Cyclic AMP-stimulated Protein Phosphorylation Research Articles

Effect of hypothyroidism on the subcellular distribution of Ca2+/calmodulin-stimulated protein kinase II in chicken brain during posthatch development.

In developing chicken brain Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T3) at 42 days. The concentrations of alpha- and beta-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca2+/calmodulin- or ZN2+-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca2+/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.

Staurosporine inhibits protein kinases activated by Ca 2+ and cyclic AMP in addition to inhibiting protein kinase C in rat islets of Langerhans

Staurosporine has been used in several studies to investigate the role of protein kinase C (PKC) in secretory responses of islets of Langerhans to insulin secretagogues. We have assessed the effect of Staurosporine on: [i]islet PKC activity in vitro; [ii]the stimulation of insulin secretion by nutrient secretagogues and [iii]the stimulation of protein phosphorylation and insulin secretion in electrically permeabilised islets. All experiments were carried out on rat isolated islets of Langerhans, either intact or permeabilised by high voltage discharge (3.4 kV/cm). The activity of PKC partially purified from rat islets was inhibited by Staurosporine (1.6–400 nM) in a concentration-dependent manner. Staurosporine also inhibited insulin secretion stimulated by both glucose and glyceraldehyde, with maximal effects at 50 nM. After prolonged exposure of islets to the tumour-promoting phorbol ester, 4β phorbol myristate acetate (4β PMA), a procedure which depletes islet PKC activity, Staurosporine still inhibited both glucose- and glyceraldehyde-stimulated insulin release. In electrically permeabilised islets, Staurosporine inhibited both Ca 2+- and cyclic AMP-stimulated protein phosphorylation and insulin secretion. These results suggest that Staurosporine should not be used as a selective inhibitor of PKC in rat islets.

Developmental changes in protein phosphorylation in chicken forebrain. I. cAMP-stimulated phosphorylation

The net level of cyclic AMP-stimulated protein phosphorylation was investigated in cytosolic and membrane fractions from chicken forebrain between embryonic day 13 (E13) and 52 days post-hatching. Throughout this period the majority of the net level of cAMP-stimulated phosphorylation of endogenous proteins was in the cytosolic fractions. Between day −8 (E13) and adult, the net level of CAMP-stimulated phosphorylation of endogenous proteins in the cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions fell by 3 and 4 fold, respectively, when expressed per mg protein and rose by 5 and 10 fold, respectively, when expressed per fraction. The changes in specific activity were completed by 6–15 days post-hatching. The occluded cytosol (P2-S) fraction showed little change in the net level of cAMP-stimulated phosphorylation of endogenous proteins per mg protein. Major changes in phosphoprotein patterns involving both decreases and increases in phosphorylation occurred in all fractions from day −8 (E13) to day 6 post-hatch; thereafter the phosphoprotein bands and their relative intensities were unchanged. Three bands (P90 in S3; P41 and P31 in P2-M) contained major cAMP-stimulated phosphoproteins in embryonic brain but were absent after hatching. When cAMP-stimulated phosphorylation activity was measured in S3 and P-2M using an exogenous peptide substrate (Kemptide) there was no change in kinase activity per mg protein between day −8 (E13) and 30 days post-hatch. This suggests that the decrease in the net level of cAMP stimulated phosphorylation of endogenous proteins was due to the decrease in levels of endogenous phosphoproteins rather than protein kinase activity. These changes in cAMP-stimulated phosphoproteins during development correlated with the period of synapse formation in chicken forebrain.

Distribution of Calmodulin‐ and Cyclic AMP‐Stimulated Protein Kinases in Synaptosomes

The subcellular location of calmodulin- and cyclic AMP stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [gamma-32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin- or cyclic AMP-stimulated protein phosphorylation. When assessed by calmodulin-stimulated autophosphorylation of the alpha subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin-stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP-stimulated phosphorylation of endogenous substrates, no cyclic AMP-stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP-stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin- and cyclic AMP-stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.

DARPP-32, a dopamine- and adenosine 3‘:5‘-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine.

