Stimulation of glycogen phosphorylase kinase by phospholipids

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.     

Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase

Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.     

Activation of membrane cholesterol by displacement from phospholipids

We tested the hypothesis that certain membrane-intercalating agents increase the chemical activity of cholesterol by displacing it from its low activity association with phospholipids. Octanol, 1,2-dioctanoyl-sn-glycerol (a diglyceride), and N-hexanoyl-D-erythrosphingosine (a ceramide) were shown to increase both the rate of transfer and the extent of equilibrium partition of human red blood cell cholesterol to methyl-beta-cyclodextrin. These agents also promoted the interaction of the sterol with two cholesterol-specific probes, cholesterol oxidase and saponin. Expanding the pool of bilayer phospholipids with lysophosphatides countered these effects. The three intercalators also protected the red cells against lysis by cholesterol depletion as if substituting for the extracted sterol. As is the case for excess plasma membrane cholesterol, treating human fibroblasts with octanol, diglyceride, or ceramide stimulated the rapid inactivation of their hydroxymethylglutaryl-CoA reductase, presumably through an increase in the pool of endoplasmic reticulum cholesterol. These data supported the stated hypothesis and point to competition between cholesterol and endogenous and exogenous intercalators for association with membrane phospholipids. We also describe simple screens using red cells in a microtiter well format to identify intercalating agents that increase or decrease the activity of membrane cholesterol.     

Regulation of receptor-mediated protein kinase C membrane trafficking by autophosphorylation

Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. In response to activation of cell-surface receptors, PKC is directed to the plasma membrane by two membrane-targeting domains, namely the C1 and C2 regions. This is followed by the return of the enzyme to the cytoplasm, a process shown recently to require PKC autophosphorylation (Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874). In the present study, we examined mechanisms of translocation and reverse translocation and the role of autophosphorylation in these processes. By visualizing the trafficking of wild-type as well as mutant PKCbetaII in live cells, we demonstrated that in response to cell-surface receptor activation, the function of the C1 region is required but not sufficient for recruitment of the enzyme to the plasma membrane. The C2 region is also critical in anchoring the enzyme to the plasma membrane. Furthermore, the inability of a kinase-deficient PKC to undergo reverse translocation was restored by the addition of intracellular calcium chelators, suggesting a role for the C2 region in the persistent phase of translocation. On the other hand, the inability of a C2 deletion mutant (C1 region intact) to translocate in response to agonist was reversed in mutants lacking kinase activity or by mutation of the Ser(660) autophosphorylation site to alanine, suggesting that autophosphorylation of this site is required for opposing the action of the C2 region. Therefore, the membrane-targeting function of the C1 region is facilitated by the C2 region and appears to be opposed by autophosphorylation. Taken together, these findings provide novel evidence of the functional regulation of reversible PKC membrane localization by autophosphorylation, and they show that the dynamic translocation of PKC in response to agonists is tightly regulated in a collaborative fashion by the C1 and C2 regions in balance with the effects of autophosphorylation.     

Stimulation of autophosphorylation of rabbit skeletal muscle phosphorylase kinase by glycogen synthase on glycogen particles

Glycogen synthase stimulated the autophosphorylation and autoactivation of phosphorylase kinase from rabbit skeletal muscle. This stimulation was additive to that by glycogen and the reaction was dependent on Ca2+. The effect by glycogen synthase was maximum within the activity ratio (the activity of enzyme without glucose-6-P divided by the activity with 10 mM glucose-6-P) of 0.3 and over 0.3 it was rather inhibitory. The results suggest that autophosphorylation of phosphorylase kinase in the presence of glycogen synthase on glycogen particles may be an important regulatory mechanism of glycogen metabolism in skeletal muscle.     

Calcium- and lipid-independent protein kinase C autophosphorylation. Activation by low pH

The activity of protein kinase C is dependent on communication between a catalytic domain and a Ca2+- and lipid-binding regulatory domain in the kinase molecule. It is shown here that acidic reaction conditions can bypass the calcium and lipid requirement in the autophosphorylation of protein kinase C. Acidic pH does not entirely deregulate the kinase, though, since only autophosphorylation is favored between pH 4 and 6 and not the phosphorylation of alternative substrate proteins. Interestingly, low pH stably activated protein kinase C: when restored to neutral pH, the autophosphorylation reaction remained independent of Ca2+ and lipid. These observations suggest that protonation of functional groups in the protein kinase C molecule, with their pKa suggestive of histidine imidazole, can produce a stable conformation where regulatory constraints on enzyme activity have been removed.     

