scholarly journals Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein

1999 ◽  
Vol 113 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Elena Molokanova ◽  
Alexei Savchenko ◽  
Richard H. Kramer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cyclic Nucleotide ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Protein Tyrosine Phosphatases ◽  
Competitive Inhibitor ◽  
Rod Photoreceptor ◽  
Protein Tyrosine ◽  
Atp Binding Site ◽  
Cng Channel

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.

scholarly journals Interactions of Cyclic Nucleotide-Gated Channel Subunits and Protein Tyrosine Kinase Probed with Genistein

2000 ◽  
Vol 115 (6) ◽  
pp. 685-696 ◽  
Author(s):  
Elena Molokanova ◽  
Alexei Savchenko ◽  
Richard H. Kramer
Keyword(s):  
Cyclic Nucleotide ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine Phosphatases ◽  
Hill Coefficient ◽  
Sensory Cells ◽  
Maximal Effect ◽  
Rod Photoreceptor ◽  
Protein Tyrosine ◽  
Α Subunits

The cGMP sensitivity of cyclic nucleotide–gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of α subunits (RETα) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETα channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing β as well as α subunits. To determine the contributions of α and β subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETα and OLFα), and heteromeric (RETα+β, OLFα+β, and OLFα+RETβ) CNG channels. We found that genistein only inhibits channels that contain either the RETα or the OLFβ subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETα and OLFβ subunits contain binding sites for the PTK, whereas RETβ and OLFα subunits do not.

scholarly journals Mechanism of Inhibition of Cyclic Nucleotide–Gated Channel by Protein Tyrosine Kinase Probed with Genistein

2001 ◽  
Vol 117 (3) ◽  
pp. 219-234 ◽  
Author(s):  
Elena Molokanova ◽  
Richard H. Kramer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cyclic Nucleotide ◽  
Tyrosine Kinases ◽  
Single Channel ◽  
Channel Gating ◽  
Open Probability ◽  
Inhibitory Interaction ◽  
Α Subunit ◽  
Protein Tyrosine ◽  
Cng Channel

Rod cyclic nucleotide–gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel α-subunit (RETα), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETα channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein–PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein–PTK. Genistein–PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein–PTK dissociation, we determined the maximal open probability for normal and genistein–PTK-bound channels. genistein–PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni2+, which potentiates RETα channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein–PTK and the channel. Studies on chimeric channels containing portions of RETα, which exhibits genistein inhibition, and the rat olfactory CNG channel α-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein–PTK binding site. Thus, genistein–PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.

scholarly journals Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in trans-Golgi

2005 ◽  
Vol 289 (3) ◽  
pp. C748-C756 ◽  
Author(s):  
Rachel J. Webb ◽  
Jacob D. Judah ◽  
Lee-Chiang Lo ◽  
Geraint M. H. Thomas
Keyword(s):  
Serum Albumin ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Protein Tyrosine Phosphorylation ◽  
Albumin Secretion ◽  
Protein Tyrosine ◽  
Constitutive Secretion

Serum albumin secretion from rat hepatocytes proceeds via the constitutive pathway. Although much is known about the role of protein tyrosine phosphorylation in regulated secretion, nothing is known about its function in the constitutive process. Here we show that albumin secretion is inhibited by the tyrosine kinase inhibitor genistein but relatively insensitive to subtype-selective inhibitors or treatments. Secretion is also blocked in a physiologically identical manner by the tyrosine phosphatase inhibitors pervanadate and bisperoxo(1,10-phenanthroline)-oxovanadate. Inhibition of either the kinase(s) or phosphatase(s) leads to the accumulation of albumin between the trans-Golgi and the plasma membrane, whereas the immediate precursor proalbumin builds up in a proximal compartment. The trans-Golgi marker TGN38 is rapidly dispersed under conditions that inhibit tyrosine phosphatase action, whereas the distribution of the cis-Golgi marker GM130 is insensitive to genistein or pervanadate. By using a specifically reactive biotinylation probe, we detected protein tyrosine phosphatases in highly purified rat liver Golgi membranes. These membranes also contain both endogenous tyrosine kinases and their substrates, indicating that enzymes and substrates for reversible tyrosine phosphorylation are normal membrane-resident components of this trafficking compartment. In the absence of perturbation of actin filaments and microtubules, we conclude that reversible protein tyrosine phosphorylation in the trans-Golgi network is essential for albumin secretion and propose that the constitutive secretion of albumin is in fact a regulated process.

scholarly journals Positive and negative regulation of T-cell activation through kinases and phosphatases

2003 ◽  
Vol 371 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Tomas MUSTELIN ◽  
Kjetil TASKÉN
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Protein Tyrosine Phosphatases ◽  
Negative Regulation ◽  
Intact Cells ◽  
Protein Tyrosine ◽  
Sequence Of Events

The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components.

scholarly journals Recent advances in PTP1B signaling in metabolism and cancer

2021 ◽  
Author(s):  
Olga Villamar-Cruz ◽  
Marco A. Loza-Mejía ◽  
Luis E. Arias-Romero ◽  
Ignacio Camacho-Arroyo
Keyword(s):  
Signaling Pathways ◽  
Tyrosine Phosphorylation ◽  
Cancer Progression ◽  
Tyrosine Kinases ◽  
Critical Role ◽  
Protein Tyrosine Phosphatases ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Promising Therapeutic Target ◽  
Molecular Aspects

Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanated from receptor tyrosine kinases. However, recent evidence demonstrates that a misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.

