Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Ablooglu, Ararat J.; Kohanski, Ronald A.

doi:10.1021/bi002292m

Cited by 45 publications

(70 citation statements)

References 51 publications

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“…Interestingly, in the presence of ATP, we found that WT-RET and 2B-RET had similar melting temperatures ( Table 1), indicating that the conformations of ATP-bound (active) WT-RET and 2B-RET enzymes were more similar than those of their autoinhibited forms. The lower melting temperature of the active forms was consistent with the thermodynamic effects of the release of autoinhibition in other kinases (24,25). Together, these data suggested that, in addition to altered kinase activity, changes in intramolecular interactions and a potential reduction in conformational rigidity might play significant roles in the functional effects of the M918T mutation in 2B-RET.…”

Section: Resultssupporting

confidence: 70%

“…In the absence of ATP, we found that WT-RET was more conformationally stable or rigid than 2B-RET, as indicated by a higher melting temperature [midpoint of denaturation transition (T m ), 65.10jC; Table 1]. The relatively higher energy requirement for denaturation is consistent with the tightly folded, more stable conformation of an autoinhibited (inactive) kinase structure (24,25). 2B-RET had a lower melting temperature (T m , 62.65jC), suggesting a relatively more flexible protein conformation with a less rigid overall structure.…”

Section: Resultsmentioning

confidence: 62%

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Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

Gujral¹,

Singh

Jia

et al. 2006

Cancer Research

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Multiple endocrine neoplasia 2B (MEN 2B) is an inherited syndrome of early onset endocrine tumors and developmental anomalies. The disease is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET); however, the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10-fold increase in ATP binding affinity and leads to a more stable receptor-ATP complex, relative to the wild-type receptor. Further, the M918T mutation alters local protein conformation, correlating with a partial loss of RET kinase autoinhibition. Finally, we show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.

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Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 62%

Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

Gujral¹,

Singh

Jia

et al. 2006

Cancer Research

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“…Enzymatic activation by ligand may represent an increase in k cat as has been shown for the EGFR (24,25) or in concomitant increases in k cat and decreases in K m as has been demonstrated for the IRK (40). In our study, we have made the novel finding that ligand can also regulate the specificity of a receptor kinase toward physiologically relevant substrates.…”

Section: Discussionmentioning

confidence: 53%

“…Many RTKs require phosphorylation of one or more conserved tyrosine in the activation loop for full catalytic activity (43). For example, IRK activation loop phosphorylation results in 25-and 40-fold decreases in K m values for ATP and peptide, respectively, and a 7-fold increase in k cat (40). Unlike IRK and other RTKs, the crystal structure of the EGFR catalytic domain revealed that the unphosphorylated A-loop exists in an open extended conformation consistent with an enzymatically active kinase (16).…”

Section: Discussionmentioning

confidence: 99%

Ligand Regulates Epidermal Growth Factor Receptor Kinase Specificity

Fan

Wong

Deb³

et al. 2004

Journal of Biological Chemistry

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The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K m and k cat , revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFRmediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k cat ϳ5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of ϳ5-fold in the specificity constants (k cat /K m ) for EGFR peptides, whereas ϳ15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins ϳ4 -6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.Protein-tyrosine kinases, which catalyze the transfer of ␥-phosphate from ATP to tyrosine side chains of peptide and protein substrates, play a central role in cellular signaling. Receptor tyrosine kinases (RTKs) 1 constitute one class of protein kinases that consist of an extracellular ligand binding, a transmembrane, an intracellular tyrosine kinase, and a Cterminal domain that contains tyrosine phosphorylation sites (1). The epidermal growth factor receptor (EGFR, ErbB1, and HER1) is the prototypical member of the ErbB family RTKs, which also includes ErbB2 (HER2, neu), ErbB3 (HER3), and ErbB4 (HER4). Binding of growth factors that are structurally related to EGF induces a process of the receptor dimerization, kinase activation and autophosphorylation (2). EGFR activation results in recruitment and phosphorylation of cytosolic downstream targets such as Grb2-associated binding protein 1 (Gab1) (3), Src homology 2 (SH2) domain, collagen-containing protein (Shc) (4), and phospholipase C␥-1 (PLC␥-1) (5, 6). EGFR functions in the proliferation, migration, survival, and differentiation of mammalian cells (2, 7), and dysregulation of signaling by EGFR ...

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“…Furthermore, kinetic evidence showed that ATP binding predisposes the unphosphorylated receptor for trans autophosphorylation (32). Given the appar-ent affinity of the kinase domain for MgATP of ϳ1 mM (1,7,18) and the intracellular ATP concentration of ϳ1 mM (possibly as high as 8 mM [37]), a significant fraction of the receptor's kinase domains should have nucleotide bound under physiological conditions and therefore should be nonlatent as an enzyme and nonlatent as a substrate (18). This raises the issue of whether latency is required to maintain the basal state and, conversely, whether the IR with nonlatent kinase domains can be kept from autophosphorylation and signaling.…”

mentioning

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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Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Cited by 45 publications

References 51 publications

Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

Ligand Regulates Epidermal Growth Factor Receptor Kinase Specificity

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

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