3 Intrasteric regulation of protein kinases

Kobe, Boštjan; Heierhorst, Jörg; Kemp, Bruce E.

doi:10.1016/s1040-7952(97)80006-7

Cited by 20 publications

(15 citation statements)

References 33 publications

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“…Many protein kinases do not bind ATP because of intrasteric inhibition (e.g., twitchin kinase [23], calcium calmodulin-dependent protein kinases [19], or cyclin-dependent protein kinases [12]). In general, relief from intrasteric inhibition is achieved by allosteric effectors acting through regulatory proteins or subunits (31), and nucleotide binding at the active site is not a typical primary regulator. The basal-state IR appears to be an exception.…”

Section: Discussionmentioning

confidence: 99%

Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

Frankel

Ablooglu

Leone³

et al. 2001

Molecular and Cellular Biology

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Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k cat and a 15-fold-lower K m ATP although K m peptide was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.Signal transduction through receptor tyrosine kinases (RTKs) is essential throughout the life of an organism. The tight control of RTK signaling required for normal cellular function is dependent on growth factor stimulation of autophosphorylation (50). Autophosphorylation is a unique regulatory motif among protein kinase signaling cascades because no other enzyme precedes or participates in autophosphorylation. Therefore the unphosphorylated RTK must have sufficient catalytic activity to perform the first phosphoryl transfer reaction while (i) avoiding autophosphorylation prior to growth factor binding and (ii) avoiding target phosphorylation prior to autophosphorylation. The first restriction is met by hormone-induced dimerization of monomeric RTKs (50), where the homodimer itself provides the enzyme-substrate pair. The second restriction is met by target or mediator recruitment sites which appear on the RTK as a consequence of autophosphorylation (44, 51). The logic of induced dimerization seems to fail when applied to the insulin receptor (IR). The IR is a heterotetramer with an ␣ 2 ␤ 2 -subunit structure. The extracellular ␣-subunits are covalently linked to each other and to the membrane-spanning ␤-subunits by disulfide bonds (36). Nev...

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Section: Discussionmentioning

confidence: 99%

Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

Frankel

Ablooglu

Leone³

et al. 2001

Molecular and Cellular Biology

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“…This vector was used as a template to perform site-directed mutagenesis using the in vitro mutagenesis system QuikChange (Stratagene) as directed by the manufacturer. MDM2 serine 17 was mutated to an aspartic acid using the following primers (bases which introduce amino acid change are in bold): amino acid 17 In vitro ubiquitination assay…”

Section: Methodsmentioning

confidence: 99%

“…This flexible motif in MDM2 has been called a pseudo-substrate motif or lid. Pseudo-substrate motifs operate in a range of allosterically regulated enzymes, including protein kinases and metabolic enzymes, and have been termed intrasteric regulatory motifs [1,16,17]. The pseudo-substrate motif of MDM2 harbors a phosphorylation site whose phosphomimetic mutation can alter the conformation of the Nterminal domain of MDM2 protein as defined by NMR studies [25,40].…”

mentioning

confidence: 99%

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

et al. 2009

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The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin-proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or "lid" in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2(S17D)) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg(97)/Lys(98), and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.

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“…The residues Lys-1085 and Gln-1208 interact with the P Ϫ1 residue of the peptide substrate, 4 shown in structures of the activated IRKD (24,25). Mutation of these residues could affect directly the value of K m, peptide .…”

Section: Relief Of Intrasteric Inhibition Revealed Through Steady-statementioning

confidence: 99%

“…Intrasteric inhibition is a regulatory feature used by many protein kinases to suppress catalytic activity (1)(2)(3)(4). This form of inhibition is achieved when a polypeptide segment of the kinase blocks or distorts the active site, thereby preventing binding or proper orientation of one or both substrates.…”

mentioning

confidence: 99%

Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

Ablooglu¹,

Frankel

Rusinova

et al. 2001

Journal of Biological Chemistry

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Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q Ϸ D1132N > D1161A > Y1162F Ϸ K1085N > Q1208S > wildtype. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple offversus-on switch for kinase activation.

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3 Intrasteric regulation of protein kinases

Cited by 20 publications

References 33 publications

Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

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