RESUMEN
Walter Cannon was a highly regarded American neurologist and physiologist with extremely broad interests. In the tradition of Cannon and his broad interests, we discuss our laboratory's multifaceted work in signal transduction over the past 40+ years. We show how our questioning of how growth hormone (GH) in the blood communicates with cells throughout the body to promote body growth and regulate body metabolism led to insight into not only body height but also important regulators of malignancy and body weight. Highlights include finding that 1) A critical initiating step in GH signal transduction is GH activating the GH receptor-associated tyrosine kinase JAK2; 2) GH activation of JAK2 leads to activation of a number of signaling proteins, including STAT transcription factors; 3) JAK2 is autophosphorylated on multiple tyrosines that regulate the activity of JAK2 and recruit signaling proteins to GH/GH receptor/JAK2 complexes; 4) Constitutively activated STAT proteins are associated with cancer; 5) GH activation of JAK2 recruits the adapter protein SH2B1 to GH/GH receptor/JAK2 complexes where it facilitates GH regulation of the actin cytoskeleton and motility; and 6) SH2B1 is recruited to other receptors in the brain, where it enhances satiety, most likely in part by regulating leptin action and neuronal connections of appetite-regulating neurons. These findings have led to increased understanding of how GH functions, as well as therapeutic interventions for certain cancer and obese individuals, thereby reinforcing the great importance of supporting basic research since one never knows ahead of time what important insight it can provide.
Asunto(s)
Hormona de Crecimiento Humana , Neoplasias , Humanos , Hormona del Crecimiento/metabolismo , Transducción de Señal/fisiología , Janus Quinasa 2/metabolismo , Hormona de Crecimiento Humana/metabolismo , Receptores de Somatotropina/metabolismo , Fosforilación , Obesidad , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance-phenotypes mimicked by mice lacking Sh2b1. SH2B1ß and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1ß driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1ß in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the ß and γ isoforms from the neurons of mice (SH2B1ßγ neuron-specific knockout [NKO] mice) or throughout the body (SH2B1ßγ knockout [KO] mice). While parameters of energy balance were normal in both male and female SH2B1ßγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1ßγ 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 ß and γ isoforms. Our work suggests that neuronal SH2B1ß and γ are not primary regulators of energy balance. Rather, non-neuronal SH2B1ß and γ in combination with neuronal SH2B1α and δ suffice for body weight maintenance. While SH2B1ß/γ and SH2B1α/δ share some functionality, SH2B1ß/γ appears to play a larger role in promoting leanness.
Asunto(s)
Obesidad Infantil , Ratones , Masculino , Niño , Humanos , Animales , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Neuronas/metabolismo , Peso Corporal , Ratones Noqueados , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
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. Surprisingly, in contrast to SH2B1α, SH2B1ß and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neuronas/citología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Ratones , Neuritas/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Mice lacking SH2B1 and humans with variants of SH2B1 display severe obesity and insulin resistance. SH2B1 is an adapter protein that is recruited to the receptors of multiple hormones and neurotrophic factors. Of the four known alternatively spliced SH2B1 isoforms, SH2B1ß and SH2B1γ exhibit ubiquitous expression, whereas SH2B1α and SH2B1δ are essentially restricted to the brain. To understand the roles for SH2B1α and SH2B1δ in energy balance and glucose metabolism, we generated mice lacking these brain-specific isoforms (αδ knockout [αδKO] mice). αδKO mice exhibit decreased food intake, protection from weight gain on standard and high-fat diets, and an adiposity-dependent improvement in glucose homeostasis. SH2B1 has been suggested to impact energy balance via the modulation of leptin action. However, αδKO mice exhibit leptin sensitivity that is similar to that of wild-type mice by multiple measures. Thus, decreasing the abundance of SH2B1α and/or SH2B1δ relative to the other SH2B1 isoforms likely shifts energy balance toward a lean phenotype via a primarily leptin-independent mechanism. Our findings suggest that the different alternatively spliced isoforms of SH2B1 perform different functions in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Encéfalo/metabolismo , Obesidad/genética , Isoformas de Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Resistencia a la Insulina/genética , Leptina/farmacología , Ratones , Ratones Noqueados , Obesidad/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1's function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Adiposidad/genética , Metabolismo Energético/genética , Resistencia a la Insulina/genética , Obesidad Infantil/genética , Dominios Homólogos a Pleckstrina/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Animales , Niño , Preescolar , Femenino , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Homeostasis/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Obesidad Infantil/metabolismoRESUMEN
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 ß 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 ß isoforms modulate SH2B1 function. We compared the actions of SH2B1α and SH2B1ß to those of the N-terminal 631 amino acids shared by both isoforms. In contrast to the ß 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 phospholipase C-gamma (PLC-γ), 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 SH2B1ß. Finally, coexpression of SH2B1α but not SH2B1α with a mutation of Y to F at position 753 (Y753F) inhibited the ability of SH2B1ß to enhance neurite outgrowth. These results suggest that the C-terminal tails of SH2B1 isoforms are key determinants of the cellular role of SH2B1. Furthermore, the function of SH2B1α is regulated by phosphorylation of the α tail.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Animales , Diferenciación Celular/fisiología , Células HEK293 , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neuritas , Células PC12 , Fosforilación , Dominios Proteicos , Isoformas de Proteínas , Ratas , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de SeñalRESUMEN
Over 20years 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 α, 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 SH2B1ß 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 GH-dependent 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.
