Abstract
We have measured nuclear protein kinase activity during the prereplicative phase of rat liver regeneration. Total nuclear protein kinase activity increased significantly 15-18 h after partial hepatectomy, with the peak of activity occurring at 16 h. DEAE-Sephacel chromatography resolved nuclear protein kinase activity into two cAMP-independent (Ib and II) and two cAMP-dependent (Ia and III) protein kinases. Sixteen h after partial hepatectomy, there was a marked increase in the activities of the nuclear cAMP-dependent protein kinases and a decrease in the activity of nuclear cAMP-independent protein kinase II. Characterization of the two nuclear cAMP-dependent protein kinases revealed them to be identical with the cytosolic type I and II isozymes. Immunotitration of nuclear catalytic subunit and densitometric analysis of autoradiographs from 8-azido-[32P]cAMP-labeled nuclear RI revealed increases in both subunits 16 h afer partial hepatectomy. Concomitantly with the observed increase in nuclear protein kinase activity, we have observed an increase in the phosphorylation of histone H1 subspecies. Administration of the beta-adrenergic antagonist DL-propranolol, which has been shown to cause delays of equal duration in both the second phase of increased intracellular cAMP levels and the initiation of DNA synthesis (MacManus, J. P., Braceland, B. M., Youdale, T., and Whitfield, J. F. (1973) J. Cell. Physiol. 82, 157-164), results in an equivalent delay of increased nuclear protein kinase activity. Colchicine, which has previously been shown to prevent the onset of DNA synthesis (Walker, P. R., and Whitfield, J. F. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1394-1398), also prevents the increased protein kinase activity normally observed 16 h after partial hepatectomy. We conclude that the onset of DNA synthesis in the regenerating rat liver is preceded by a cAMP-mediated translocation of type I and type II cAMP-dependent protein kinase to the nucleus and phosphorylative modification of histone H1 subspecies. The inhibitory effects of propranolol and colchicine suggest a common cAMP-mediated, colchicine-sensitive link between protein kinase translocation and the initiation of DNA synthesis.
Highlights
From the *Cancer Center, Biochemistry Division, Northwestern University Medical School, Chicago, Illinois 60611 and the Slnstitut fiir Physiologische Chemie, Universitats-KrankenhausEppendori Hamburg, Federal Republic of Germany
Sixteen h cell division [1,2,3,4]T. his seeming contradiction is reflected after partial hepatectomy, there was a marked increase by the reported effects on cellular proliferation of the target in the activities of the nuclear CAMP-dependentprotein enzyme for CAMP,CAMP-dependent protein kinase
II. kinases anda decrease inthe activity of nuclear CAMP- reports provide evidence that CAMP-dependent protein kiindependent protein kinase Characterization of the nase mediates the effect of cAMP on growth inhibition
Summary
Antiserum against the Catalytic Subunit of Cyclic AMP-depend- from rat liver and in isolated purified nuclei. The per cent activity ent Protein Kinase-Preparation of the antiserum against bovine shown representsthe nuclear activityas compared to the total activity heart catalytic subunit in rabbits and its characterization and cross- present in the total liver homogenate. Comparison of the DNA content either diluted cytosol or diluted nuclear 0.35 M NaCl extract were of the homogenate with that present in isolated nuclei reincubatedin the presence of 5 p~ cAMPwith the dilutionsof antiserum listed in the text. Two h later the animals were killed by cervical dislocation.For the isolation of nuclei,liver was homogenized as described above in buffer A containing 2.3 M sucrose, 15 mM NaF, and 5 m~ ZnC12.Nuclear0.35 M NaCl extracts were prepared as.
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