins that assemble the components of different kinase tiers into a sequential phosphorylation cascade. In the prevailing model scaffold proteins function as isolated entities, where the flux of phosphorylation events progresses downstream linearly, to achieve ERK phosphorylation. We show that different types of scaffold proteins, specifically KSR1 (kinase suppressor of Ras 1) and IQGAP1 (IQ motif-containing guanosine triphosphatase activating protein 1), can bind to each other, forming a complex whereby phosphorylation reactions occur across both species. MEK (mitogen-activated protein kinase kinase) bound to IQGAP1 can phosphorylate ERK docked at KSR1, a process that we have named "trans-phosphorylation." We also reveal that ERK trans-phosphorylation participates in KSR1-regulated adipogenesis, and it also underlies the modest cytotoxicity exhibited by KSR-directed inhibitors. Overall, we identify interactions between scaffold proteins and trans-phosphorylation as an additional level of regulation in the ERK cascade, with broad implications in signaling and the design of scaffold protein-aimed therapeutics.