Asunto(s)
Hormona del Crecimiento/metabolismo , Janus Quinasa 2/metabolismo , Receptores de Somatotropina/metabolismo , Citoesqueleto de Actina , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Movimiento Celular/genética , Humanos , Resistencia a la Insulina/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Obesidad Infantil/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de SeñalRESUMEN
We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1ß enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1α. SH2B1α variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1α, like SH2B1ß, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1α enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1α to enhance GH-induced cell motility. In contrast to SH2B1ß, SH2B1α does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1ß may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Obesidad/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Adulto , Empalme Alternativo , Niño , Femenino , Humanos , Insulina/metabolismo , Leptina/metabolismo , Masculino , Mutación Missense , Obesidad/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
The tyrosine kinase Janus kinase 2 (JAK2) is activated by many cytokine receptors, including receptors for GH, leptin, and erythropoietin. However, very few proteins have been identified as binding partners for JAK2. Using a yeast 2-hybrid screen, we identified steroid-sensitive gene-1 (SSG1)/coiled-coil domain-containing protein 80 (Ccdc80) as a JAK2-binding partner. We demonstrate that Ccdc80 preferentially binds activated, tyrosyl-phosphorylated JAK2 but not kinase-inactive JAK2 (K882E) in both yeast and mammalian systems. Ccdc80 is tyrosyl phosphorylated in the presence of JAK2. The binding of Ccdc80 to JAK2 occurs via 1 or more of the 3 DUDES/SRPX (DRO1-URB-DRS-Equarin-SRPUL/sushi repeat containing protein, x-linked) domain 5 domains of Ccdc80. Mutagenesis of the second DUDES domain suggests that the N-terminal third of the DUDES domain is sufficient for JAK2 binding. Ccdc80 does not alter the kinase activity of JAK2. However, Ccdc80 increases GH-dependent phosphorylation of Stat (signal transducer and activator of transcription) 5b on Tyr699 and substantially enhances both basal and GH-dependent phosphorylation/activation of Stat3 on Tyr705. Furthermore, Ccdc80 belongs to the group of proteins that function both in the intracellular compartment and are secreted. Secreted Ccdc80 associates with the extracellular matrix and is also found in the medium. A substantial portion of the Ccdc80 detected in the medium is cleaved. Finally, consistent with the DUDES domain serving as a JAK2-binding domain, we also demonstrate that another protein that contains a DUDES domain, SRPX2, binds preferentially to the activated tyrosyl-phosphorylated form of JAK2.
