Magnetic resonance and kinetic studies of the manganese(II) ion and substrate complexes of the catalytic subunit of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart

Armstrong, Richard N.; Kondo, Hiroki; Granot, Joseph; Kaiser, E. T.; Mildvan, Albert S.

doi:10.1021/bi00574a018

Cited by 127 publications

(147 citation statements)

References 36 publications

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“…Affinities of the Mn 2ϩ for the two binding sites in a binary PKA-AMP-PCP complex were 6 -10 M and 50 -60 M respectively. Kinetic analysis revealed that the high affinity Mn 2ϩ binding has activation properties, while the lower affinity has an inhibitory effect [26]. In the same study, two binding sites of Mg 2ϩ were detected with apparent much lower binding affinity of 1.6 mM for both sites.…”

Section: Discussionmentioning

confidence: 71%

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Pinkse

Heck

Rumpel

et al. 2004

J. Am. Soc. Mass Spectrom.

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Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog ␤,␥-imidoadenosine 5=-triphosphate (AMPPNP) and Mn 2ϩ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg 2ϩ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl 2 to the spray solution. This is in agreement with binding of AMPPNP/Mn 2ϩ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the ␤ and ␥ phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substratedocking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP) 4 (AMPPNP/Mn 2ϩ ) 2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3-4 phosphates per subunit. (J Am Soc Mass Spectrom 2004, 15, 1392-1399

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Section: Discussionmentioning

confidence: 71%

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Pinkse

Heck

Rumpel

et al. 2004

J. Am. Soc. Mass Spectrom.

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“…Kinases have a similar requirement, but they also have an additional site of interaction that can be distinguished from the primary site by affinity. Kinases can be activated or inactivated by binding of a cation to this second site depending upon the specific kinase and identity of the cation (18,19). The effect of divalent cation concentration on the rate of peptide substrate phosphorylation catalyzed by the type 1 receptor kinases is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Comparison of the Biochemical and Kinetic Properties of the Type 1 Receptor Tyrosine Kinase Intracellular Domains

Brignola¹,

Lackey²,

Kadwell³

et al. 2002

Journal of Biological Chemistry

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Epidermal growth factor receptor (EGFR), ErbB-2, and ErbB-4 are members of the type 1 receptor tyrosine kinase family. Overexpression of these receptors, especially ErbB-2 and EGFR, has been implicated in multiple forms of cancer. Inhibitors of EGFR tyrosine kinase activity are being evaluated clinically for cancer therapy. The potency and selectivity of these inhibitors may affect the efficacy and toxicity of therapy. Here we describe the expression, purification, and biochemical comparison of EGFR, ErbB-2, and ErbB-4 intracellular domains. Despite their high degree of sequence homology, the three enzymes have significantly different catalytic properties and substrate kinetics. For example, the catalytic activity of ErbB-2 is less stable than that of EGFR. ErbB-2 uses ATP-Mg as a substrate inefficiently compared with EGFR and ErbB-4. The three enzymes have very similar substrate preferences for three optimized peptide substrates, but differences in substrate synergies were observed. We have used the biochemical and kinetic parameters determined from these studies to develop an assay system that accurately measures inhibitor potency and selectivity between the type 1 receptor family. We report that the selectivity profile of molecules in the 4-anilinoquinazoline series can be modified through specific aniline substitutions. Moreover, these compounds have activity in whole cells that reflect the potency and selectivity of target inhibition determined with this assay system.The type 1 receptor tyrosine kinase family contains four members, epidermal growth factor receptor (EGFR 1 or ErbB-1), ErbB-2, ErbB-3, and ErbB-4 (reviewed in Ref. 1). These receptors consist of an extracellular ligand binding domain, a single transmembrane domain, an intracellular tyrosine kinase catalytic domain, and a tyrosine-rich cytoplasmic tail. The biological activity of these receptors is mediated through the following signal transduction mechanism. A complex pattern of ligand/receptor interactions results in the formation of homoand heterodimers among type 1 receptor family members. Dimerization activates the catalytic domains and allows one receptor monomer to phosphorylate tyrosine residues on the cytoplasmic tail of the other monomer in the pair. Phosphorylation of tyrosine residues on the cytoplasmic tail creates specific binding sites for Src homology 2 or phosphotyrosine binding domain containing proteins. The recruitment of these proteins to the receptor activates signal transduction pathways that produce the response of the cell to the ligand. EGFR, ErbB-2, and ErbB-4 all possess active tyrosine kinase catalytic domains. ErbB-3 is catalytically impaired (2), but it is biologically active because it can form heterodimers with the other family members.Members of the type 1 family of receptors have been implicated in cancer. Specifically, overexpression of ErbB-2 and EGFR is correlated with poor prognosis and reduced overall survival (3). The role of ErbB-3 and ErbB-4 in cancer is not as well described. In pre-clinical models of ...

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“…The substrate sits at the edge of the cleft on the surface of the large C-lobe. PKAc requires one or two divalent metal ions to bind to the active site to be active (5,6). The physiological metal is magnesium, although others can support phosphotransferase activity (7,8).…”

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confidence: 99%

Phosphoryl Transfer Reaction Snapshots in Crystals

Gerlits

Tian

Das

et al. 2015

Journal of Biological Chemistry

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Background: PKAc (catalytic subunit) catalyzes phosphorylation of protein substrates thereby regulating a myriad of cellular processes. Results: X-ray structures of PKAc complexes along the phosphoryl transfer reaction have been obtained. Conclusion:The phosphotransfer follows a multistep mechanism, including conformational changes of the substrate and product groups, a loose transition state, and metal movement. Significance: Mechanistic knowledge about the phosphorylation by PKAc will contribute to understanding of the kinase function and regulation.

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Magnetic resonance and kinetic studies of the manganese(II) ion and substrate complexes of the catalytic subunit of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart

Cited by 127 publications

References 36 publications

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Comparison of the Biochemical and Kinetic Properties of the Type 1 Receptor Tyrosine Kinase Intracellular Domains

Phosphoryl Transfer Reaction Snapshots in Crystals

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