BioSystems, 22, (1988)
Role of divalent metals in the activation and regulation
of insulin receptor tyrosine kinase
Vicario, P.P., Saperstein, R. and Bennun, A.
Multiple equilibrium equations were solved to separate the
individual effects of ionic divalent metals, free nucleotides
and their chelated species on insulin receptor tyrosine kinase
(IRTK). Basal IRTK is activated by divalent metal cations
when present in excess of that required for substrate formation,
indicating the presence of a divalent cation-dependent regulatory
site on the kinase. The activatory order for basal activity
was Mn2+ > CO2+ > Mg2+ and Ca2+ = 0. The insulin-dependent
activation of IRTK was minimal in the absence of excess free
divalent metal, even when the concentration of MnATP or MgATP
substrate present exceeded the apparent K of the kinase. The
activatory order for insulin-dependent activation of IRTK
changed to Mg2+ > Mn2+ and Co2+ = 0. The titration of the
MnCl2 saturation response at several concentrations of MgCl2
revealed that the insulin-dependent response of IRTK increases
as a function of increasing MgCl2´ while basal activity
was unaffected. This enhancement of the responsiveness to
insulin in the presence of both cations was not due to differing
affinities of the kinase for substrate, as evidenced by nearly
identical apparent Km values for MnATP and MgATP. The Mg2+-dependent
increase in the response of the kinase to insulin may be due
to Mg2+ inducing a stronger coupling between receptor and
kinase than that observed with Mn2+ alone. The plotting of
the effect of several concentrations of free divalent metals
on substrate saturation curves revealed that an increase in
either of the reactants increased the affinity of the insulin-activated
kinase for the other respective reactant. Accordingly, free
divalent metal and metal-ATP substrate interact with IRTK
in a mutually inclusive manner. CaCl2 saturation curves in
the presence of constant MnCl2 and increasing MgCl2 showed
that the affinity of IRTK for Ca2+ decreases and the affinity
for CaATP increased with increasing Mg2+. Our data suggests
that IRTK contains three sites for interaction with divalent
metal cations: a MeATP (active) site, a regulatory site, and
a metal-dependent site acting to couple the receptor with
the kinase.