Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells.

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter.     

Endothelins Stimulate c-fos and Nerve Growth Factor Expression in Astrocytes and Astrocytoma

Abstract: Endothelin receptors have been identified on astrocytes and astrocytoma, but their physiological significance has remained elusive. It is shown here that endothelins induce c- fos in primary cultures of mouse embryo astrocytes, as well as in two subclones of rat astrocytoma C6 cells, although with different kinetics. In addition, nerve growth factor expression is stimulated, as seen by mRNA accumulation and protein secretion, in primary astrocytes and one of the two C6 subclones, with an apparent correlation with the transience of c- fos induction. The activation of protein kinase C appears as an obligatory step during these processes, because (a) inhibition of protein kinase C by staurosporine blocks the induction by endothelin or phorbol esters of both c- fos and nerve growth factor, and (b) phorbol esterevoked down-regulation of protein kinase C completely abolishes the c- fos induction by endothelin, but not that by the β-adrenergic agonist isoproterenol, a known activator of the cyclic AMP-dependent pathway. Our results support the hypothesis that c- fos product might be implicated in nerve growth factor expression by astrocytes, and also suggest that endothelins may participate in vivo in the modulation of the glial neurotrophic activity during brain development or wound healing.     

Nerve growth factor-induced increase in the cell-free phosphorylation of a nuclear protein in PC12 cells

In previous studies from this laboratory (Yu, M.W., Tolson, N. W., and Guroff, G. (1980) J. Biol. Chem. 255, 10481-10492) nerve growth factor treatment of PC12 cells was shown to increase the phosphorylation of a specific nonhistone nuclear protein. In the present work these whole-cell observations have been pursued and a cell-free system developed, based on the detergent treatment devised by Lenk et al. (Lenk, R., Ransom, L., Kaufmann, Y., and Penman, S. (1977) Cell 10, 67-78), in order to explore the nerve growth factor-sensitive phosphorylation system in biochemical detail. Using this preparation it has been shown that treatment of the whole cells with nerve growth factor for 30 min or more leads to a marked increase in the subsequent cell-free phosphorylation of the same nonhistone nuclear protein. A characterization of this phosphorylation indicates that it is quite labile to heat and to structural disruption, that it prefers ATP as phosphate donor, and that it requires Mg2+, but is inhibited by high Mg2+ levels as well as by certain other divalent cations. The site of phosphorylation appears to be on serine residues of the protein, as was the phosphorylation observed previously in whole cells. The use of various inhibitors and stimulators suggests that the kinase catalyzing this phosphorylation is not cAMP-dependent, nor is it similar to protein kinase C or casein kinase. The increased phosphorylation produced by nerve growth factor is not transient, the stimulation being constant for at least 3 days in the continuous presence of nerve growth factor. Increases in the phosphorylation of the same nuclear protein can be seen upon treatment of the cells with other effectors such as epidermal growth factor and dibutyryl cyclic AMP, the latter in spite of the fact that cAMP-dependence could not be established in the cell-free system. Finally, a similar system, with a similar stimulation of phosphorylation due to nerve growth factor treatment, can be prepared from sympathetic ganglia from neonatal animals.     

Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.     

Nerve growth factor, a differentiating agent, and epidermal growth factor, a mitogen, increase the activities of different S6 kinases in PC12 cells

Treatment of PC12 cells with either nerve growth factor, a differentiating agent, or epidermal growth factor, a mitogen, leads to an increase in the phosphorylation of the ribosomal protein S6. The soluble fraction of PC12 cells contains two S6 kinases, separable on heparin-Sepharose. Treatment of the cells with nerve growth factor results in an increase in the activity of one of these kinases; treatment of the cells with epidermal growth factor results in an increase in the activity of the other one. The data suggest that the patterns of phosphorylation and, in turn, the functional properties of S6 are different in cells instructed to differentiate from those in cells instructed to divide.     

The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.     

