TY - JOUR
T1 - Mn2+-binding properties of a recombinant protein-tyrosine kinase derived from the human insulin receptor
AU - Wente, Susan R.
AU - Villalba, Mayte
AU - Schramm, Vern L.
AU - Rosen, Ora M.
PY - 1990/4
Y1 - 1990/4
N2 - The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd > 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in α-helical content. The addition of ATP to Mn2+-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.
AB - The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd > 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in α-helical content. The addition of ATP to Mn2+-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.
KW - Circular dichroism
KW - Conformational change
KW - Electron paramagnetic resonance
KW - Secondary structure
UR - http://www.scopus.com/inward/record.url?scp=0025195547&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025195547&partnerID=8YFLogxK
U2 - 10.1073/pnas.87.7.2805
DO - 10.1073/pnas.87.7.2805
M3 - Article
C2 - 2157215
AN - SCOPUS:0025195547
SN - 0027-8424
VL - 87
SP - 2805
EP - 2809
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 7
ER -