Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase

Vicario, Pasquale P.; Saperstein, Richard; Bennun, Alfred

doi:10.1016/0003-9861(88)90349-9

Cited by 21 publications

(16 citation statements)

References 26 publications

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“…M1 is directly bound to the β- and γ-phosphate oxygens of ATP while M2 is bound to the β-phosphate oxygen of ATP and two amino acid residues, E1047 and D1150, either directly or indirectly through water molecules. This finding confirmed our earlier kinetic studies on the requirement of two Mg 2+ by PTKs and the interaction of M2 with ATP is consistent with the evidence that the second Mg 2+ activates IRK by increasing its affinity for ATP−Mg ( , ). But this structural information with regard to M2 is unlikely to extend to PTKs utilizing an apparently different mechanism of M2 activation, such as Csk and the Src family.…”

supporting

confidence: 92%

“…We recently demonstrated that, in addition to using Mg 2+ to form ATP-Mg, PTKs also require an additional Mg 2+ as an essential activator (8,9). Kinetic evidence suggests that the second Mg 2+ appears to activate PTKs by one of two mechanisms: increasing the affinity for ATP-Mg or increasing the catalytic efficiency without affecting the binding of ATP-Mg. IRK and v-Fps utilize the first mechanism (10,11) while Src and Csk utilize the second (8). Both mechanisms are still poorly understood.…”

mentioning

confidence: 99%

“…Furthermore, there is increasing evidence that the concentration of free Mg 2+ in cells is tightly regulated ( , ) and that Mg 2+ influx is an important step in the stimulatory action of growth factors (). The sensitivity of PTK activity to Mg 2+ fluctuation ( , ) may provide a mechanism for extracellular signals to modulate the activity of these enzymes (). For these reasons, it is important that we further understand the interaction of free Mg 2+ with PTKs and how that interaction accelerates the phosphorylation reaction.…”

mentioning

confidence: 99%

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Substitution Studies of the Second Divalent Metal Cation Requirement of Protein Tyrosine Kinase CSK

Sun

Budde

1999

Biochemistry

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supporting

confidence: 92%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Substitution Studies of the Second Divalent Metal Cation Requirement of Protein Tyrosine Kinase CSK

Sun

Budde

1999

Biochemistry

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“…Previously reported inhibitors include amino acid derivatives (Tamura et al, 1984;Begum et al, 1985), peptides (Ueno et al, 1987;Walker et al, 1987), tyrosine-containing synthetic 1 Abbreviations: hIR, human insulin receptor; (hlR)CHO, chínese hamster ovary cells transfected with human insulin receptor; IRTK, insulin receptor tyrosine kinase; EDAc, 1 -ethyl-3-[3-(dimethylamino)propyljcarbodiimide hydrochloride; CHO, Chinese hamster ovary; Hepes, N-(2-hydroxyethyl)piperazine-VV-2-ethananesulfomc acid; ICM, concentration of inhibitor yielding half-maximal activity; Km, Michaelis-Menten constant; Vm, maximum velocity; EGFTK, epidermal growth factor receptor tyrosine kinase. polymers (Braun et al, 1984;Sahat et al, 1988;Vicario et al, 1988a), ATP-competitive inhibitors (Davis & Czech, 1985; Kruse et al, 1988a,b), and divalent metal ions (Pang & Shafer, 1985;Vicario et al, 1988b). In addition, catecholamines (Haring et al, 1986a;Obermaier et al, 1987) and phorbol esters (Bollag et al, 1986;Haring et al, 1986b;Obermaier et al, 1987;Takayama et al, 1984) have been shown to reduce IRTK activity.…”

mentioning

confidence: 99%

Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

Saperstein

Vicario²,

Strout³

et al. 1989

Biochemistry

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100

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An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-naphthalenyl-methyl) phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa beta-subunit of the insulin receptor (IC50 = 200 microM). The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 microM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa beta-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. Our data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.

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“…For example, M2 activates Csk and Src by increasing the k cat without affecting the K m for ATP [ 7 ]. However, M2 activates IRK [ 18 ] and v-Fps [ 11 ] by decreasing the K m for ATP without affecting the k cat . The mechanistic basis for such kinetic differences has not been determined.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

2005

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BackgroundProtein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator.ResultsExperiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number.ConclusionThese studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

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Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase

Cited by 21 publications

References 26 publications

Substitution Studies of the Second Divalent Metal Cation Requirement of Protein Tyrosine Kinase CSK

Substitution Studies of the Second Divalent Metal Cation Requirement of Protein Tyrosine Kinase CSK

Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

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