Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase

1. We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the cDNA sequence [López-Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85, 5784-5788] shows that this site corresponds to Ser1215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region. 3. Prior phosphorylation of Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP-activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Ser1200 by the AMP-activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino-terminal region containing Ser77/79 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77.     

Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity

Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.     

Negative interactions between phosphorylation of acetyl-CoA carboxylase by the cyclic AMP-dependent and AMP-activated protein kinases

We have reported previously that cyclic AMP-dependent protein kinase phosphorylates two sites on acetyl-CoA carboxylase (site 1: Arg-Met-Ser(P)-Phe, and site 2: Ser-Ser(P)-Met-Ser-Gly-Leu), while the AMP-activated protein kinase also phosphorylates site 1, plus site 3 (Ser-Ser-Met-Ser(P)-Gly-Leu), the latter being two residues C-terminal to site 2. We now report that prior phosphorylation of site 2 by cyclic AMP-dependent protein kinase prevents the subsequent phosphorylation of site 3 and the consequent large decrease in Vmax produced by the AMP-activated protein kinase. Similarly, prior phosphorylation of site 3 by the AMP-activated protein kinase prevents subsequent phosphorylation of site 2 by cyclic AMP-dependent protein kinase.     

Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay

1. We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for the AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). 2. Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. 3. The Km of AMP-activated protein kinase for the SAMS peptide is higher than that for acetyl-CoA carboxylase, but the Vmax for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. 4. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a post-mitochondrial supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMP-activated protein kinase in this tissue. We could not demonstrate AMP-dependence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br8AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. 5. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mouse macrophage cell line, and find that there is a correlation between tissues expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. 6. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase.     

Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.     

Roles of the AMP-activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes.

1. In isolated rat adipocytes, acetyl-CoA carboxylase is inactivated by treatment of the cells with adrenaline or the beta-agonist isoproterenol, but not by the alpha-agonist phenylephrine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30-40% increase in the labelling of enzyme isolated from 32P-labelled cells. 2. Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser-Ser77-Met-Ser79-Gly-Leu-His-Leu-Val-Lys, containing Ser-77 (phosphorylated by cyclic-AMP-dependent protein kinase) and Ser-79 (phosphorylated by the AMP-activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser-79 in both basal and hormone- or agonist-stimulated cells. Treatment of adipocytes with various agents which activate cyclic-AMP-dependent protein kinase by receptor-independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl-CoA carboxylase and increased phosphorylation at Ser-79. 3. The (Rp)-[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic-AMP-dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl-CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic-AMP-dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl-CoA carboxylase. 4. As shown by okadaic acid inhibition, greater than 95% of the acetyl-CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase-2A, with less than 5% attributable to protein phosphatase-1. Inhibition of protein phosphatase-1 via phosphorylation of inhibitor-1 is therefore unlikely to be the mechanism by which cyclic-AMP-dependent protein kinase indirectly increases phosphorylation of acetyl-CoA carboxylase. Various other potential mechanisms are discussed.     

Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism

Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantly at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMP-dependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo.     

Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets.

1. Acetyl-CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze-clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase-2A. Amino acid sequencing and fast-atom-bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell-free assays by the AMP-activated protein kinase. 2. The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N-terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP-activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220-kDa core fragment (which contains Ser1200 and Ser1215). 3. Analysis of the phosphorylation state of Ser79 in acetyl-CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze-clamping is not used. The higher phosphorylation observed in extracts made without freeze-clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP-activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re-emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl-CoA carboxylase (as well as 3-hydroxy-3-methylglutaryl-CoA reductase). 4. Using the freeze-clamping procedure, the ratio of 'expressed' activity (measured in the presence of protein phosphatase inhibitors) to 'total' activity (measured after complete dephosphorylation) of rat liver acetyl-CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase, another substrate for the AMP-activated protein kinase. Neither the activity of the AMP-activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of WASP by the Cdc42-associated kinase ACK1: dual hydroxyamino acid specificity in a tyrosine kinase

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.     

