Site-specific dephosphorylation and deactivation of the human insulin receptor tyrosine kinase by particulate and soluble phosphotyrosyl protein phosphatases.
Ontology highlight
ABSTRACT:
SUBMITTER: King MJ
PROVIDER: S-EPMC1150069 | biostudies-other | 1991 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Insulin stimulates autophosphorylation of the insulin receptor on multiple tyrosines in three domains: tyrosines 1316 and 1322 in the C-terminal tail, 1146, 1150 and 1151 in the tyrosine-1150 domain, and possibly 953, 960 or 972 in the juxtamembrane domain. In the present work the sequence of dephosphorylation of the various autophosphorylation sites by particulate and cytosolic preparations of phosphotyrosyl-protein phosphatase from rat liver was studied with autophosphorylated human placental insulin receptor as substrate. Both phosphatase preparations elicited a broadly similar pattern of dephosphorylation. The tyrosine-1150 domain in triphosphorylated form was found to be exquisitely sensitive to dephosphorylation, and was dephosphorylated 3-10-fold faster than the di- and monophosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains. The major route for dephosphorylation of the triphosphorylated tyrosine-1150 domain involved dephosphorylation of one of the phosphotyrosyl pair, 1150/1151, followed by phosphotyrosyl 1146 to generate a species monophosphorylated mainly (greater than 80%) at tyrosine 1150 or 1151. Insulin receptors monophosphorylated in the tyrosine-1150 domain disappeared slowly, and overall the other domains were completely dephosphorylated faster than the tyrosine-1150 domain. Dephosphorylation of the diphosphorylated C-terminal domain yielded insulin receptor in which the domain was singly phosphorylated at tyrosine 1322. Triphosphorylation of the insulin receptor in the tyrosine-1150 domain appears important in activating the receptor tyrosine kinase to phosphorylate other proteins. The extreme sensitivity of the triphosphorylated form of the tyrosine-1150 domain to dephosphorylation may thus be important in terminating or regulating insulin-receptor tyrosine kinase action and insulin signalling.
| S-EPMC1131122 | biostudies-other
Project description:Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.
| S-EPMC1135500 | biostudies-other
Project description:The insulin-like growth factor-I (IGF-I) receptor exhibits structural and functional similarities to the insulin receptor. Although the regulation of the insulin-receptor tyrosine kinase has been extensively investigated, the mechanisms involved in phosphorylation/dephosphorylation of the IGF-I receptor have received only little attention. To obtain a better understanding of the mode of IGF-I action, we have investigated the effects of protein phosphotyrosine phosphatases (PTPases) on the phosphorylation status of the IGF-I receptor. The dephosphorylation of the human IGF-I receptor by membrane-associated tyrosine phosphatases was studied by an immuno-enzymic assay based on the recognition of phosphotyrosine residues by anti-phosphotyrosine antibodies. Using intact IGF-I receptors as substrates, we show that they could be completely dephosphorylated by different cellular PTPases. Three pieces of evidence indicate that receptor dephosphorylation takes place on phosphotyrosine, i.e. the inhibition profile of phosphatase activity by zinc and vanadate, its absolute requirement for thiol compounds and the diminution of [32P]phosphotyrosine labelling of the beta subunit assessed by SDS/PAGE and phosphoamino acid analysis. Tyrosine kinase activity and autophosphorylation of the IGF-I receptor were decreased in a dose-dependent manner by PTPases, indicating that partial dephosphorylation of the receptor was associated with a decrease in its intrinsic activity. The sensitivity of the activated human IGF-I receptor to dephosphorylation on tyrosine leads to the speculation that IGF-I receptor activity might be regulated by mechanisms such as those described for the insulin receptor. Further investigation of the pathways of IGF-I receptor dephosphorylation will contribute to define the role(s) of PTPases in the overall mechanism of IGF-I signalling.
| S-EPMC1132746 | biostudies-other
Project description:Nitric oxide (NO) exerts its biological function through S-nitrosylation of cellular proteins. Due to the labile nature of this modification under physiological condition, identification of S-nitrosylated residue in enzymes involved in signaling regulation remains technically challenging. The present study investigated whether intrinsic NO produced in endothelium-derived MS-1 cells response to insulin stimulation might target endogenous protein tyrosine phosphatases (PTPs). For this, we have developed an approach using a synthetic reagent that introduces a phenylacetamidyl moiety on S-nitrosylated Cys, followed by detection with anti-phenylacetamidyl Cys (PAC) antibody. Coupling with sequential blocking of free thiols with multiple iodoacetyl-based Cys-reactive chemicals, we employed this PAC-switch method to show that endogenous SHP-2 and PTP1B were S-nitrosylated in MS-1 cells exposed to insulin. The mass spectrometry detected a phenylacetamidyl moiety specifically present on the active-site Cys463 of SHP-2. Focusing on the regulatory role of PTP1B, we showed S-nitrosylation to be the principal Cys reversible redox modification in endothelial insulin signaling. The PAC-switch method in an imaging format illustrated that a pool of S-nitrosylated PTP1B was colocalized with activated insulin receptor to the cell periphery, and that such event was endothelial NO synthase (eNOS)-dependent. Moreover, ectopic expression of the C215S mutant of PTP1B that mimics the active-site Cys215 S-nitrosylated form restored insulin responsiveness in eNOS-ablated cells, which was otherwise insensitive to insulin stimulation. This work not only introduces a new method that explores the role of physiological NO in regulating signal transduction, but also highlights a positive NO effect on promoting insulin responsiveness through S-nitrosylation of PTP1B's active-site Cys215.