Activation of endogenous phosphorylase kinase in liver glycogen pellet by cAMP-dependent protein kinase

Liver glycogen phosphorylase associated with the glycogen pellet was activated by a MgATP-dependent process. This activation was reduced by 90% by ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, not affected by the inhibitor of the cAMP-dependent protein kinase, and increased 2.5-fold by the catalytic subunit of cAMP-dependent protein kinase. Low levels of free Ca2+ (8 x 10(-8) M) completely prevented the effects of the chelator. The activation of phosphorylase by MgATP was shown not to be due to formation of AMP. DEAE-cellulose chromatography of the glycogen pellet separated phosphorylase from phosphorylase kinase. The isolated phosphorylase was no longer activated by MgATP in the presence or absence of the catalytic subunit of cAMP-dependent protein kinase. The isolated phosphorylase kinase phosphorylated and activated skeletal muscle phosphorylase b and the activation was increased 2- to 3-fold by the catalytic subunit of cAMP-dependent protein kinase. Mixing the isolated phosphorylase and phosphorylase kinase together restored the effects of MgATP and the catalytic subunit of cAMP-dependent protein kinase on phosphorylase activity. These findings demonstrate that the phosphorylase kinase associated with liver glycogen has regulatory features similar to those of muscle phosphorylase kinase.     

Activation/dephosphorylation of muscle glycogen synthase phosphorylated by phosphorylase kinase

1. Glycogen synthase from rabbit skeletal muscle was phosphorylated by phosphorylase kinase to yield synthase b2. 2. Dephosphorylation and activation of synthase b2 by the catalytic subunits of protein phosphatase-1 (PP-1c) and protein phosphatase-2A (PP-2Ac) was studied. The apparent Km of PP-1c and PP-2Ac were 3.3 microM and 6.2 microM, respectively. The apparent Vmax of PP-1c was about two times larger than that of PP-2Ac. 3. Ligands with phosphate moiety (AMP, glucose-6-P at high concentration) caused an inhibition in dephosphorylation by both phosphatases. Spermine inhibited the dephosphorylation by PP-1c and stimulated the action of PP-2Ac. Therefore it can be employed to distinguish the phosphatases using synthase b2 as substrate.     

Activation of phosphorylase kinase from rabbit muscle by actin and calmodulin

The activation of different forms of muscle phosphorylase kinase by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase b by actin is not due to the presence of trace amounts of calmodulin in actin preparations: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation by actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of V. F-actin has no stimulating effect on the phosphorylated form (a) of phosphorylase kinase or on the form a previously activated by proteolysis.     

Activation of phosphorylase kinase from brain by small amounts of calcium ion

E lectrical stimulation of brain results in an enhancement of glycogen breakdown (K ing , L owry , P assonneau and V enson , 1967) and one should therefore expect exitation to be linked in some way to glycogen metabolism. Stimulation of muscle cells, which induces contraction, also increases glycogen breakdown. In this case it has been shown that a minute amount of Ca²⁺, which couples excitation and contraction, also serves as a link between excitation and glycogenolysis (O zawa , H osoi and E bashi , 1967). A small amount of Ca²⁺, released into the sarcoplasm as a result of excitation of the cell membrane, activates phosphorylase kinase. The activated kinase, in turn, converts phosphorylase α (the inactive form of phosphorylase) to phosphorylasea (the active form). The present communication summarizes the evidence that a similar mechanism operates in brain.     

Control of platelet glycogenolysis; Activation of phosphorylase kinase by calcium.

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels; Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca-2+ markedly activate phosphorylase b kinase from human platelets, with a Ka of 0i muM Ca-2+, which is similar to that for the skeletal muscle enzyme; The kinase activity is unstable, and on enzyme ageing is a 50% loss in activity with the Ka decreasing to 0.33 muM Ca-2+. In unstilulated platelets, phosphorylase a was 13.3% of toal measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes; These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca-2+, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved;     

Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-kappaB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-kappaB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.     

Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation

MST1 is a member of the Sterile-20 family of cytoskeletal, stress, and apoptotic kinases. MST1 is activated by phosphorylation at previously unidentified sites. This study examines the role of phosphorylation at several sites and effects on kinase activation. We define Thr(183) in subdomain VIII as a primary site of phosphoactivation. Thr(187) is also critical for kinase activity. Phosphorylation of MST1 in subdomain VIII was catalyzed by active MST1 via intermolecular autophosphorylation, enhanced by homodimerization. Active MST1 (wild-type or T183E), but not inactive Thr(183)/Thr(187) mutants, was also highly autophosphorylated at the newly identified Thr(177) and Thr(387) residues. Cells expressing active MST1 were mostly detached, whereas with inactive MST1, adhesion was normal. Active MKK4, JNK, caspase-3, and caspase-9 were detected in the detached cells. These cells also contained all autophosphorylated and essentially all caspase-cleaved MST1. Similar phenotypes were elicited by a caspase-insensitive D326N mutant, suggesting that kinase activity, but not cleavage of MST1, is required. Interestingly, an S327E mutant mimicking Ser(327) autophosphorylation was also caspase-insensitive, but only when expressed in caspase-3-deficient cells. Together, these data suggest a model whereby MST1 activation is induced by existing, active MST kinase, which phosphorylates Thr(183) and possibly Thr(187). Dimerization promotes greater phosphorylation. This leads to induction of the JNK signaling pathway, caspase activation, and apoptosis. Further activation of MST1 by caspase cleavage is best promoted by caspase-3, although this appears to be unnecessary for signaling and morphological responses.     

Acidic phospholipids stimulate the autophosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase

The autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase was stimulated by the acidic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol. Other phospholipids (phosphatidylethanolamine, phosphatidylcholine, sphingomyelin), acidic compounds (dextran sulfate, polyglutamic acid, chondroitin sulfate, hyaluronic acid) and calciumcalmodulin were essentially inactive. Sodium dodecyl sulfate also stimulated the catalytic subunit autophosphorylation, but other detergents (Triton X-100 and deoxycholic acid) did not. The combination of phosphatidic acid and sodium dodecyl sulfate was as effective as each agent alone, suggesting similar stimulation mechanisms. The data suggest that acidic membrane phospholipids might have a role in regulating the autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.     

Ganglioside-modulated protein phosphorylation in muscle. Activation of phosphorylase b kinase by gangliosides

Gangliosides have profound effects on protein phosphorylation in skeletal muscle. Addition of GT1b to guinea pig muscle extract stimulated the phosphorylation of a 98-kDa protein 4-8-fold. In contrast, Ca2+ stimulated the phosphorylation of this protein and two other proteins with apparent Mr of 107,000 and 145,000, respectively. Addition of GT1b in the presence of Ca2+ further enhanced the phosphorylation of the 98-kDa protein but completely inhibited the phosphorylation of both the 107- and the 145-kDa proteins. The nature of the ganglioside-modulated 98-kDa protein has been characterized. Results on the pH activity profiles and the requirements of Ca2+ for phosphorylation suggest that this phosphoprotein may correspond to glycogen phosphorylase. Phosphorylation of purified rabbit muscle phosphorylase b by nonactivated phosphorylase kinase was stimulated by GT1b. This stimulation was in part due to an activation of the kinase activity. Autophosphorylation of highly purified phosphorylase kinase was increased 4-10-fold in the presence of GT1b. Polysialogangliosides were more potent than monosialogangliosides in stimulating the autocatalytic activity, whereas asialo-GM1, colominic acid, N-acetylneuraminic acid, and phosphatidylserine were ineffective. The effects of gangliosides were dose-dependent. At physiological pH, the concentrations of GT1b required for half-maximal stimulation of the autophosphorylation of phosphorylase kinase were 6.4 microM in the absence of Ca2+ and 1.3 microM when the divalent cation was present. These findings suggest that gangliosides may play a role as biomodulators in the regulation of glycogenolysis in muscle.     

Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.