The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase

In addition to acetyl-CoA carboxylase and HMG-CoA reductase, the AMP-activated protein kinase phosphorylates glycogen synthase, phosphorylase kinase, hormone-sensitive lipase and casein. A number of other substrates for the cyclic AMP-dependent protein kinase, e.g., L-pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, are not phosphorylated at significant rates. Examination of the sites phosphorylated on acetyl-CoA carboxylase, hormone-sensitive lipase, glycogen synthase and phosphorylase kinase suggests a consensus recognition sequence in which the serine residue phosphorylated by the AMP-activated protein kinase has a hydrophobic residue on the N-terminal side (i.e., at -1) and at least one arginine residue at -2, -3 or -4. Substrates for cyclic AMP-dependent protein kinase which lack the hydrophobic residue at -1 are not substrates for the AMP-activated protein kinase.     

Evidence for a protein kinase cascade in higher plants. 3-Hydroxy-3-methylglutaryl-CoA reductase kinase.

Protein phosphorylation is well established as a regulatory mechanism in higher plants, but only a handful of plant enzymes are known to be regulated in this manner, and relatively few plant protein kinases have been characterized. AMP-activated protein kinase regulates key enzymes of mammalian fatty acid, sterol and isoprenoid metabolism, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. We now show that there is an activity in higher plants which, by functional criteria, is a homologue of the AMP-activated protein kinase, although it is not regulated by AMP. The plant kinase inactivates mammalian HMG-CoA reductase and acetyl-CoA carboxylase, and peptide mapping suggests that it phosphorylates the same sites on these proteins as the mammalian kinase. However, with the target enzymes purified from plant sources, it inactivates HMG-CoA reductase but not acetyl-CoA carboxylase. The kinase is located in the soluble, and not the chloroplast, fraction of leaf cells, consistent with the idea that it regulates HMG-CoA reductase, and hence isoprenoid biosynthesis, in vivo. The plant kinase also appears to be part of a protein kinase cascade which has been highly conserved during evolution, since the kinase is inactivated and reactivated by mammalian protein phosphatases (2A or 2C) and mammalian kinase kinase, respectively. This contrasts with the situation for many other mammalian protein kinases involved in signal transduction, which appear to have no close homologue in higher plants. To our knowledge, this represents the first direct evidence for a protein kinase cascade in higher plants.     

Regulation of the renal-specific Na+-K+-2Cl- co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.     

Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase

Glycogen synthase kinase-3 phosphorylates three serine residues on glycogen synthase (sites 3a, 3b and 3c) which are all located in the same nine-amino-acid segment of the polypeptide chain. The sequence in this region is: Arg-Tyr-Pro-Arg-Pro-Ala-Ser(P)-Val-Pro-Pro-Ser(P)-Pro-Ser-Leu-Ser(P)-Arg-. These serine residues are distinct from the sites phosphorylated preferentially by cyclic-AMP-dependent protein kinase (sites 1a and 1b) and phosphorylase kinase (site 2). The N-terminal sequence of glycogen synthase containing the serine residue phosphorylated by phosphorylase kinase has been extended. The sequence in this region is: Pro-Leu-Ser-Arg-Thr-Leu-Ser(P)-Val-Ser-Ser-Leu-Pro-Gly-Leu-Glu-Asp-Trp-Glu-Asp- Glu-Phe-Asp-Leu-Glu-Asn-Ser-Val-Leu-Phe-(Asx2,Glx2,Ala2,Val2,Lys)-. The similarity to the N-terminal sequence of phosphorylase is confined to the immediate vicinity of the phosphorylation site (residues 4--15). The relationship of glycogen synthase kinase-3 to glycogen synthase kinases that have been described by other laboratories is discussed.     

The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase

Acetyl-CoA carboxylase purified from isolated hepatocytes is activated dramatically by protein phosphatase treatment, concomitant with a reduction of the phosphate content from 3.7 to 1.1 mol/subunit. Glucagon treatment of the cells produces a further inactivation of the enzyme that is totally reversed by phosphatase treatment, and is associated with an increase in phosphate content of 0.8 mol/subunit, distributed in two peptides which contain the sites phosphorylated in vitro by the cyclic AMP-dependent and AMP-activated protein kinases. Sequencing of these peptides shows that the low activity of acetyl-CoA carboxylase is due to phosphorylation by the AMP-activated protein kinase, and not cyclic AMP-dependent protein kinase, even after glucagon treatment.     

AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser2?2 and Ser3??, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1-42) leads to increased phosphorylation of tau at Ser2?2/Ser3?? and Ser33??. Activation of AMPK by Aβ-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1-42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.     

Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion.

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.     

Insulin and phorbol ester stimulate phosphorylation of acetyl-CoA carboxylase at similar sites in isolated adipocytes. Lack of correspondence with sites phosphorylated on the purified enzyme by protein kinase C

1. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates fatty acid synthesis from glucose in isolated adipocytes with a half-maximal effect at 0.72 microM. In seven batches of cells, the maximal effects of TPA and insulin were 8.5 +/- 1.1-fold and 27.1 +/- 2.1-fold respectively. Insulin also stimulated fatty acid synthesis from acetate 8.9 +/- 0.5-fold (three experiments), but TPA did not significantly increase fatty acid synthesis from this precursor. 2. In contrast to insulin, TPA treatment of isolated adipocytes did not produce an activation of acetyl-CoA carboxylase which was detectable in crude cell extracts. 3. The total phosphate content of acetyl-CoA carboxylase, isolated from adipocytes in the presence of protein phosphatase inhibitors, was estimated by 32P-labelling experiments to be 2.6 +/- 0.1 (5), 3.4 +/- 0.2 (5), and 3.8 +/- 0.2 (3) mol/mol subunit for enzyme from control, insulin- and TPA-treated cells respectively. Insulin and TPA stimulated phosphorylation within the same two tryptic peptides. 4. Purified acetyl-CoA carboxylase is phosphorylated in vitro by protein kinase C at serine residues which are recovered in three tryptic peptides, i.e. peptide T1, which appears to be identical with the peptide Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys phosphorylated by cyclic-AMP-dependent protein kinase, and peptides Ta and Tb, which have the sequences Ile-Asp-Ser(P)-Gln-Arg and Lys-Ile-Asp-Ser(P)-Gln-Arg respectively, and which appear to be derived from a single site by alternative cleavages. None of these correspond to the peptides whose 32P-labelling increase in response to insulin or TPA. Peptides Ta/Tb are not significantly phosphorylated in isolated adipocytes, even after insulin or TPA treatment. Peptide T1 is phosphorylated in isolated adipocytes, but this phosphorylation is not altered by insulin or TPA. 5. These results show that TPA mimics the effect of insulin on phosphorylation, but not activation, of acetyl-CoA carboxylase, i.e. that these two events can be dissociated. In addition, phorbol ester stimulates phosphorylation of acetyl-CoA carboxylase in isolated adipocytes, but this is not catalyzed directly by protein kinase C, and acetyl-CoA carboxylase does not appear to be a physiological substrate for this kinase.     

Identification and role of the basal phosphorylation site on hormone-sensitive lipase

Phosphorylation site 2 on bovine hormone-sensitive lipase (HSL), which is phosphorylated in vitro by the AMP-activated protein kinase, has been found also to be phosphorylated in vitro by glycogen synthase kinase-4. Peptide mapping of HSL phosphorylated in vitro and in isolated adipocytes demonstrates that this site corresponds to the basal phosphorylation site on HSL, which is phosphorylated in intact adipocytes in the absence of lipolytic stimuli. Site 2 has been proposed to have an antilipolytic role in that phosphorylation at this site greatly reduces subsequent phosphorylation (at site 1) and activation of HSL by cyclic-AMP-dependent protein kinase. Further evidence for an antilipolytic role of site 2 has been obtained using a synthetic peptide based on the sequence around sites 1 and 2. Phosphorylation of the peptide at site 2 totally prevents the subsequent phosphorylation of site 1 and vice versa.     

Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.     

AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-alpha1 and -alpha2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser(79) (AMPK phosphorylation site) approximately 8.5-fold. Under these conditions, there is also an approximately 5.5-fold increase in phosphorylation of neuronal NO synthase-mu (nNOSmu;) at Ser(1451). These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSmu to protect the muscle from ischemia/metabolic stress.