Biochimica et Biophysica Acta, 539 ( 1 9 7 8 ) 2 9 4 - - 3 0 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
SUBCELLULAR ALTERATIONS IN CYCLIC AMP-BINDING AND PROTEIN KINASE ACTIVITIES DURING ONTOGENY IN THE RAT CEREBELLUM
G E O R G E E. S H A M B A U G H III, M A R Y H U N Z I C K E R - D U N N , A N T H O N Y B. D e A N G E L O * and R I C H A R D A. J U N G M A N N
Department of Medicine, V.A. Lakeside Hospital and Department of Biochemistry and Medicine, Northwestern University Medical School, Chicago, Ill. 60611 (U.S.A.) ( R e c e i v e d J u n e 8th, 1977)
Summary Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP binding by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permitted distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 + 6 to 176 -+ 16 and 79 + 12 to 340 -+ 12 pmol/ min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMPbinding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4--20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th--12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between dif* Present address: Frederick Cancer Research Center, PO Box B, Frederick, Md. 21701, U.S.A.
295 ferent subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-binding activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.
Several lines of inquiry have implicated a physiologic role for cyclic AMP in the brain [1--5]. Hormonal action mediated by cyclic AMP includes the following sequential events: hormone binding to a membrane receptor; activation of adenylyl cyclase to generate cyclic AMP; binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase with release of an active catalytic subunit; and phosphorylation of cellular proteins . As component parts of this system have been demonstrated in adult nervous tissue [7--9] and may function to mediate neurotransmission [2,10], studies have been directed to the ontogeny of these component parts to gain insight into functional concomitants of brain development. Adenylyl cyclase activity increases in both cerebellum and cerebral cortex of the rat between birth and day 15. Adenylyl cyclase levels then plateau in the cerebellum, but fall steadily in the cerebrum [11,12]. These differences are reflected in the progressive enhancement of cyclic AMP levels in the cerebellum during the first 5 weeks of age, and in an inital rise followed by a fall in the levels of this cyclic nucleotide in the cerebrum . Unlike the adenylyl cyclase and phosphodiesterase systems, ontogenic studies in the brain of both the rat and guinea pig [13,14] have indicated that cyclic AMP- and cyclic GMP-dependent protein kinase activities are already present in late fetal life . As generation of intracellular cyclic AMP by exogenous hormones is dependent upon adenylyl cyclase, these differences can be interpreted to mean that protein kinases may function independently of intracellular cyclic AMP generation during late fetal life, with implications for cellular development unrelated to neurotransmission. Alternatively, development of the cyclic AMP-dependent protein kinase may antecede the development of adenylyl cyclase and/or membrane receptors which could limit functional integrity of the adenylyl cyclase-protein kinase system until neonatal life. High levels of adenylyl cyclase in the cerebellum [9,12] and increased levels of cyclic AMP in cerebral cortical and cerebellar slices following exposure to norepinephrine [1,16] indicate an active role of the cyclic AMP system in this structure. We therefore chose the cerebellum as a discrete region of the brain that develops during the first 3 weeks of neonatal life  to clarify ontogenic interrelationships between the several components of the cyclic AMP-protein kinase system. In the present study endogenous cyclic AMP levels were determined and taken as indirect evidence for a functioning adenylyl cyclase system, and both cyclic AMP-dependent and -independent protein kinase activities were determined in subcellular fractions of the cerebellum. Herein are shown data indicating that acquisition of a functional cyclic AMP-dependent protein kinase system in the cerebellum occurs as a unit, but that subcellular fractions manifest qualitatively different patterns of protein kinase activity during maturation of this structure.