scholarly journals Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Jun Sumaoka ◽  
Hiroki Akiba ◽  
Makoto Komiyama
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
High Selectivity ◽  
Protein Tyrosine Phosphatases ◽  
Protein Tyrosine Kinases ◽  
Selective Detection ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Recent Developments ◽  
Efficient Screening

Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclearTbIIIcomplexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to theTbIIIion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding theseTbIIIcomplexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

scholarly journals Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

2021 ◽  
Vol 22 (9) ◽  
pp. 4417
Author(s):  
Lester J Lambert ◽  
Stefan Grotegut ◽  
Maria Celeridad ◽  
Palak Gosalia ◽  
Laurent JS De Backer ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Synaptic Function ◽  
Label Free ◽  
Protein Tyrosine

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

Protein tyrosine phosphatase-α complexes with the IGF-I receptor and undergoes IGF-I-stimulated tyrosine phosphorylation that mediates cell migration

2009 ◽  
Vol 297 (1) ◽  
pp. C133-C139 ◽  
Author(s):  
Shirley C. Chen ◽  
Ranvikram S. Khanna ◽  
Darrell C. Bessette ◽  
Lionel A. Samayawardhena ◽  
Catherine J. Pallen
Keyword(s):  
Cell Migration ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Phosphorylation Site ◽  
Protein Tyrosine ◽  
Fibroblast Migration ◽  
Igf I ◽  
In Cells

Protein tyrosine phosphatase-α (PTPα) is a widely expressed receptor-type phosphatase that functions in multiple signaling systems. The actions of PTPα can be regulated by its phosphorylation on serine and tyrosine residues, although little is known about the conditions that promote PTPα phosphorylation. In this study, we tested the ability of several extracellular factors to stimulate PTPα tyrosine phosphorylation. The growth factors IGF-I and acidic FGF induced the highest increase in PTPα phosphorylation at tyrosine 789, followed by PMA and lysophosphatidic acid, while EGF had little effect. Further investigation of IGF-I-induced PTPα tyrosine phosphorylation demonstrated that this occurs through a novel Src family kinase-independent mechanism that does not require focal adhesion kinase, phosphatidylinositol 3-kinase, or MEK. We also show that PTPα physically interacts with the IGF-I receptor. In contrast to IGF-I-induced PTPα phosphorylation, this association does not require IGF-I. The interaction of PTPα and the IGF-I receptor is independent of PTPα catalytic activity, and expression of exogenous PTPα does not promote IGF-I receptor tyrosine dephosphorylation, indicating that PTPα does not act as an IGF-I receptor phosphatase. However, PTPα mediates IGF-I signaling, because IGF-I-stimulated fibroblast migration was reduced by ∼50% in cells lacking PTPα or in cells with mutant PTPα lacking the tyrosine 789 phosphorylation site. Our results suggest that PTPα tyrosine phosphorylation can occur in response to diverse stimuli and can be mediated by various tyrosine kinases. In the case of IGF-I, we propose that IGF-I-induced tyrosine 789 phosphorylation of PTPα, possibly catalyzed by the PTPα-associated IGF-I receptor tyrosine kinase, is required for efficient cell migration in response to this growth factor.

Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcγRIIA and αIIbβ3

2005 ◽  
Vol 93 (05) ◽  
pp. 932-939 ◽  
Author(s):  
Caroline Pampolina ◽  
Archibald McNicol
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Lag Phase ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Streptococcus Sanguis

SummaryThe low-affinity IgG receptor, FcγRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcγRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017–78 caused the tyrosine phosphorylation of FcγRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCγ2. These early events were dependent on Src family kinases but independent of either TxA2 or the engagement of the αIIbβ3 integrin. During the lag phase prior to platelet aggregation, FcγRIIA, Syk, LAT, and PLCγ2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017–78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcγRIIA, Syk, LAT, and PLCγ2 were rephosphorylated in an RGDS-sensitive, and therefore αIIbβ3-dependent, manner. Additionally, TxA2 release, 5-hydro-xytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited αIIbβ3-mediated re-phosphorylation. Therefore, S.sanguis-bound IgG cross links FcγRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of αIIbβ3 leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA2 release and consequent platelet aggregation.

scholarly journals Protein tyrosine phosphatases and signalling

2005 ◽  
Vol 185 (1) ◽  
pp. 19-33 ◽  
Author(s):  
Andrew W Stoker
Keyword(s):  
Tyrosine Kinases ◽  
Current Knowledge ◽  
Insulin Signalling ◽  
Cell Signalling ◽  
Protein Tyrosine Phosphatases ◽  
Protein Tyrosine Kinases ◽  
Tyrosine Phosphatases ◽  
Large Protein ◽  
Protein Tyrosine ◽  
Wide Range

A cornerstone of many cell-signalling events rests on reversible phosphorylation of tyrosine residues on proteins. The reversibility relies on the coordinated actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), both of which exist as large protein families. This review focuses on the rapidly evolving field of the PTPs. We now know that rather than simply scavenging phosphotyrosine, the PTPs specifically regulate a wide range of signalling pathways. To illustrate this and to highlight current areas of agreement and contention in the field, this review will present our understanding of PTP action in selected areas and will present current knowledge surrounding the regulatory mechanisms that control PTP enzymes themselves. It will be seen that PTPs control diverse processes such as focal adhesion dynamics, cell–cell adhesion and insulin signalling, and their own actions are in turn regulated by dimerisation, phosphorylation and reversible oxidation.

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