Asunto(s)
Janus Quinasa 2/metabolismo , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Activación Enzimática , Matriz Extracelular/metabolismo , Humanos , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Factores de Transcripción STAT , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos HíbridosRESUMEN
Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1ß to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicating SH2B1ß in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1ß releases SH2B1ß from the plasma membrane. Here, we examined the role of SH2B1ß in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cultures of peritoneal and bone marrow-derived macrophages. SH2B1ß overexpression enhances, whereas SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1ß-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1ß are phosphorylated. Mutating these tyrosines in SH2B1ß decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1ß or creating the phosphomimetics SH2B1ß(S161E) or SH2B1ß(S165E), all of which release SH2B1ß from the plasma membrane, enhances macrophage motility. Conversely, SH2B1ß(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serine residues does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1ß. Mutating tyrosines 439 and 494 does not affect localization of SH2B1ß at the plasma membrane or movement of SH2B1ß into focal adhesions. Taken together, these results suggest that SH2B1ß enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1ß on tyrosines 439 and 494 and movement of SH2B1ß out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Hormona del Crecimiento/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Animales , Línea Celular , Movimiento Celular/fisiología , Adhesiones Focales/metabolismo , Ratones , FosforilaciónRESUMEN
Src homology 2 B adapter protein 1 (SH2B1) modulates signaling by a variety of ligands that bind to receptor tyrosine kinases or JAK-associated cytokine receptors, including leptin, insulin, growth hormone (GH), and nerve growth factor (NGF). Targeted deletion of Sh2b1 in mice results in increased food intake, obesity, and insulin resistance, with an intermediate phenotype seen in heterozygous null mice on a high-fat diet. We identified SH2B1 loss-of-function mutations in a large cohort of patients with severe early-onset obesity. Mutation carriers exhibited hyperphagia, childhood-onset obesity, disproportionate insulin resistance, and reduced final height as adults. Unexpectedly, mutation carriers exhibited a spectrum of behavioral abnormalities that were not reported in controls, including social isolation and aggression. We conclude that SH2B1 plays a critical role in the control of human food intake and body weight and is implicated in maladaptive human behavior.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Mutación del Sistema de Lectura , Mutación Missense , Obesidad/genética , Adolescente , Adulto , Agresión , Secuencia de Bases , Estudios de Casos y Controles , Movimiento Celular , Niño , Preescolar , Análisis Mutacional de ADN , Ingestión de Energía/genética , Femenino , Estudios de Asociación Genética , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Transporte de Proteínas , Aislamiento Social , Adulto JovenRESUMEN
GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na(+)/H(+) exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly broadens our thinking of GH-regulated cell functions.
Asunto(s)
Hormona del Crecimiento/fisiología , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Células 3T3 , Secuencias de Aminoácidos , Animales , Cromatografía por Intercambio Iónico , Secuencia de Consenso , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Proteoma/química , Proteoma/aislamiento & purificación , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Somatotropina/metabolismo , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
The adaptor protein SH2B1ß participates in regulation of the actin cytoskeleton during processes such as cell migration and differentiation. Here, we identify SH2B1ß as a new focal adhesion protein. We provide evidence that SH2B1ß is phosphorylated in response to phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation and show that PMA induces a rapid redistribution of SH2B1ß out of focal adhesions. We also show that growth hormone (GH) increases cycling of SH2B1ß into and out of focal adhesions. Ser161 and Ser165 in SH2B1ß fall within consensus PKC substrate motifs. Mutating these two serine residues into alanine residues abrogates PMA-induced redistribution of SH2B1ß out of focal adhesions, decreases SH2B1ß cycling into and out of focal adhesions in control and GH-stimulated cells, and increases the size of focal adhesions. By contrast, mutating Ser165 into a glutamate residue decreases the amount of SH2B1ß in focal adhesions and increases the number of focal adhesions per cell. These results suggest that activation of PKC regulates SH2B1ß focal adhesion localization through phosphorylation of Ser161 and/or Ser165. The finding that phosphorylation of SH2B1ß increases the number of focal adhesions suggests a mechanism for the stimulatory effect on cell motility of SH2B1ß.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular/metabolismo , Adhesiones Focales/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Movimiento Celular , Citoesqueleto/metabolismo , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/patología , Hormona del Crecimiento/farmacología , Ratones , Mutagénesis Sitio-Dirigida , Mutación/genética , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Serina/genética , Transducción de Señal/efectos de los fármacos , Ciclo del Sustrato/efectos de los fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/metabolismoAsunto(s)
Quinasa de la Caseína II/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Trastornos Mieloproliferativos/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/fisiología , Quinasa de la Caseína II/antagonistas & inhibidores , Humanos , Trastornos Mieloproliferativos/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
An intriguing question in cell biology is what targets proteins to, and regulates their translocation between, specific cellular locations. Here we report that the polybasic nuclear localization sequence (NLS) required for nuclear entry of the adapter protein and candidate human obesity gene product SH2B1ß, also localizes SH2B1ß to the plasma membrane (PM), most probably via electrostatic interactions. Binding of SH2B1ß to the PM also requires its dimerization domain. Phosphorylation of serine residues near this polybasic region, potentially by protein kinase C, releases SH2B1ß from the PM and enhances nuclear entry. Release of SH2B1ß from the PM and/or nuclear entry appear to be required for SH2B1ß enhancement of nerve growth factor (NGF)-induced expression of urokinase plasminogen activator receptor gene and neurite outgrowth of PC12 cells. Taken together, our results provide strong evidence that the polybasic NLS region of SH2B1 serves the dual function of localizing SH2B1 to both the nucleus and the PM, the latter most probably through electrostatic interactions that are enhanced by SH2B1ß dimerization. Cycling between the different cellular compartments is a consequence of the phosphorylation and dephosphorylation of serine residues near the NLS and is important for physiological effects of SH2B1, including NGF-induced gene expression and neurite outgrowth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Inmunoprecipitación , Espectrometría de Masas , Ratones , Células PC12 , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Ratas , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1beta (SH2B1beta), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e.g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation.