| S-EPMC5514559 | biostudies-literature
Project description:Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of phosphotyrosine, a central control element in mammalian signal transduction. Small-molecule inhibitors that are specific for each cellular PTP would be valuable tools in dissecting phosphorylation networks and for validating PTPs as therapeutic targets. However, the common architecture of PTP active sites impedes the discovery of selective PTP inhibitors. Our laboratory has recently used enzyme/inhibitor-interface engineering to generate selective PTP inhibitors. The crux of the strategy resides in the design of "inhibitor-sensitized" PTPs through protein engineering of a novel binding pocket in the target PTP. "Allele-specific" inhibitors that selectively target the sensitized PTP can be synthesized by modifying broad-specificity inhibitors with bulky chemical groups that are incompatible with wild-type PTP active sites; alternatively, specific inhibitors that serendipitously recognize the sensitized PTP's non-natural pocket may be discovered from panels of "non-rationally" designed compounds. In this review, we describe the current state of the PTP-sensitization strategy, with emphases on the methodology of identifying PTP-sensitizing mutations and synthesizing the compounds that have been found to target PTPs in an allele-specific manner. Moreover, we discuss the scope of PTP sensitization in regard to the potential application of the approach across the family of classical PTPs.
| S-EPMC1950444 | biostudies-literature
Project description:NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesized that NO-induced inactivation of endogenous protein-tyrosine phosphatases (PTPs) would enhance insulin receptor-mediated signaling. To test this hypothesis, we devised a new method of the PTP labeling using a cysteine sulfhydryl-reacted probe. Under the acidic conditions employed in this study, the probe recognized the reduced and active forms but not the S-nitrosylated and inactive forms of endogenous PTPs. Our data suggest that phosphatases SHP-1, SHP-2, and PTP1B, but not TC-PTP, are likely S-nitrosylated at the active site cysteine residue concomitantly with a burst of NO production in signaling response to insulin stimulation. These results were further confirmed by phosphatase activity assays. We investigated further the role of NO as a regulator of insulin signaling by RNA interference that ablates endogenous eNOS expression in endothelial MS-1 cells. We have shown that eNOS-dependent NO production is essential for the activation of insulin signaling. Our findings demonstrate that NO mediates enhancement of insulin responsiveness via the inhibition of insulin receptor phosphatases.
| S-EPMC2832942 | biostudies-literature
Project description:Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na(3)VO(4) desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.
| S-EPMC2858656 | biostudies-literature
Project description:The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner.
| S-EPMC1479551 | biostudies-literature
Project description:Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of signal transduction mediated by reversible protein-tyrosine phosphorylation. In order to characterize individual rat hepatic PTPases that might have specificity for autophosphorylated receptor tyrosine kinases, we isolated cDNA segments encoding three PTPases (PTPase 1B, LAR and LRP) that are expressed in insulin-sensitive liver and skeletal muscle tissue, and evaluated their catalytic activity in vitro. The intrinsic PTPase activities of the full-length PTPase 1B protein and the cytoplasmic domains of LAR and LRP were studied by expression of recombinant cDNA constructs in the inducible bacterial vector pKK233-2 using extracts of a host strain of Escherichia coli that lacks endogenous PTPase activity. Each of the cloned cDNAs dephosphorylated a cognate phosphopeptide derived from the regulatory region of the insulin receptor. Despite having only 30-39% sequence identity in their catalytic domains, LAR and PTPase 1B had similar relative activities between the peptide substrate and intact insulin receptors, and also displayed similar initial rates of simultaneous dephosphorylation of insulin and epidermal growth factor (EGF) receptors. In contrast, LRP exhibited a higher rate of dephosphorylation of both intact receptors relative to the peptide substrate, and also dephosphorylated EGF receptors more rapidly than insulin receptors. These studies indicate that three PTPases with markedly divergent structures have the catalytic potential to dephosphorylate both insulin and EGF receptors in intact cells and that redundant PTPase activity may occur in vivo. For these PTPases to have specific physiological actions in intact cells, they must be influenced by steric effects of the additional protein segments of the native transmembrane enzymes, cellular compartmentalization and/or interactions with regulatory proteins.
| S-EPMC1132676 | biostudies-other
Project description:Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic "switch" residue to an internal position when the ?4-?4 loop adopts an active-site proximal conformation.
| S-EPMC3035606 | biostudies-literature