J. Biol. Chem., Vol. 265, Issue 1, 227-234, Jan, 1990
Purification of a novel insulin-stimulated protein kinase from rat liver
JK Klarlund, AP Bradford, MG Milla and MP Czech
Program of Molecular Medicine, University of Massachusetts Medical Center, Worcester 01655.
We previously described a novel insulin-stimulated protein kinase activity
that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic
extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J.
Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic
extracts of livers from insulin-treated rats also exhibited a 30-100%
increase in this Kemptide kinase activity and served as an abundant source
for purification. The Kemptide kinase was purified in parallel from liver
extracts of insulin-treated or control rats through five chromatographic
steps and one polyethylene glycol precipitation. The chromatographic
behavior of the insulin-stimulated Kemptide kinase differed significantly
from the control kinase on Mono Q and heparin- Sepharose resins. The
purified kinase preparations retain insulin stimulations of 2-10-fold.
Analysis of the purified control and insulin- stimulated kinases by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands
with similar silver staining intensity and apparent molecular masses of 52
kDa. The insulin- stimulated Kemptide phosphorylating activity also
coincided with the major silver-stained band following isoelectric focusing
in polyacrylamide gels. The stimulation of kinase activity in response to
administration of insulin is due to an increase in Vmax, whereas the Km for
Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native
kinase determined by gel filtration is approximately 50 kDa, suggesting
that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the
kinase which phosphorylates a variety of basic substrates including a
number of peptides and histones. The activity of the Kemptide kinase is not
changed by several compounds that have been shown to modulate other
kinases. Based on these data, we conclude 1) a novel insulin-sensitive
Kemptide kinase in liver cytosol has been purified to near homogeneity, and
2) insulin administration acutely modulates the specific activity of this
Kemptide kinase in livers of intact rats.
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