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.

Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Endogenous cyclic AMP-stimulated phosphorylation of a 49700-Mr Wolfgram protein component in rabbit central nervous system was investigated by using photoaffinity labelling and 2',3'-cyclic nucleotide 3'-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffinity labelling with 8'-azidoadenosine 3',5'-cyclic monophosphate showed a cyclic AMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cyclic AMP-dependent protein kinase. This photoaffinity-labelled protein was of larger apparent Mr than the protein showing cyclic AMP-stimulated phosphorylation. Blotting of one-dimensional sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms followed by staining for 2',3'-cyclic nucleotide 3'-phosphodiesterase activity showed two activity bands corresponding to the two components of the Wolfgram protein doublet. Cyclic AMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of two-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cyclic AMP-stimulated protein phosphorylation with enzyme activity. It is proposed that central-nervous-system myelin contains an endogenous type I cyclic-AMP dependent protein kinase that phosphorylates the larger subunit of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Localization of an endogenous substrate for cyclic AMP-stimulated protein phosphorylation in retina

Incubation of mouse or rabbit whole retina homogenates in the presence of [γ 32P]-ATP and Mg 2+ leads to the phosphorylation of various proteins, as demonstrated using SDS-polyacrylamide gel electrophoresis and autoradiography. The phosphorylation of one protein (ca. ≈ 31 000 mol. wt) was increased by cyclic AMP in both species, with half-maximal stimulation at 5 × 10 −7 m. Cyclic GMP was also active, but much less potent. Protein phosphorylation patterns were compared in retina homogenates from normal mice (C57BL/6J), from adult C57BL/6J mice homozygous for the retinal degeneration gene (rd/rd) in which rod photoreceptor cells are absent, and from 21-day-old 020/Cpb mice homozygous for the retinal degeneration slow gene (rds/rds) in which only the outer segments of the rod photoreceptors are missing. The 31 K protein was only present in normal and in 21-day-old rds/rds mice. When rabbit retina was microdissected into outer segment, inner segment plus outer nuclear, and inner retina layers, cyclic AMP-stimulated phosphorylation of the 31 K protein was evident only in the inner segment plus outer nuclear layer. These data indicated the presence of a specific, endogenous substrate for a cAMP-dependent protein kinase which is found in the inner portions of rod photoreceptor cells.

Protein phosphorylation in isolated synaptic junctional structures: changes during development

Endogenous phosphorylation in subcellular fractions enriched in synaptic plasma membranes (SPM) and purified synaptic junction (SJ) has been examined in vitro at various stages of postnatal development. Protein kinase activity was measured using both endogenous and exogenous (histones) proteins as substrates. Protein phosphorylation that is regulated by cyclic AMP also was investigated. SPM and SJ fractions displayed large increments in kinase activities and cyclic AMP-stimulated protein phosphorylation between postnatal days 5 and 15. SJ fractions exhibited a dramatic age-dependent change in the cyclic AMP-stimulated phosphorylation of endogenous proteins of molecular weights 73,000 (1a), 68,000 (1b), 65,000 (1c) and 55,000. Experiments in which isolated SJs were phosphorylated with soluble cyclic AMP-dependent kinase showed that the phosphorylation of proteins 1a, 1b and 1c resulted from their de novo appearance in newborn SJ fractions and not from a maturation-dependent coupling of kinases and substrates that were already present in newborn SJ fractions. The levels of regulatory (R) subunits of cyclic AMP-dependent kinases in synaptic fractions were measured by [ 3H]cyclic AMP binding and photoaffinity labeling with [ 32P]8-N 3-cyclic AMP and were observed to change little during postnatal development. These findings show that there is a strong correlation between the in situ appearance of synapses and the enzymatic maturation of endogenous, cyclic AMP-stimulated protein phosphorylation in SJ fractions isolated at different stages of development.

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg 2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.

Protein Phosphorylation of the Smooth and Rough Endoplasmic Reticulum in Normal and Neoplastic Liver of the Rat

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.