cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators

The relationship between cAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios greater than 0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A-kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the cAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC50 for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligand-free environment achieved with adenosine deaminase; insulin IC50 values were approximately 120 and 600 pM, respectively. Such data establish a link between insulin actions in modifying cAMP concentrations and in modifying events apparently independent of changes in cAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase.     

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.     

Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents

This paper examines the modulation of insulin-stimulated glucose transport activity in rat adipose cells by ligands for receptors (R) that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changes in glucose transport activity and cAMP, as assessed by 3-O-methylglucose uptake and (-/+) cAMP-dependent protein kinase (A-kinase) activity ratios, respectively, were monitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C., Dhillon, G. S., and Londos, C. (1985) J. Biol. Chem. 260, 15122-15129). Removal of endogenous adenosine with adenosine deaminase decreased insulin-stimulated glucose transport activity by approximately 30%, which was prevented or restored with Ri agonists such as phenylisopropyladenosine, nicotinic acid, and prostaglandin E1. These changes in transport activity were not accompanied by changes in A-kinase activity ratios, indicating that Ri-mediated effects on transport are independent of cAMP changes. Addition of an Rs ligand, isoproterenol, in the presence of adenosine increased kinase activity but did not change glucose transport activity. Conversely, upon removal of adenosine, addition of Rs ligands such as isoproterenol, adrenocorticotropic hormone, or glucagon strongly inhibited transport (approximately 50%) and stimulated kinase activity. However, subsequent addition of phenylisopropyladenosine nearly restored transport activity without alteration of A-kinase activity. These data and additional kinetic experiments suggest that Rs-mediated glucose transport modulations are also independent of cAMP. The interchangeability of ligands for both Rs and Ri receptors in modulating transport activity suggests that these cAMP-independent effects are mediated by the stimulatory (Ns) and inhibitory (Ni) guanyl nucleotide-binding regulatory proteins of adenylate cyclase. All Rs-and Ri-induced changes in transport activity occurred without a change in glucose transporter distribution, as assessed by D-glucose-inhibitable cytochalasin B binding, suggesting that Rs and Ri ligands modulate the intrinsic activity of the glucose transporter present in the plasma membrane.     

Isoproterenol-induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium

The effect of beta-adrenergic stimulation on sarcolemmal protein phosphorylation was examined in intact ventricular myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with 32Pi after which membrane vesicles enriched 3-5-fold in sarcolemma were isolated by differential centrifugation followed by sucrose gradient centrifugation. Perfusion of hearts with isoproterenol stimulated 32P incorporation into a protein of apparent molecular weight of 15,000, which copurified with sarcolemmal vesicles. The increase in 32P incorporation was rapid in onset and elevated 2.5-3.0-fold after 30-45 s exposure of hearts to 100 nM isoproterenol. A positive correlation was found between stimulation of phosphorylation of the 15-kDa protein and the increase in the maximal rate of developed tension in intact ventricles after administration of isoproterenol. Phosphorylated phospholamban (most likely present as a contaminant) was also identified in the same sarcolemmal preparations. However, phospholamban and the 15-kDa sarcolemmal substrate were different proteins. Boiling of the membrane samples in sodium dodecyl sulfate prior to electrophoresis dissociated the high Mr form of phospholamban into the form of lower Mr but did not alter the mobility of the 15-kDa protein in sodium dodecyl sulfate-polyacrylamide gels. The 15-kDa protein did not undergo the electrophoretic mobility shift that is characteristic of phospholamban after cAMP-dependent phosphorylation nor did it cross-react with a highly specific phospholamban antibody. In vitro phosphorylation experiments conducted with the unmasking agent Triton X-100 suggested that the 15-kDa protein was localized to the cytoplasmic surfaces of sarcolemmal vesicles. These results demonstrate phosphorylation of a sarcolemmal protein, distinct from phospholamban, in response to beta-adrenergic stimulation of the heart. Phosphorylation of the sarcolemmal 15-kDa protein may play a role in mediating the effects of beta-adrenergic agonists on cardiac contractile force.     

Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells

The counterregulatory action of catecholamines on insulin-stimulated glucose transport and its relation to glucose transporter phosphorylation were studied in isolated rat adipose cells. Plasma membranes exhibiting reduced glucose transport activity were prepared as described previously (Joost, H. G., Weber, T. M., Cushman, S. W., and Simpson, I. A. (1986) J. Biol. Chem. 261, 10033-10036) from cells treated with insulin, and subsequently with isoproterenol and adenosine deaminase. In these membranes, transporter affinity for cytochalasin B binding was significantly reduced (KD = 133.5 +/- 14 versus 89.8 +/- 11 nM, means +/- S.E.) with no change in number of sites or immunoreactivity of the transporter on Western blots. Reconstituted plasma membrane transport was significantly lower with isoproterenol treatment (0.50 +/- 0.12 versus 0.97 +/- 0.27 nmol/mg protein/10 s). In contrast, transport activity reconstituted from corresponding intracellular transporters (from low density microsomes) was unchanged (5.4 +/- 2.2 versus 6.9 +/- 1.2 nmol/mg protein/10 s). Thus, the intrinsic activity change of the transporter produced by catecholamines appears to reflect a structural modification that is confined to the plasma membrane and not recycled into the intracellular compartment. In cells equilibrated with [32P]phosphate, neither insulin nor isoproterenol induced [32P]phosphate incorporation into the glucose transporter immunoprecipitated from plasma membranes. Conversely, phorbol 12-myristate 13-acetate stimulated significant incorporation of [32P]phosphate into the glucose transporter in insulin-stimulated cells without any change in plasma membrane transport activity or transporter concentration. Thus, the phosphorylation state of the glucose transporter does not seem to be involved in either signaling transporter translocation or triggering changes in transporter intrinsic activity.     

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.     

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.     

The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin

At maximally active concentrations with 20-min exposure, insulin and phorbol myristate acetate (PMA) stimulated hexose transport in 3T3-L1 adipocytes by 11- and 2-fold, respectively. The potential role of phosphorylation of the glucose transporter (GT) in these stimulations was investigated by the isolation of GT through immunoprecipitation from ortho[32P]phosphate-labeled 3T3-L1 adipocytes. It was found that there was no significant 32P incorporation into GT from basal adipocytes after 2- or 18 h-labeling in the presence of 0.5 mCi of 32Pi/ml. Furthermore, under these labeling conditions, insulin treatment for 1, 4, or 30 min failed to stimulate the phosphorylation of GT. Also, there was no detectable phosphate incorporation into GT upon reversal of insulin-stimulated hexose transport by the removal of insulin (half-time for reversal approximately 8 min). In contrast to these results, exposure of adipocytes to PMA (1 microM) for 20 min elicited a phosphorylation of GT to the extent of about 0.1 phosphate/GT molecule. Exposure of cells to both insulin and PMA resulted in a 3-fold increase in the level of phosphate in GT compared to that seen with PMA alone. Possibly this increase is due to the translocation of GT to the plasma membrane where it is a better substrate for activated protein kinase C. Stimulation of hexose transport was the same with the combined treatment of insulin and PMA compared to that seen with insulin alone. These results indicate that neither a change in the phosphorylation state of the GT nor activation of protein kinase C is involved in the mechanism by which the insulin receptor stimulates glucose transport.     

Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes

Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4 alpha-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. The catecholamine isoproterenol induced a 51% desensitization. Incubation of 32Pi labeled erythrocytes with TPA promoted a 3- to 4-fold increase in phosphorylation of the beta-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating beta-adrenergic receptor function.     

Hormone-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. Regulation of insulin- and cAMP-dependent activation by phosphorylation.