296 Materials and Methods
Chemicals. Biochemical reagents were obtained from Sigma Chemical Co., St. Louis, Mo. Adenosine 5'-[~,-32P]triphosphate, a m m o n i u m salt (91 Ci/mmol) and [8-3H]adenosine 3',5'-monophosphate (30 Ci/mmol) were purchased from Amersham/Searle, Arlington Heights, Ill. Animals. Rats bred at 6 weeks of age were obtained from Charles River Animal Farms on the 13th day of gestation. All rats were allowed continuous access to food and water. Following delivery litters were adjusted to 8--10 pups per mother. For each experiment, pups were obtained at random from at least three litters. All experiments were carried out between 8:00 a.m. and 11:00 a.m. Preparation o f subcellular fractions. Cerebella were placed in two volumes of ice-cold buffer A (0.05 M Tris, pH 7.4, containing 0.25 M sucrose and 3 mM MgC12) and homogenized in a Dounce homogenizer. The homogenate was centrifuged at 800 X g for 10 min at 4°C to derive a crude nuclear pellet I and a supernatant fraction I. For preparation of the nuclear fraction, pellet I was washed twice in buffer A and subsequently resuspended in 0.05 M Tris (pH 7.4) containing 0.25 M sucrose, 0.15 M NaC1 and 3 mM MgC12. The suspension was mixed with 1.9 M sucrose and centrifuged at 50 000 X g for 30 min. The resulting pellet was resuspended in buffer A containing 0.2% Triton X-100 and designated 'nuclear fraction'. Utilizing DNA as a marker the recovery of nuclei from the homogenate was routinely 70%. No DNA was detected in other particulate fractions. Electrophotomicrographs of the nuclei showed essentially no contamination with extranuclear organelles. The protein/ DNA ratio of nuclei was 3.1 and did n o t change with the age of the rat. For the preparation of the mitochondrial fraction, supernatant fraction I was centrifuged at 27 000 X g for 30 min to yield a pellet which was resuspended in buffer A and resedimented by centrifugation. The pellet was resuspended in buffer A, and used for further experimentation as the "lysosomal-mitochondrial fraction". Triton X-100 was not used to prepare this fraction. Recovery of mitochondria using glutamate dehydrogenase as marker enzyme averaged 56% of the activity present in the homogenate. No glutamate dehydrogenase activity was identified in the nuclear or microsomal fractions. The 27 000 X g supernatant fraction was centrifuged at 105 000 X g for 60 min. The 105 000 X g pellet was resuspended in buffer A and resedimented at 105 000 X g for 60 min. The sedimented pellet was then homogenized in buffer A containing 0.1% Triton X-100 to derive the 'microsomal fraction'. Microsome recovery utilizing glucose-6-phosphatase as a marker averaged 44%. The 105 000 X g supernatant fraction was designated " c y t o s o l " and used for futher experimentation. Utilizing lactic acid dehydrogenase as a marker, it could be shown that less than 3% of the total cellular lactic acid dehydrogenase was present in the mitochondrial fraction and none was detectable in either the microsomal or nuclear fractions. Determination o f protein kinase activity. The protein kinase assay was carried out as described previously  in a total incubation volume of 0.2 ml containing: 10--15 pg of protein to be assayed for protein kinase activity, 100 pg of histone FI, 22 mM a-glycerol phosphate (pH 7.0), 4.5 mM NaF, 4.5
297 mM magnesium acetate, 0.45 mM dithiothreitol, 0.9 mM theophylline, 5 pM [7-32P]ATP (0.5 pCi), in the absence and presence of cyclic AMP. Since maximal enhancement of protein kinase activity was always obtained with 5 . 10 -6 M cyclic AMP, this nucleotide concentration was routinely employed in the assays. Incubation was started by the addition of enzyme and carried o u t for 10 min at 37°C. The reaction was terminated by the addition of 2 ml of 20% trichloroacetic acid containing 1% sodium dodecyl sulfate (SDS) and samples were filtered on Millipore filters (0.3 pro). The filters were washed with 20 ml of trichloroacetic acid/SDS solution, dissolved in 10 ml of phase-combining system (Amersham/Searle), and analyzed for radioactivity. Under these experimental conditions 32p incorporation into substrate protein proceeded linearly with respect to incubation time and protein concentration. The