Papers by Lawrence Argetsinger
Endocrinology, Feb 17, 2023
Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperph... more Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance—phenotypes mimicked by mice lacking Sh2b1. SH2B1β and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1β driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1β in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the β and γ isoforms from the neurons of mice (SH2B1βγ neuron-specific knockout [NKO] mice) or throughout the body (SH2B1βγ knockout [KO] mice). While parameters of energy balance were normal in both male and female SH2B1βγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1βγ KO mice. Analysis of long-read single-cell RNA seq data from wild-type mouse brain revealed that neurons express almost exclusively the α and δ isoforms, whereas neuroglial cells express almost exclusively the β and γ isoforms. Our work suggests that neuronal SH2B1β and γ are not primary regulators of energy balance. Rather, non-neuronal SH2B1β and γ in combination with neuronal SH2B1α and δ suffice for body weight maintenance. While SH2B1β/γ and SH2B1α/δ share some functionality, SH2B1β/γ appears to play a larger role in promoting leanness.
When the catalytically active, tyrosyl-phosphorylated form of insulin receptor was isolated from ... more When the catalytically active, tyrosyl-phosphorylated form of insulin receptor was isolated from human placenta and treated with ADP, only partial dephosphorylation was observed. This observation suggests the existence of two distinct classes of phosphotyrosyl residues of the phosphorylated insulin receptor: one in which the phosphoryl groups undergo reversible transfer to ADP and one in which they do not. There were 8.8 +/- 2.2 phosphorylation sites per tetrameric insulin receptor, of which 2.4 +/- 0.5 were irreversible (mean +/- S.D., n = 6). The reversible sites were determined to be equivalent and noninteracting and to have an equilibrium constant for the transfer of a phosphoryl group from ATP to a site of reversible phosphorylation of 8.7. Surprisingly, both phosphorylation and dephosphorylation at the reversible sites were relatively insensitive to the presence of insulin. Since only minimal autophosphorylation of insulin receptor was detected in the absence of insulin, the results suggest that the incorporation of phosphate into the two to three irreversible phosphorylation sites is insulin dependent. Phosphorylation of the irreversible phosphorylation sites then renders the insulin receptor relatively insensitive to the continued presence of insulin and facilitates rapid reversible phosphorylation of a second group of tyrosyl residues. The dependence of the degree of phosphorylation of insulin receptor on the ATP:ADP ratio may provide a mechanism for modulating the cellular response to insulin.
Journal of Biological Chemistry, Nov 1, 1992
Cellular Signalling, May 1, 2004
During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whos... more During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whose products are destined for the synapse, is suppressed by muscle activity. One of the best-studied examples of activity-dependent gene regulation in muscle are those encoding nicotinic acetylcholine receptor (nAChR) subunits. We recently showed that nAChR gene expression is inhibited by calcium/calmodulin-dependent protein kinase II (CaMKII) and CaMKII inhibitors block activity-dependent suppression of these genes. Here we report results investigating the mechanism by which CaMKII suppresses nAChR gene expression. We show that the muscle helix-loop-helix transcription factor, myogenin, is necessary for activity-dependent control of nAChR gene expression in cultured rat myotubes and is a substrate for CaMKII both in vitro and in vivo. CaMKII phosphorylation of myogenin is induced by muscle activity and this phosphorylation influences DNA binding and transactivation. Thus we have identified a signaling mechanism by which muscle activity controls nAChR gene expression in developing muscle.
Growth hormone & IGF research, Jun 1, 2016
Over 20 years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor... more Over 20 years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein , a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1 is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GHdependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight.
Journal of Animal Science, 1997
Page 1. Molecular Mechanisms of Growth Hormone Action I Christin Carter-Su 2, Anthony PJ King, Li... more Page 1. Molecular Mechanisms of Growth Hormone Action I Christin Carter-Su 2, Anthony PJ King, Lisa S. Smit, Joyce A. VanderKuur, Lawrence S. Argetsinger, George S. Campbell, and Wei-hua Huo Department of Physiology ...
Journal of Biological Chemistry, Apr 1, 2003
SH2-B binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent ... more SH2-B binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-B may act as an adapter protein to recruit downstream signaling proteins to kinase⅐SH2-B complexes. SH2-B is tyrosyl-phosphorylated in response to GH and interferon-␥, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-B, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-B as targets of JAK2 both in vitro and in intact cells. SH2-B lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-B and that PDGF receptor phosphorylates SH2-B on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-B may provide a binding site for one or more proteins linking cytokine receptor⅐JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.
The Endocrine Society's 93rd Annual Meeting & Expo, June 4–7, 2011 - Boston, Jun 1, 2011
The FASEB Journal, Apr 1, 2008
Biochimica Et Biophysica Acta - Proteins And Proteomics, Mar 1, 1990
A procedure has been developed for the isolation of a catalytically competent phosphorylated tyro... more A procedure has been developed for the isolation of a catalytically competent phosphorylated tyrosine kinase (RSV Y-kinase) from avian sarcoma virus-induced rat tumors. The procedure involves reaction of partially purified RSV Y-kinase with ATP to effect tyrosyl phosphorylation of catalytically competent RSV Y-kinase. Tyrosyl phosphorylated RSV Y-kinase was isolated from the heterogeneous reaction mixture by immunoadsorption on immobilized phosphotyrosyl binding antibodies and elution with the hapten p-nitrophenyl phosphate. Estimation of the phosphate content of the purified phosphorylated RSV Y-kinase indicated that 1-3 tyrosyl groups had been phosphorylated upon reaction with ATP. The specific activity toward histone 2B of the purified phosphorylated RSV Y-kinase was at least 30-fold greater than that estimated for the RSV Y-kinase prepared previously by immunoadsorption on immobilized antiserum from tumor bearing rabbits.
