scholarly journals Caveolin-1 Interacts with the Insulin Receptor and Can Differentially Modulate Insulin Signaling in Transfected Cos-7 Cells and Rat Adipose Cells

1999 ◽  
Vol 13 (12) ◽  
pp. 2013-2024 ◽  
Author(s):  
Fredrik H. Nystrom ◽  
Hui Chen ◽  
Li-Na Cong ◽  
Yunhua Li ◽  
Michael J. Quon
Keyword(s):  
Insulin Receptor ◽  
Insulin Signaling ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Binding Motif ◽  
Intact Cells ◽  
Caveolin 1 ◽  
Mitogen Activated Protein ◽  
Adipose Cells

Abstract Caveolae may function as microdomains for signaling that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a major component of caveolae, contains a scaffolding domain (SD) that binds to a caveolin-1 binding motif in the kinase domain of the IR in vitro. To investigate the potential role of caveolin-1 in insulin signaling we overexpressed wild-type (Cav-WT) or mutant (Cav-Mut; F92A/V94A in SD) caveolin-1 in either Cos-7 cells cotransfected with IR or rat adipose cells (low and high levels of endogenous caveolin-1, respectively). Cav-WT coimmunoprecipitated with the IR to a much greater extent than Cav-Mut, suggesting that the SD is important for interactions between caveolin-1 and the IR in intact cells. We also constructed several IR mutants with a disrupted caveolin-1 binding motif and found that these mutants were poorly expressed and did not undergo autophosphorylation. Interestingly, overexpression of Cav-WT in Cos-7 cells significantly enhanced insulin-stimulated phosphorylation of Elk-1 (a mitogen-activated protein kinase-dependent pathway) while overexpression of Cav-Mut was without effect. In contrast, in adipose cells, overexpression of either Cav-WT or Cav-Mut did not affect insulin-stimulated phosphorylation of a cotransfected ERK2 (but did significantly inhibit basal phosphorylation of ERK2). Furthermore, we also observed a small inhibition of insulin-stimulated translocation of GLUT4 when either Cav-WT or Cav-Mut was overexpressed in adipose cells. Thus, interaction of caveolin-1 with IRs may differentially modulate insulin signaling to enhance insulin action in Cos-7 cells but inhibit insulin’s effects in adipose cells.

Exercise-associated differences in an array of proteins involved in signal transduction and glucose transport

2001 ◽  
Vol 90 (1) ◽  
pp. 29-34 ◽  
Author(s):  
Mei Yu ◽  
Eva Blomstrand ◽  
Alexander V. Chibalin ◽  
Harriet Wallberg-Henriksson ◽  
Juleen R. Zierath ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Insulin Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Signaling Cascade ◽  
Vastus Lateralis Muscle ◽  
Glut 4 ◽  
Mitogen Activated Protein ◽  
Kinase Expression

Vastus lateralis muscle biopsies were obtained from endurance-trained (running ∼50 km/wk) and untrained (no regular physical exercise) men, and the expression of an array of insulin-signaling intermediates was determined. Expression of insulin receptor and insulin receptor substrate-1 and -2 was decreased 44% ( P < 0.05), 57% ( P < 0.001), and 77% ( P < 0.001), respectively, in trained vs. untrained muscle. The downstream signaling target, Akt kinase, was not altered in trained subjects. Components of the mitogenic signaling cascade were also assessed. Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase expression was 190% greater ( P < 0.05), whereas p38 mitogen-activated protein kinase expression was 32% lower ( P < 0.05), in trained vs. untrained muscle. GLUT-4 protein expression was twofold higher ( P < 0.05), and the GLUT-4 vesicle-associated protein, the insulin-regulated aminopeptidase, was increased 4.7-fold ( P < 0.05) in trained muscle. In conclusion, the expression of proteins involved in signal transduction is altered in skeletal muscle from well-trained athletes. Downregulation of early components of the insulin-signaling cascade may occur in response to increased insulin sensitivity associated with endurance training.

scholarly journals MSK1 activity is controlled by multiple phosphorylation sites

2005 ◽  
Vol 387 (2) ◽  
pp. 507-517 ◽  
Author(s):  
Claire E. McCOY ◽  
David G. CAMPBELL ◽  
Maria DEAK ◽  
Graham B. BLOOMBERG ◽  
J. Simon C. ARTHUR
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase ◽  
In Cells

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

scholarly journals Stepwise autophosphorylation regulates biased agonism of the insulin receptor

2019 ◽  
Author(s):  
Na-Oh Yunn ◽  
Mangeun Park ◽  
Jungeun Noh ◽  
Sung Ho Ryu
Keyword(s):  
Insulin Receptor ◽  
Mapk Pathway ◽  
Insulin Binding ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Metabolic Effects ◽  
Intermediate Step ◽  
Peripheral Tissues ◽  
Biased Agonism ◽  
Mitogen Activated Protein