In 32PO4-labeled adipocytes, isoproterenol (ISO) or physiologically relevant concentrations of insulin rapidly increased phosphorylation of a particulate 135-kDa protein which has been identified as a cGMP-inhibited "low Km" cAMP phosphodiesterase (CGI-PDE) by several criteria, including selective immunoprecipitation with anti-CGI-PDE IgG (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 533-537). The time courses and concentration dependences for phosphorylation of CGI-PDE by ISO and insulin correlated with CGI-PDE activation in the presence of these agents; effects of ISO were somewhat more rapid than those of insulin. Adenosine deaminase, which metabolizes the adenylate cyclase inhibitor adenosine, also rapidly induced phosphorylation and activation of CGI-PDE. Phenylisopropyladenosine (an adenosine deaminase-resistant adenosine analog) prevented or reversed both adenosine deaminase-stimulated phosphorylation and activation of CGI-PDE (IC50 approximately 0.2 nM). Incubation of adipocytes with 0.1 nM insulin in the presence of ISO rapidly produced 30-200% greater activation and phosphorylation of CGI-PDE than the expected added effects of insulin and ISO individually; both effects preceded the insulin-induced decreases in protein kinase A activity and inhibition of lipolysis. These and other results indicate that CGI-PDE can be phosphorylated at distinct sites and activated by cAMP-dependent and insulin-dependent serine kinase(s), that the activation state of CGI-PDE reflects its relative phosphorylation state, and that synergistic phosphorylation/activation of CGI-PDE may be important in the antilipolytic action of insulin.     

Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells.

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.     

Ca2+ (calmodulin)-dependent phosphorylation of plasma membranes of pig myometrium

The high-purified vesicles of pig myometrium sarcolemma closed, mainly, so that the cytoplasmatic side is outside possess the Ca2+ (calmodulin)-dependent protein kinase activity. The initial rate of the endogenic phosphorylation without exogenic calmodulin is 6.3 and with its presence--10.7 pmol of 32Pi 1 min per 1 mg of protein. Km for ATP is equal to 164 microM, and Vmax--0.27 nmol of 32Pi 1 min per 1 mg of protein. Exogenic calmodulin increases the affinity to ATP (50 microM), Vmax being unchanged. Under optimal concentrations of calmodulin (10(-7)-10(-6) M) and 10(-4) M Ca2+ the protein kinase activity is 0.132 nmol of 32Pi min per 1 mg of protein. Electrophoresis in DS-PAAG has shown that membrane proteins with molecular weight of 105, 58, 25, 12 and 2 kDa are basic substrates of Ca2+ (calmodulin)-dependent phosphorylation. Trifluoperazine++ in the concentration of 40 microM inhibits phosphorylation of all five proteins. Ca2+ (calmodulin)-dependent phosphorylation is supposed to be a regulator of Ca2+-transport processes of sarcolemma.     

Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.     

Protein phosphorylation in a myogenic cell line: effects of insulin, epinephrine and glucose

Protein phosphorylation was studied in L6 cultured muscle cells by incubating cells with Na 32Pi and subsequently exposing them to external agents. L6 cells readily incorporated 32Pi into a number of peptides approaching steady-state incorporation by 2 h. Insulin stimulated the phosphorylation of one peptide of molecular mass 29,000 daltons by 37% with an ED50 of 3 mU/ml. This peptide was located in the high-speed pellet (105,000 g 60 min) which is consistent with an S6 ribosomal protein. Epinephrine (10(-5) M) led to only a modestly stimulated (less than 14%) phosphorylation of three peptides of molecular masses 39,000, 29,000 and 21,000 daltons. Glucose (5-50 mM) stimulated the phosphorylation of one peptide of molecular mass 19,000 daltons by 24%.     

Serine protein kinase activity associated with rotavirus phosphoprotein NSP5.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.     

Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.     

