The aetiology and molecular landscape of insulin resistance.

Insulin resistance, defined as a defect in insulin-mediated control of glucose metabolism in tissues - prominently in muscle, fat and liver - is one of the earliest manifestations of a constellation of human diseases that includes type 2 diabetes and cardiovascular disease. These diseases are typically associated with intertwined metabolic abnormalities, including obesity, hyperinsulinaemia, hyperglycaemia and hyperlipidaemia. Insulin resistance is caused by a combination of genetic and environmental factors. Recent genetic and biochemical studies suggest a key role for adipose tissue in the development of insulin resistance, potentially by releasing lipids and other circulating factors that promote insulin resistance in other organs. These extracellular factors perturb the intracellular concentration of a range of intermediates, including ceramide and other lipids, leading to defects in responsiveness of cells to insulin. Such intermediates may cause insulin resistance by inhibiting one or more of the proximal components in the signalling cascade downstream of insulin (insulin receptor, insulin receptor substrate (IRS) proteins or AKT). However, there is now evidence to support the view that insulin resistance is a heterogeneous disorder that may variably arise in a range of metabolic tissues and that the mechanism for this effect likely involves a unified insulin resistance pathway that affects a distal step in the insulin action pathway that is more closely linked to the terminal biological response. Identifying these targets is of major importance, as it will reveal potential new targets for treatments of diseases associated with insulin resistance.

Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes.

Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulin's ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D.

MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that regulates cell survival.

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.

Natural and inducible TH17 cells are regulated differently by Akt and mTOR pathways.

Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.

Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development.

Generation of CD8(+) memory T cells requires metabolic reprogramming that is characterized by enhanced mitochondrial fatty-acid oxidation (FAO). However, where the fatty acids (FA) that fuel this process come from remains unclear. While CD8(+) memory T cells engage FAO to a greater extent, we found that they acquired substantially fewer long-chain FA from their external environment than CD8(+) effector T (Teff) cells. Rather than using extracellular FA directly, memory T cells used extracellular glucose to support FAO and oxidative phosphorylation (OXPHOS), suggesting that lipids must be synthesized to generate the substrates needed for FAO. We have demonstrated that memory T cells rely on cell intrinsic expression of the lysosomal hydrolase LAL (lysosomal acid lipase) to mobilize FA for FAO and memory T cell development. Our observations link LAL to metabolic reprogramming in lymphocytes and show that cell intrinsic lipolysis is deterministic for memory T cell fate.

Tolerability, safety and pharmacodynamics of oral, small-molecule glucagon-like peptide-1 receptor agonist danuglipron for type 2 diabetes: A 12-week, randomized, placebo-controlled, Phase 2 study comparing different dose-escalation schemes.

AIM: To evaluate the tolerability, safety and pharmacodynamics of different dose-escalation schemes of the oral small-molecule glucagon-like peptide-1 receptor (GLP-1R) agonist danuglipron. MATERIALS AND METHODS: This Phase 2a, double-blind, placebo-controlled, parallel-group study randomly assigned adults with type 2 diabetes (T2D) treated with metformin to placebo or danuglipron (low [5-mg] or high [10-mg] starting dose, with 1- or 2-week dose-escalation steps, to target doses of 80, 120 or 200 mg twice daily [BID]) and adults with obesity without diabetes to placebo or danuglipron 200 mg BID. RESULTS: Participants with T2D (n = 123, mean glycated haemoglobin [HbA1c] 8.19%) or obesity without diabetes (n = 28, mean body mass index 37.3 kg/m2 ) were randomly assigned and treated. Discontinuation from study medication occurred in 27.3% to 72.7% of participants across danuglipron groups versus 16.7% to 18.8% for placebo, most often due to adverse events. Nausea (20.0%-47.6% of participants across danuglipron groups vs. 12.5% for placebo) and vomiting (18.2%-40.9% danuglipron vs. 12.5% placebo, respectively) were most commonly reported in participants with T2D. Gastrointestinal adverse events were generally related to danuglipron target dose and were not substantially affected by starting dose. In participants with T2D, least squares mean changes from baseline in HbA1c (-1.04% to -1.57% across danuglipron groups vs. -0.32% for placebo), fasting plasma glucose (-23.34 mg/dL to -53.94 mg/dL danuglipron vs. -13.09 mg/dL placebo) and body weight (-1.93 to -5.38 kg danuglipron vs. -0.42 kg placebo) at Week 12 were generally statistically significant for danuglipron compared with placebo (P < 0.05). CONCLUSIONS: Danuglipron resulted in statistically significant reductions in HbA1c, FPG and body weight over 12 weeks, in the setting of higher discontinuation rates and incidence of gastrointestinal adverse events with higher target doses. CLINICALTRIALS: gov identifier: NCT04617275.

Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP.

Glucose production by the liver is essential for providing a substrate for the brain during fasting. The inability of insulin to suppress hepatic glucose output is a major aetiological factor in the hyperglycaemia of type-2 diabetes mellitus and other diseases of insulin resistance. For fifty years, one of the few classes of therapeutics effective in reducing glucose production has been the biguanides, which include phenformin and metformin, the latter the most frequently prescribed drug for type-2 diabetes. Nonetheless, the mechanism of action of biguanides remains imperfectly understood. The suggestion a decade ago that metformin reduces glucose synthesis through activation of the enzyme AMP-activated protein kinase (AMPK) has recently been challenged by genetic loss-of-function experiments. Here we provide a novel mechanism by which metformin antagonizes the action of glucagon, thus reducing fasting glucose levels. In mouse hepatocytes, metformin leads to the accumulation of AMP and related nucleotides, which inhibit adenylate cyclase, reduce levels of cyclic AMP and protein kinase A (PKA) activity, abrogate phosphorylation of critical protein targets of PKA, and block glucagon-dependent glucose output from hepatocytes. These data support a mechanism of action for metformin involving antagonism of glucagon, and suggest an approach for the development of antidiabetic drugs.