microsomal fraction exhibited maximum protein kinase activity only after solubilization for 20 min in the presence of 0.1% Triton X-100. The final concentration of Triton X-100 in the kinase assays (0.01%) did not significantly affect protein kinase activity of this fraction. Preparation of heat-stable protein kinase inhibitor protein. Heat-stable inhibitor protein was prepared from rabbit skeletal muscle by the m e t h o d of Walsh et al. . The inhibitor, contained in 10 mM potassium phosphate buffer, pH 7.0, at a concentration of 1 mg of protein per ml, did not exhibit protein kinase activity and did n o t accept 32p label when used as a substrate in a protein kinase assay. Beef heart cyclic AMP-dependent protein kinase (10 pg) was inhibited more than 95% when 50 pg of the inhibitor preparation was included in the protein kinase assay. Assay of cyclic [3H]AMP-binding activity. Binding of cyclic [3H]AMP to protein was determined b y membrane filter assay as described previously  in a total incubation volume of 0.35 ml containing: 6.7 mM Tris (pH 7.4), 6.7 mM theophylline, 10 mM MgCl2, 5 • 10 -8 M cyclic [3H]AMP (0.38 pCi), and 20--25 pg of protein to be assayed for cyclic AMP-binding activity. Following incubation for 60 min at 4°C, the samples were passed through Millipore filters (0.3 pm) which had been presoaked in ice-cold 0.25 mM Tris buffer, pH 7.4, containing 10 mM MgC12. Filters were washed five times each with 5 ml of 2.25 mM Tris/10 mM MgC12, dissolved in 10 ml of PCS, and analyzed for radioactivity. Under the conditions of the assay, binding of cyclic [3H]AMP to protein was linear up to 0.1 mg of protein. Cyclic AMP assay. Cyclic AMP was assayed using a commercially available competitive binding assay (Amersham/Searle) in samples of cerebella prepared by trichloroacetic acid precipitation and acid ether extraction. Duplicate assays varied by less than 5%. Protein concentrations were determined by the m e t h o d of Lowry et al.  with bovine serum albumin, fraction V, as reference standard. Student's t-test was utilized to estimate the probability P. Results
Total cyclic AMP and cyclic AMP binding in subcellular fractions Experiments were carried o u t to assess the ontogenic changes in cerebellar cyclic AMP levels as an indirect estimate of adenylyl cyclase activity. Cyclic
298 AMP levels, examined sequentially in cerebella from the second to 25th postnatal day, indicated a 6-fold rise from 3.39 + 0.24 to 22.14 + 0 . 7 8 p m o l / g (Fig. 1). These observations in the cerebellum are similar to the reported changes in cerebellum, cerebral cortex and whole brain reported by others [12, 16,21]. Ontogeny of cyclic AMP-binding proteins was subsequently assessed by measuring cyclic AMP-binding activity in subcellular fractions of the cerebellum. The results are shown for the cytosol, nuclear, mitochondrial and microsomal fractions in Figs. 2A, 2B, 2C and 2D, respectively. In the cytosol fraction (Fig. 2A) cyclic AMP-binding activity increased from 1.1 -+ 0.2 to 2.8 + 0.2 pmol per mg protein, P < 0.001. A similar pattern was apparent in the nuclear fraction where cyclic AMP-binding activity increased from 0.5 + 0.1 to 2.1 -+ 0.2 pmol per mg protein (Fig. 2B). In the mitochondrial fraction a reciprocal pattern was apparent. Binding activity was greatest on day 4, b u t declined thereafter to reach a significantly lower level by day 14 (Fig. 2C). Cyclic AMPbinding activity in the microsomal fraction (Fig. 2D) increased progressively after day 7 to reach significantly higher levels on day 20, and fell thereafter to levels on the 26th day that were not significantly different from the levels observed on the 4th and 7th days. Protein kinase activity Protein kinase activity was determined in subcellular fractions of the cerebellum as a function of postnatal development. The results of these studies in cytosol, nuclear, mitochondrial and microsomal fractions are shown in Figs. 3A, 3B, 3C, and 3D, respectively. In the cytosol fraction (Fig. 3A) total protein kinase activity increased progressively between the 4th and 20th postnatal day. In the nuclear fraction (Fig. 3B) total protein kinase activity increased from 64-+ 6 to 176-+ 16 pmol/min per mg protein, P < 0.001, a