Journal of Cell Science, 2022
The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as N... more The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α–SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1−/− [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisin...
Isolation of a catalytically competent phosphorylated tyrosine kinase from Rous sarcoma virus-ind... more Isolation of a catalytically competent phosphorylated tyrosine kinase from Rous sarcoma virus-induced rat tumor by immunoadsorption to and hapten elution from phosphotyrosine binding antibodies
Molecular and cellular biology, Jan 11, 2017
The scaffold protein SH2B1, a major regulator of body weight, is recruited to the receptors of mu... more The scaffold protein SH2B1, a major regulator of body weight, is recruited to the receptors of multiple cytokines and growth factors, including nerve growth factor (NGF). The β isoform, but not the α isoform, of SH2B1 greatly enhances NGF-dependent neurite outgrowth of PC12 cells. Here we asked how the unique C-terminal tails of the α and β isoforms modulate SH2B1 function. We compared the actions of SH2B1α and SH2B1β to those of the N-terminal 631 amino acids shared by both isoforms. In contrast to the β-tail, the α-tail inhibited the ability of SH2B1 to both cycle through the nucleus and enhance NGF-mediated neurite outgrowth, gene expression, phosphorylation of Akt and PLCγ and autophosphorylation of the NGF receptor TrkA. These functions were restored when Tyr753 in the α-tail was mutated to phenylalanine. We provide evidence that TrkA phosphorylates Tyr753 in SH2B1α, as well as tyrosines 439 and 55 in both SH2B1α and SH2B1β. Finally, co-expression of SH2B1α, but not SH2B1α Y753...
Journal of Clinical Investigation, 2013
Journal of the Endocrine Society, Apr 1, 2019
Abstract The scaffold protein SH2B1 plays a critical role in the regulation of energy balance and... more Abstract The scaffold protein SH2B1 plays a critical role in the regulation of energy balance and glucose homeostasis—humans with mutations in SH2B1 are obese and diabetic, as are SH2B1KO mice. The 4 known isoforms of SH2B1 (α, β, γ, δ), which differ only in their C-terminal tails, have distinct expression profiles: β and γ are ubiquitously expressed whereas α and δ are expressed almost exclusively in brain. Some human obesity-associated mutations of SH2B1 are specific to the α and δ isoforms. However, the contributions of the latter isoforms to the regulation of energy balance are not understood. Knowing that the brain is a major coordinator of energy homeostasis, we hypothesized that the brain-specific α and δ isoforms of SH2B1 would be critical players in the regulation of energy balance. To investigate the potential contribution of these isoforms to the control of energy balance, we used CRISPR-Cas9 to delete SH2B1 α and δ in mice (SH2B1αδKO). On a standard chow diet, SH2B1αδKO mice exhibit decreased body weight, fat content, and circulating leptin levels, as well as increased lean content for their body weight. They are hypophagic yet have normal energy expenditure, suggesting that their decreased body weight and fat mass result from reduced food intake. To determine whether deletion of these isoforms could protect against extreme weight gain, we challenged SH2B1αδKO mice with a 60% high fat diet. SH2B1αδKO mice are protected against the high fat diet: compared to WT littermates, they weigh less, have decreased fat mass and leptin levels, are leaner for their body weight, and are protected against impairments to glucose control. Because SH2B1 is recruited to the activated leptin receptor/JAK2 complex, we tested whether deletion of the α and δ isoforms of SH2B1 enhance leptin sensitivity. SH2B1αδKO mice exhibit normal expression levels of hypothalamic genes that are associated with leptin-dependent regulation of feeding behavior, as well as normal sensitivity to leptin in vivo. Together these data suggest that the α and/or δ isoforms of SH2B1, acting in a leptin-independent manner, are potent regulators of energy balance. To explain the lean phenotype of SH2B1αδKO mice, we considered the unique actions of these isoforms. At the cellular level, the β, γ, and δ isoforms enhance neurite outgrowth and/or neurotrophic factor-dependent gene expression in neuron-like PC12 cells. In contrast, SH2B1α does not enhance these actions, and even inhibits the ability of SH2B1β to enhance these activities. We propose that the removal of SH2B1α in SH2B1αδKO mice enables the remaining β and/or γ isoforms to enhance the function of neurons that contribute to the regulation of energy balance, as they are now free from SH2B1α’s inhibitory influence. Funding: NIH R01, NSF Graduate Research Fellowship
The Endocrine Society's 93rd Annual Meeting & Expo, June 4–7, 2011 - Boston, 2011
Analytical Chemistry, 2005
Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophor... more Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-B (SH2-B (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K d) 82 (7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-B (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K d (101 (12 nM in good agreement with FA measurements) and dissociation rate (k off) 0.95 (0.02 s-1 corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K d or k off ; however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics. Cellular chemistry is controlled by affinity interactions between biomolecules. Quantitative analysis of such interactions is important in developing an understanding of how reactions are organized within cells and for developing drugs or chemical probes of cellular processes. A variety of affinity methods based on
Endocrinology
Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperph... more Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance - phenotypes mimicked by mice lacking Sh2b1. SH2B1β and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1β driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1β in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the β and γ isoforms from the neurons of mice (SH2B1βγ NKO mice) or throughout the body (SH2B1βγ KO mice). While parameters of energy balance were normal in both male and female SH2B1βγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1βγ KO mice. Analysis of long-read single cell RNA seq data from wild-type mouse brain revealed that neurons express almost exclusively the α and δ isoforms, ...
Journal of Clinical Investigation, 2013
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Papers by Lawrence Argetsinger