Oxalic acid and diacylglycerol 36:3 are cross-species markers of sleep debt.

Sleep is an essential biological process that is thought to have a critical role in metabolic regulation. In humans, reduced sleep duration has been associated with risk for metabolic disorders, including weight gain, diabetes, obesity, and cardiovascular disease. However, our understanding of the molecular mechanisms underlying effects of sleep loss is only in its nascent stages. In this study we used rat and human models to simulate modern-day conditions of restricted sleep and addressed cross-species consequences via comprehensive metabolite profiling. Serum from sleep-restricted rats was analyzed using polar and nonpolar methods in two independent datasets (n = 10 per study, 3,380 measured features, 407 identified). A total of 38 features were changed across independent experiments, with the majority classified as lipids (18 from 28 identified). In a parallel human study, 92 metabolites were identified as potentially significant, with the majority also classified as lipids (32 of 37 identified). Intriguingly, two metabolites, oxalic acid and diacylglycerol 36:3, were robustly and quantitatively reduced in both species following sleep restriction, and recovered to near baseline levels after sleep restriction (P < 0.05, false-discovery rate < 0.2). Elevated phospholipids were also noted after sleep restriction in both species, as well as metabolites associated with an oxidizing environment. In addition, polar metabolites reflective of neurotransmitters, vitamin B3, and gut metabolism were elevated in sleep-restricted humans. These results are consistent with induction of peroxisome proliferator-activated receptors and disruptions of the circadian clock. The findings provide a potential link between known pathologies of reduced sleep duration and metabolic dysfunction, and potential biomarkers for sleep loss.

Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Akt-mediated foxo1 inhibition is required for liver regeneration.

UNLABELLED: Understanding the hepatic regenerative process has clinical interest as the effectiveness of many treatments for chronic liver diseases is conditioned by efficient liver regeneration. Experimental evidence points to the need for a temporal coordination between cytokines, growth factors, and metabolic signaling pathways to enable successful liver regeneration. One intracellular mediator that acts as a signal integration node for these processes is the serine-threonine kinase Akt/protein kinase B (Akt). To investigate the contribution of Akt during hepatic regeneration, we performed partial hepatectomy in mice lacking Akt1, Akt2, or both isoforms. We found that absence of Akt1 or Akt2 does not influence liver regeneration after partial hepatectomy. However, hepatic-specific Akt1 and Akt2 null mice show impaired liver regeneration and increased mortality. The major abnormal cellular events observed in total Akt-deficient livers were a marked reduction in cell proliferation, cell hypertrophy, glycogenesis, and lipid droplet formation. Most importantly, liver-specific deletion of FoxO1, a transcription factor regulated by Akt, rescued the hepatic regenerative capability in Akt1-deficient and Akt2-deficient mice and normalized the cellular events associated with liver regeneration. CONCLUSION: The Akt-FoxO1 signaling pathway plays an essential role during liver regeneration.

Proteolytic Cleavage of AMPKα and Intracellular MMP9 Expression Are Both Required for TLR4-Mediated mTORC1 Activation and HIF-1α Expression in Leukocytes.

LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPKα is cleaved and its relevance to human health. Our data indicate that AMPKα cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1α expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.

Glucagon: acute actions on hepatic metabolism.

Type 2 diabetes mellitus is the result of impaired systemic control of glucose homeostasis, in part through the dysregulation of the hormone glucagon. Glucagon acts on the liver to increase glucose production through alterations in hepatic metabolism, and reducing the elevated glucagon signalling in diabetic patients is an attractive strategy for the treatment of hyperglycaemia. Here we review the actions of the hormone in the liver, focusing on the acute alterations of metabolic pathways. This review summarises a presentation given at the 'Novel data on glucagon' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Mona Abraham and Tony Lam, DOI: 10.1007/s00125-016-3950-3 , and by Young Lee and colleagues, DOI: 10.1007/s00125-016-3965-9 ) and an overview by the Session Chair, Isabel Valverde (DOI: 10.1007/s00125-016-3946-z ).

The role of mouse Akt2 in insulin-dependent suppression of adipocyte lipolysis in vivo.

AIM/HYPOTHESIS: The release of fatty acids from adipocytes, i.e. lipolysis, is maintained under tight control, primarily by the opposing actions of catecholamines and insulin. A widely accepted model is that insulin antagonises catecholamine-dependent lipolysis through phosphorylation and activation of cAMP phosphodiesterase 3B (PDE3B) by the serine-threonine protein kinase Akt (protein kinase B). Recently, this hypothesis has been challenged, as in cultured adipocytes insulin appears, under some conditions, to suppress lipolysis independently of Akt. METHODS: To address the requirement for Akt2, the predominant isoform expressed in classic insulin target tissues, in the suppression of fatty acid release in vivo, we assessed lipolysis in mice lacking Akt2. RESULTS: In the fed state and following an oral glucose challenge, Akt2 null mice were glucose intolerant and hyperinsulinaemic, but nonetheless exhibited normal serum NEFA and glycerol levels, suggestive of normal suppression of lipolysis. Furthermore, insulin partially inhibited lipolysis in Akt2 null mice during an insulin tolerance test (ITT) and hyperinsulinaemic-euglycaemic clamp, respectively. In support of these in vivo observations, insulin antagonised catecholamine-induced lipolysis in primary brown fat adipocytes from Akt2-deficient mice. CONCLUSIONS/INTERPRETATION: These data suggest that suppression of lipolysis by insulin in hyperinsulinaemic states can take place in the absence of Akt2.

Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule.

Construction of human activity-based phosphorylation networks.

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.