Phosphorylation Of Endogenous Substrate Proteins Research Articles

Selective Changes in Protein Kinase C Isoforms and Phosphorylation of Endogenous Substrate Proteins in Rat Cerebral Cortex during Pre- and Postnatal Ethanol Exposure

The effect of pre- and postnatal ethanol exposure on protein kinase C (PKC) activity, immunochemical analysis of PKC α, βI, βII, γ, δ, ϵ, η, and ζ by isoform-specific antibodies, andin vitrophosphorylation of endogenous substrate proteins was investigated in rat cerebral cortex. The PKC activity was increased throughout the development. However, the activity at the age of 8 days was significantly high in cytosolic and membrane fractions from ethanol-treated rats. Immunochemical analysis showed increased levels of PKC βI and βII at the age of 8 days, and a decrease in δ isoform at 8, 30, and 90 days of age. PKC isoforms α, γ, ϵ, and η showed no appreciable change in ethanol-treated rats. PKC ζ levels were high in the cytosolic fraction from ethanol-treated samples of 90 days age.In vitrophosphorylation of endogenous substrate proteins in the presence of Ca2+/phospholipid showed increased phosphorylation of selective membrane and cytosolic proteins with 87, 65, 50, 43, 36, and 29 kDa in ethanol-treated rats. The phosphorylation of these proteins decreased in the presence of staurosporine, which also supported PKC-mediated phosphorylation. Increased PKC activity, activation of βI and βII isoforms, decreased levels of δ isoform, and phosphorylation of selective substrate proteins in cerebral cortex due to alcohol exposure might be relevant in ethanol-induced central nervous system dysfunction and fetal alcohol syndrome.

Pentylenetetrazole-induced chemoshock affects protein kinase C and substrate proteins in mouse brain.

Protein kinase C (PKC) activity, western blot analysis of PKC alpha, beta, gamma, epsilon, and zeta by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentylenetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 mM KCl. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 mM KCl eluates from DE-52 columns contain more PKC alpha and beta in both cytosol and membrane than the 200 mM KCl eluates, whereas PKC gamma, epsilon, and zeta appear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 mM KCl eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC gamma isozyme. As for novel-type epsilon and zeta isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 mM KCl from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 mM KCl eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKC gamma isoform were involved in the pentylenetetrazole-induced chemoshock.

Protein kinase C and dopamine release—II: Effect of dopamine acting drugs in vivo

The hypothesis that protein kinase C (PKC) plays a role in the release of dopamine (DA) in the nigrostriatal pathway was examined. It was found that injections of apomorphine, SKF 38393 (D1 agonist), LY 171555 (D2 agonist) or γ-butyrolactone (GBL) (which decreases impulse-induced release of DA) resulted in a decrease in particulate, and an increase in soluble, PKC activity. Injections of fluphenazine, haloperidol, SCH 23390 (D1 antagonist), sulpiride (D2 antagonist) or picrotoxin (γ-aminobutyric acid antagonist which increases DA release transneuronally) had the opposite effect of increasing particulate and decreasing soluble PKC activity. The total activity was not changed. These effects were receptor mediated since the effect of each agonist could be reversed by its specific antagonist. These drugs influenced PKC in the striatum in a dose-dependent manner. In contrast, no effects were seen in the cerebellum, a region with sparse dopaminergic innervations. The change in PKC activity was mediated via a change in the K m for calcium, while the V max was unchanged. The phosphorylation of endogenous substrate proteins by PKC was also altered by injections of these drugs. Besides affecting PKC, these DA acting drugs also affected the calmodulin-dependent protein kinase activity, but the direction of change was opposite to that for PKC. In a synaptosomal preparation, PKC acting drugs also affected the depolarization-induced release of DA. Adriamycin and melittin decreased the potassium-induced release of DA, whereas tetradecanoyl-phorbol-13-acetate (TPA) enhanced this release. These results showed that there was a good correlation between the ability of drugs to alter the impulseinduced release of DA in vivo and their ability to affect changes in particulate and soluble PKC activity. They lend support to the hypothesis that PKC, together with calmodulin, plays a key role in the release of DA in the nigrostriatal pathway.

Linoleic acid is a potent activator of protein kinase C type III- α isoform in pancreatic acinar cells; its role in amylase secretion

Linoleic acid, an unsaturated-long chain fatty acid, was found to maximally activate protein kinase C (PKC) more effectively than arachidonic or linolenic acid, while the saturated fatty acids palmitic or arachidic had no stimulatory effect. Treatment of intact pancreatic acinar cells with linoleic acid resulted in dose-dependent phosphorylation of endogenous substrate proteins for this kinase and simultaneously stimulated amylase secretion in a dose- and time-dependent fashion. During chromatographic separation of pancreas protein kinase C activity, utilizing hydroxylapatite (HTP), Type III-alpha PKC isoform was detected. These data are consistent with a role for PKC in the regulation of pancreatic exocrine secretion.