Enhanced phosphorylation of a coated vesicle polypeptide in response to insulin stimulation of rat adipocytes

The effect of insulin to increase the cell surface concentration of various receptors is accompanied by an increase in the concentration of clathrin assembled on the plasma membrane (Corvera, S. (1990) J. Biol. Chem. 265, 2413-2416). In the present study, clathrin-coated membranes were purified from isolated adipocytes labeled isotopically with [32P]orthophosphate. Analysis of the coated vesicle preparation by polyacrylamide gel electrophoresis and autoradiography revealed the presence of a cluster of phosphopeptides of 90-100 kDa as well as other phosphorylated species of 125, 70, 58, 50, 43, and 32 kDa. Incubation of the coated vesicles in alkaline pH resulted in the elution of the majority of the phosphopeptides, suggesting that these components are part of the clathrin coat and not integral membrane proteins. A pronounced increase in the amount of phosphate incorporated into the 125-kDa species was observed in response to stimulation of labeled cells by low concentrations of insulin. Phosphoamino acid analysis of an acid hydrolysate of this band revealed that its phosphorylation occurred exclusively on serine residues. The increased serine phosphorylation of this protein was apparent after only 2 min of exposure of cells to insulin and persisted for at least 60 min. The effect of insulin to increase the cell surface concentration of receptors and the assembly of clathrin on the plasma membrane displays a similar time course. Phorbol esters or dibutyryl cyclic AMP did not mimic the effects of insulin to stimulate the incorporation of [32P]phosphate into the 125-kDa polypeptide. Phosphorylation of the 125-kDa polypeptide was not observed after incubation of purified adipocyte-coated vesicles with [gamma-32P]ATP, suggesting that the kinase responsible for this reaction may not be contained within the clathrin-coated vesicle itself. These results suggest that phosphorylation of this 125-kDa polypeptide in intact cells may play a role in the regulation of clathrin-coated membrane formation and receptor-mediated endocytosis in response to insulin.     

Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications

Previously, we reported that protein phosphorylation plays an important role in regulating soluble l-glutamic acid decarboxylase (GAD) [Bao, J. (1995) J. Biol. Chem. 270, 6464-6467] and membrane-associated GAD activity [Hsu, C. C. (1999) J. Biol. Chem. 274, 24366-24371]. Here, we report the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrate that protein kinase A (PKA) and protein kinase C isoform epsilon are the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. Direct phosphorylation of GAD65 and GAD67 was demonstrated by incorporation of [(32)P] from [gamma-(32)P]ATP into purified GAD65 and GAD67 and immunoblotting assay using anti-phosphoserine/threonine antibodies. We have identified one specific phosphorylation site, threonine 91 (T91), in hGAD67 that can be phosphorylated by PKA using MALDI-TOF. Site-directed mutation of T91 to alanine abolished PKA-mediated phosphorylation and inhibition of GAD activity. Furthermore, mutation of T91 to aspartic acid or glutamic acid mimics the effect of phosphorylation. A model depicting the effect of phosphorylation on GAD activity upon neuronal stimulation is also proposed.     

An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells

Insulin stimulates the phosphorylation of the 40 S ribosomal subunit protein, S6, in intact 32P-labeled H4IIE-C3 cells, a rat hepatoma line. Cell-free cytosolic extracts from H4 cells exhibit a 5- to 10-fold increase in S6 protein kinase activity (measured by transfer of 32P to exogenous 40 S rat liver ribosomal subunits) when prepared from cells exposed to insulin prior to homogenization. Stimulation of S6 phosphorylation in intact cells and activation of S6 protein kinase in cell-free extracts are both detectable within 2 min after insulin, and are maximally stimulated by 10 min. Half-maximal stimulation is observed at 10(-11) M insulin. The stimulated S6 kinase activity requires ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to be present during the kinase assay for full expression. Despite the presence of a 5- to 10-fold increase in S6 protein kinase activity, the extracts from insulin-treated cells exhibit no stimulated kinase activity toward casein, histone, or ATP-citrate lyase assayed under the conditions employed for S6. Thus, insulin mediates the rapid activation of protein kinase specific for ribosomal protein S6 by an as yet unidentified mechanism.