SummaryInsulin is a key regulator of energy metabolism in peripheral tissues but also functions as a growth factor. Insulin binding to the insulin receptor (IR) leads to autophosphorylation of intracellular tyrosine residues, which simultaneously initiates a multitude of signals and functions. In contrast, some artificial (non-insulin) ligands for the IR result in biased agonism, selectively activating the PI3K/AKT pathway and metabolic effects without activating mitogen-activated protein kinase (MAPK) pathway and mitogenic effects. However, the precise mechanism of biased agonism at the receptor level remains unclear. The biased agonist IR-A48 aptamer selectively induces mono-phosphorylation of Tyr1150 residue (m-pY1150) in the kinase domain of IR. Hence, we hypothesized that IR autophosphorylation is a stepwise process in which formation of m-pY1150 represents an intermediate step. To explore this idea, we used hybrid receptors in which insulin can bind only one of the two binding sites of the dimeric receptor. Asymmetric insulin binding selectively induced symmetric m-pY1150 in both kinase domains. Moreover, the juxtamembrane domain, which interacts with the kinase domain, restricted the full activation of IR, and symmetric m-pY1150 played a crucial role in the rearrangement of intracellular domains to release this restriction. Our findings demonstrate that the symmetry of insulin binding to a dimeric receptor determines the stepwise autophosphorylation of IR. Furthermore, considering that the degree of ligand symmetry on a receptor depends mainly on ligand concentration, our results suggest that IR may play metabolic-biased roles in peripheral tissues dependent on local insulin concentrations.

scholarly journals c-Jun N-terminal kinase–mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks

2012 ◽  
Vol 23 (10) ◽  
pp. 1943-1954 ◽  
Author(s):  
Laura R. Barkley ◽  
Komaraiah Palle ◽  
Michael Durando ◽  
Tovah A. Day ◽  
Aditi Gurkar ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ectopic Expression ◽  
Mitogen Activated Protein Kinase ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Dependent Manner ◽  
Replication Forks ◽  
Mitogen Activated Protein ◽  
Stalled Replication Forks

The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1–dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage–independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη−PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.

scholarly journals Direct Activation of Mitogen-Activated Protein Kinase Kinase Kinase MEKK1 by the Ste20p Homologue GCK and the Adapter Protein TRAF2

2002 ◽  
Vol 22 (3) ◽  
pp. 737-749 ◽  
Author(s):  
Deborah N. Chadee ◽  
Takashi Yuasa ◽  
John M. Kyriakis
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Adapter Protein ◽  
Vitro Assay ◽  
Mapk Kinase ◽  
Surface Receptors ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) → MAPK kinase (MEK) → MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.

scholarly journals Mechanistic Studies of the Mitotic Activation of Mos

2006 ◽  
Vol 26 (14) ◽  
pp. 5300-5309 ◽  
Author(s):  
Jianbo Yue ◽  
James E. Ferrell
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Tyrosine Kinases ◽  
Spindle Assembly Checkpoint ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Egg Extracts ◽  
Mitogen Activated Protein

ABSTRACT The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2β. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix αC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix αC.

scholarly journals Mitogen-activated Protein Kinase (MAPK) Phosphatase 3-mediated Cross-talk between MAPKs ERK2 and p38α

2011 ◽  
Vol 286 (18) ◽  
pp. 16150-16162 ◽  
Author(s):  
Yuan-Yuan Zhang ◽  
Jia-Wei Wu ◽  
Zhi-Xin Wang
Keyword(s):  
Cross Talk ◽  
Molecular Basis ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
Intact Cells ◽  
Catalytic Constant ◽  
Mitogen Activated Protein ◽  
Enzymatic Activation ◽  
Mapk Phosphatase

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.

scholarly journals Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping

1988 ◽  
Vol 252 (2) ◽  
pp. 607-615 ◽  
Author(s):  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Thin Layer ◽  
Insulin Receptor ◽  
Peptide Mapping ◽  
Beta Subunit ◽  
Two Dimensional ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Domain 3 ◽  
Receptor Beta

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

scholarly journals Coordinated Regulation of Insulin Signaling by the Protein Tyrosine Phosphatases PTP1B and TCPTP

2005 ◽  
Vol 25 (2) ◽  
pp. 819-829 ◽  
Author(s):  
Sandra Galic ◽  
Christine Hauser ◽  
Barbara B. Kahn ◽  
Fawaz G. Haj ◽  
Benjamin G. Neel ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Protein Tyrosine ◽  
Akt Activation ◽  
Protein Levels ◽  
Mitogen Activated Protein

ABSTRACT The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP−/− and PTP1B−/− immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR β-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B−/− MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP−/− MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B−/− MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell.

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