The role of calmodulin in hormone-stimulated lipolysis

To explore the role of calmodulin (CaM) in lipolysis, studies were carried out on effects of CaM inhibitors on hormone-stimulated lipolysis, the activity of cAMP-dependent protein kinase, and phosphorylation of endogenous substrate proteins. When adipocytes were incubated with trifluoerazine (TFP) and W-7 but not with W-5, stimulation of lipolysis by epinephrine was blunted. W-7 also inhibited lipolysis induced by ACTH, 1-methyl-3-isobutylxanthine (MIX) or (Bu) 2 cAMP. The binding of 3H-cAMP to its receptor protein (the regulatory subunit of protein kinase) as well as the activity of cAMP-dependent protein kinase was suppressed by W-7, and the anti-CaM antibody, but not by W-5. The CaM-dependence of the protein kinase was also proved by the fact that the protein kinase activity that was markedly reduced in CaM-depleted cell extracts, was significantly restored by addition of exogenous CaM to them. Furthermore, W-7 decreased cAMP-stimulated phosphorylation of endogenous substrate proteins (mol wt 230k, 200k, 130k, 85k, 75k, and 50kdalton), among which the one of 85kdalton is most likely to be the hormone-sensitive lipase. These findings suggest that CaM is involved in the mechanism of hormone-induced lipolysis by exerting stimulatory effects on the activation of cAMP-dependent protein kinase in cell extracts capable of phosphorylating substrate proteins including hormone-sensitive lipase.

Ca2+-Calmodulin-Dependent Phosphorylation of Soluble and Nuclear Proteins in the Rat Ovary*

Studies were undertaken to determine if calmodulin-regulated Ca2+-dependent protein kinase system(s) exist in the prepubertal rat ovary. Phosphorylation studies were performed with ovarian cytosol, calmodulin-depleted cytosol, and nuclear extracts. In vitro phosphorylation of endogenous substrate proteins was accomplished by incubation of tissue fractions with [gamma-32P]ATP followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Calmodulin-dependent phosphorylations of a cytosol protein, mol wt, 95,000, and of three nuclear proteins in the mol wt range of 50,000-60,000, were established by demonstrating: Ca2+ requirement; inhibition by the phenothiazine derivative chlorpromazine; and dependence upon addition of exogenous calmodulin to the calmodulin-depleted cytosol, or to the nuclear extract. These findings demonstrate the presence of rat ovarian cytosol and nuclear Ca2+-calmodulin-dependent protein kinase activities capable of recognizing endogenous substrate proteins.

Ca2+-dependent phosphorylation and Ca2+ uptake in membrane fractions of the mesenteric artery.

Ca2+-dependent phosphorylation of endogenous substrate proteins (mol. wt 30 800, 35 500, 38 600 and 53 200) is found in a membrane subcellular fraction from rabbit mesenteric arteries. Characteristics of 32P incorporation are suggestive of a phosphoester-type phosphorylation produced by a Ca2+-dependent protein kinase. Ca2+-dependent phosphorylation and Ca2+ uptake rate show comparable affinities for Ca2+ of 3.5 x 10(-7) M and 2.4 x 10(-7) M, respectively. The dependence of both phenomena on the MgATP concentration is also similar. Ca2+-dependent phosphorylation and Ca2+ uptake are inhibited by trifluoperazine with an IC50 of 3 x 10(-5) M and 5 x 10(-5) M, respectively. These results suggest that Ca2+ uptake might be modulated by a Ca2+-dependent protein kinase, which is possibly regulated by membrane-bound calmodulin. Endogenous Ca2+-dependent phosphorylation is stimulated up to 300% by the addition of boiled cytosol. This stimulation is due to phosphorylation of proteins of molecular weight 21 000 and 81 500 and is reversed by trifluoperazine. Since this stimulation cannot be mimicked by addition of calmodulin or phosphatidylserine, and since boiled cytosol does not stimulate Ca2+ uptake, it is proposed that an unknown cytosolic factor stimulates a second Ca2+ pump. Since cAMP-dependent protein kinase is shown to cause little phosphorylation and has no effect on Ca2+ uptake, it is concluded that a Ca2+-dependent rather than a cAMP-dependent protein kinase might modulate Ca2+ transport in vascular smooth muscle.

Stimulation by phosphatidylserine and calmodulin of calcium-dependent phosphorylation of endogenous proteins from cerebral cortex.

The Ca2+-dependent phosphorylation of a number of proteins in the cytosol of the rat or guinea pig cerebral cortex was profoundly stimulated by phosphatidylserine; calmodulin, on the other hand, had only a minimal effect. The Ca2+-dependent phosphorylation of different proteins from the total particulate fraction of the same tissue, in comparison, was specifically stimulated by either phosphatidylserine or calmodulin. The present findings, in line with the phospholipid-sensitive Ca2+-dependent protein kinase recently recognized, suggest an involvement of phospholipid in regulating Ca2+-dependent phosphorylation of endogenous substrate proteins. This new system presumably functions independent or in a complementary manner with the calmodulin-sensitive Ca2+-dependent protein phosphorylation system previously reported by others.

Guanosine 3':5'-cyclic monophosphate-dependent phosphorylation of endogenous substrate proteins in membranes of mammalian smooth muscle.

Guanosine 3':5'-cyclic monophosphate (cyclic GMP) stimulated the endogenous phosphorylation of two proteins in isolated membrane fractions from mammalian organs rich in smooth muscle, including ductus deferens, uterus, and small intestine. The apparent molecular weights of the substrate proteins were 130,000 and 100,000. In the presence of 10 mM MnCl(2), a half-maximal increase in phosphorylation of these proteins was achieved with 20-30 nM cyclic GMP. Approximately 10-fold higher concentrations of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were required to produce the same increase in phosphorylation of these two proteins. Cyclic AMP, but not cyclic GMP, regulated the phosphorylation of a third protein present in these same membrane fractions; the apparent molecular weight of this protein was 50,000. Cyclic GMP-dependent phosphorylation of endogenous protein was not observed in the cell sap of any of the three preparations of smooth muscle studied. The finding of endogenous cyclic GMP-dependent protein kinase activity and associated substrate proteins in membrane fractions from several mammalian organs containing smooth muscle raises the possibility that physiological actions of cyclic GMP in smooth muscle may be mediated by the phosphorylation of membrane proteins.