Regulation of Nerve Growth Factor Action on Nsp100 Phosphorylation in PC12h Cells by Calcium

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

Purification of a hepatic S6 kinase from cycloheximide-treated Rats

Cycloheximide injection of rats results in the activation of a protein kinase that phosphorylates 40 S ribosomal protein S6. This Ca2+/cyclic nucleotide-independent kinase exhibits chromatographic properties that are indistinguishable from the S6 kinase in H4 hepatoma cells whose activity is stimulated by insulin and growth factors and the S6 kinase that is activated during liver regeneration. The enzyme has been purified 50,000-fold to near homogeneity: a critical step in purification employs a peptide affinity column using a synthetic peptide corresponding to the carboxyl-terminal 32-amino acid residues of mouse liver S6, which encompasses all S6 phosphorylation sites. The purified enzyme is a 70,000-dalton polypeptide that is reactive with azido-ATP. In addition to 40 S ribosomal S6 and the synthetic peptide, the S6 kinase catalyzes rapid phosphorylation of a number of other protein substrates including histone H2b, glycogen synthase, and ATP citrate lyase; this last protein is phosphorylated by S6 kinase in vitro on the same serine residue that is phosphorylated in response to insulin and epidermal growth factor in intact hepatocytes. Moreover, the S6 kinase catalyzes the phosphorylation of a number of hepatic nonhistone nuclear proteins. This S6 kinase probably underlies the increased hepatic S6 phosphorylation observed after cycloheximide treatment, which in turn corresponds to the mitogen-activated S6 kinase.     

Neuromodulator-Mediated Phosphorylation of Specific Proteins in a Neurotumor Hybrid Cell Line (NCB-20)

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.     

Partial purification and characterization of a nerve growth factor-sensitive kinase and its substrate from PC12 cells

The cell-free, nerve growth factor-sensitive incorporation of radioactive phosphate into a 100,000-dalton protein (Nsp100), observed in a previous study (End,D., Tolson, N., Hashimoto, S., and Guroff, G. (1983) J. Biol. Chem. 258, 6549-6555), has been characterized and the system fractionated. It is shown here that the decrease in incorporation due to treatment of the cells with nerve growth factor is transient, even in the continued presence of nerve growth factor. The decrease in radioactive phosphate incorporation is due to an inhibition of phosphorylation, not to a stimulation of a dephosphorylation. Evidence is presented to suggest that no soluble cofactors are needed for the phosphorylation and no soluble second messengers are responsible for the inhibition. It is demonstrated that the phosphorylation requires divalent cations; both Mg2+ and Mn2+ are effective in this regard. ATP is the preferred phosphate donor, the phosphorylation is maximal at pH values between 5 and 6, and Na+, K+, and Zn2+ are rather specific inhibitors. The system has been partially purified and the resolved components have been used to show that the kinase and the substrate are separate molecules, that the kinase, not the substrate, is the heat-labile portion, and that the kinase has a molecular weight of 110,000-130,000. Finally, evidence is presented to indicate that the kinase, not the substrate, is the component responsible for the decrease in phosphorylation seen after treatment of the cells with nerve growth factor.     

Induction of a Nerve Growth Factor-Sensitive Kinase that Phosphorylates the DNA-Binding Domain of the Orphan Nuclear Receptor NGFI-B

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI-B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA-binding domain inhibits its binding to the NGFI-B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI-B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA-binding domain with this purified preparation inhibits its binding to the NGFI-B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg²⁺ but will use Mn²⁺. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90 ^rsk . Comparison with a partially purified preparation of cyclic AMP response element-binding protein kinase, however, indicates that the two are either very similar or identical.     

Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.     

6-Methylmercaptopurine Riboside Is a Potent and Selective Inhibitor of Nerve Growth Factor-Activated Protein Kinase N

Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine analogs, such as 6-thioguanine and 2-aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a findings suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6-methylmercaptopurine riboside (6-MMPR) inhibits NGF-stimulated PKN activity in vitro with an apparent Ki of approximately 5 nM. This is approximately 1,000-fold lower than the Ki of the most potent purine inhibitor of PKN. Compounds similar to 6-MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. A survey of six additional purified protein kinases shows no inhibitory effect of 6-MMPR, thus indicating a good degree of specificity of this compound for PKN. In contrast to NGF-stimulated PKN, a PKN-like activity stimulated in PC12 cells in response to activation of cyclic AMP-dependent protein kinase was nearly insensitive to 6-MMPR. Application of 6-MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6-MMPR is a potent and selective agent for characterizing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.