scholarly journals Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain.

1995 ◽  
Vol 15 (5) ◽  
pp. 2500-2508 ◽  
Author(s):  
T A Gustafson ◽  
W He ◽  
A Craparo ◽  
C D Schaub ◽  
T J O'Neill
Keyword(s):  
Insulin Receptor ◽  
Mutational Analysis ◽  
Sh2 Domain ◽  
Binding Domain ◽  
Homologous Region ◽  
Juxtamembrane Domain ◽  
Atp Binding Site ◽  
Protein Protein Interaction ◽  
Binding Domains ◽  
Src Homology 2 Domain

The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.

scholarly journals The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies

2007 ◽  
Vol 408 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Gina E. Sosinsky ◽  
Joell L. Solan ◽  
Guido M. Gaietta ◽  
Lucy Ngan ◽  
Grace J. Lee ◽  
...  
Keyword(s):  
Gap Junction ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Rat Kidney ◽  
Intracellular Localization ◽  
Protein Protein Interaction ◽  
Binding Domains ◽  
C Terminus ◽  
Junction Protein

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein–protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375–Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375–Asp379 is essential in targeting and regulation of gap junctional connexin43.

Src Homology 2 Domain Binding as a Phosphoproteomic Approach to Signaling in Acute Lymphoblastic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4442-4442
Author(s):  
Martin A. Horstmann ◽  
Peter Nollau
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Effector Proteins ◽  
Sh2 Domains ◽  
Binding Domains ◽  
Src Homology 2 ◽  
Src Homology ◽  
Src Homology 2 Domain

Abstract A general theme of orchestrated signal transduction is played by activated receptor phosphotyrosine kinases (PTK) and receptor PTK targets which propagate signals via recognition of sequence-specific phoshorylated tyrosines by so-called Src homology 2 (SH2) domains. SH2 domain interactions are used as a means of recruiting target proteins to activated PTKs and to translocate them to the plasma membrane where many effector proteins activated by receptor PTKs such as phospholipase C-γ or PI-3 kinase have their substrates. SH2 domains make up the most prevalent type of phosphotyrosine binding domains involved in signaling downstream of activated PTKs. SH2 domains are not only present in proteins with intrinsic enzymatic activity but also in adaptor proteins which shuttle effector enzymes to target signaling complexes. Increasing numbers of diseases are known to involve phosphotyrosine specific kinases and/or phosphatases going awry exemplified by the notorious ErbB2 receptor PTK in breast cancer or the Bcr-Abl PTK in CML. Currently, the tyrosine phosphorylation state in most acute lymphoblastic leukemias is undefined which is predicted to differ among the various subgroups and to be distinct from the signaling state of normal hematopoietic cells. To identify aberrant tyrosine kinase or phosphatase activity in the various types of acute lymphoblastic leukemia is of great interest since enzymes in general make good targets for drugs. A novel SH2 domain binding approach is presented which can detect distinctive profiles of tyrosine-phosphorylated proteins in complex mixtures of cellular proteins. A battery of SH2 domains is employed as probes in a competitive far-Western blot based assay to identify specific tyrosine-phosphorylated sites which reflect active signaling pathways in a cell. A further refinement of this technology is under way with DNA-tagged probes being developed which allow for multiplexing and high throughput quantitative assessment of SH2-domain binding by quantitative PCR or microarray technologies.

scholarly journals Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

1994 ◽  
Vol 14 (10) ◽  
pp. 6433-6442 ◽  
Author(s):  
T J O'Neill ◽  
A Craparo ◽  
T A Gustafson
Keyword(s):  
Amino Acids ◽  
Insulin Receptor ◽  
Yeast Cells ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Yeast Saccharomyces Cerevisiae ◽  
Atp Binding Site ◽  
Protein Protein Interaction ◽  
Amino Acid Region ◽  
Two Hybrid

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.

Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system

1994 ◽  
Vol 14 (10) ◽  
pp. 6433-6442
Author(s):  
T J O'Neill ◽  
A Craparo ◽  
T A Gustafson
Keyword(s):  
Amino Acids ◽  
Insulin Receptor ◽  
Yeast Cells ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Yeast Saccharomyces Cerevisiae ◽  
Atp Binding Site ◽  
Protein Protein Interaction ◽  
Amino Acid Region ◽  
Two Hybrid

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.

scholarly journals Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP.

1994 ◽  
Vol 269 (44) ◽  
pp. 27186-27192
Author(s):  
P A Staubs ◽  
D R Reichart ◽  
A R Saltiel ◽  
K L Milarski ◽  
H Maegawa ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Receptor Binding ◽  
Binding Sites ◽  
Sh2 Domain ◽  
Insulin Receptor Binding

scholarly journals Mutational analysis of the peptide segment linking phosphorylation and Ca2+ -binding Domains in the Sarcoplasmic Reticulum Ca2+ -ATPase*

1995 ◽  
Vol 270 (27) ◽  
pp. 16283-16290
Author(s):  
Ziyu Zhang ◽  
Carlota Sumbilla ◽  
David Lewis ◽  
Stephen Summers ◽  
Michael G. Klein ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Mutational Analysis ◽  
Binding Domains

Mutational Analysis of the Nucleotide Binding Domain of the Mismatch Repair Enzyme hMSH-2

1996 ◽  
Vol 229 (1) ◽  
pp. 147-153 ◽  
Author(s):  
Adrian Whitehouse ◽  
Rekha Parmar ◽  
Jayne Deeble ◽  
Graham R. Taylor ◽  
Simon E.V. Phillips ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mutational Analysis ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Repair Enzyme

scholarly journals Serine Phosphorylation Proximal to Its Phosphotyrosine Binding Domain Inhibits Insulin Receptor Substrate 1 Function and Promotes Insulin Resistance

2004 ◽  
Vol 24 (21) ◽  
pp. 9668-9681 ◽  
Author(s):  
Yan-Fang Liu ◽  
Avia Herschkovitz ◽  
Sigalit Boura-Halfon ◽  
Denise Ronen ◽  
Keren Paz ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Insulin Signaling ◽  
Insulin Treatment ◽  
Binding Domain ◽  
Serine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Control Mechanisms ◽  
Insulin Receptor Substrate 1 ◽  
Phosphotyrosine Binding Domain

ABSTRACT Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-17A), unlike wild-type IRS-1 (IRS-1WT), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-17A to remain complexed with the insulin receptor (IR), unlike IRS-1WT, which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-17A and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

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