Asunto(s)
Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Tirosina/metabolismo , Animales , Western Blotting , Línea Celular , Hormona del Crecimiento/metabolismo , Humanos , Inmunoprecipitación , Janus Quinasa 2/genética , Ratones , Mutagénesis Sitio-Dirigida , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Transducción de Señal/genética , Transducción de Señal/fisiología , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Tirosina/químicaRESUMEN
Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr(317) in the FERM domain and Tyr(637) in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr(317) promotes increased Jak2 activity, and the phosphorylation of Tyr(317) during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr(317) in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr(317) in the attenuation of Jak2 signaling. In contrast, mutation of Tyr(637) decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr(637) phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr(317) and Tyr(637) act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.
Asunto(s)
Citocinas/metabolismo , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Retroalimentación Fisiológica , Humanos , Janus Quinasa 2/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem , Tirosina/químicaRESUMEN
OBJECTIVE: Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure. RESEARCH DESIGN AND METHODS: Kidney biopsies were obtained from 74 patients (control subjects, early and progressive type 2 diabetic nephropathy). Glomerular and tubulointerstitial mRNAs were microarrayed, followed by bioinformatics analyses. Gene expression changes were confirmed by real-time RT-PCR and immunohistological staining. Samples from db/db C57BLKS and streptozotocin-induced DBA/2J mice, commonly studied murine models of diabetic nephropathy, were analyzed. RESULTS: In human glomeruli and tubulointerstitial samples, the Janus kinase (Jak)-signal transducer and activator of transcription (Stat) pathway was highly and significantly regulated. Jak-1, -2, and -3 as well as Stat-1 and -3 were expressed at higher levels in patients with diabetic nephropathy than in control subjects. The estimated glomerular filtration rate significantly correlated with tubulointerstitial Jak-1, -2, and -3 and Stat-1 expression (R(2) = 0.30-0.44). Immunohistochemistry found strong Jak-2 staining in glomerular and tubulointerstitial compartments in diabetic nephropathy compared with control subjects. In contrast, there was little or no increase in expression of Jak/Stat genes in the db/db C57BLKS or diabetic DBA/2J mice. CONCLUSIONS: These data suggest a direct relationship between tubulointerstitial Jak/Stat expression and progression of kidney failure in patients with type 2 diabetic nephropathy and distinguish progressive human diabetic nephropathy from nonprogressive murine diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Expresión Génica , Adulto , Animales , Western Blotting , Femenino , Humanos , Inmunohistoquímica , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Janus Quinasa 3/genética , Janus Quinasa 3/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/farmacología , Fosfoserina/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Células 3T3 , Alanina/genética , Animales , Butadienos/farmacología , Células COS , Células Cultivadas , Chlorocebus aethiops , Activación Enzimática/efectos de los fármacos , Humanos , Janus Quinasa 2 , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Espectrometría de Masas , Ratones , Mutación/genética , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Factor de Transcripción STAT5/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
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-Bbeta (SH2-Bbeta (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-Bbeta (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.