Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Phosphorylation of atrial natriuretic factor R1 receptor by serine/threonine protein kinases: evidences for receptor regulation

The 130 kDa atrial natriuretic factor receptor (ANF-R₁) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99–126) and there is no detectable intrinsic kinase activity associated with the ANF-R₁ receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for ¹²⁵I-ANF. However, we have demonstrated a significant ³²P incorporation in the ANF-R₁ receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-R₁ receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension.Abbreviations ANF Atrial Natriuretic Factor - ANF-R₁ Atrial Natriuretic Factor Receptor, subtype 1 - ATP Adenosine Triphosphate - CaCl₂ Calcium Chloride - cAMP Adenosine cyclic 3,5-Monophosphate acid - cGMP Guanosine cyclic 35-Monophosphate acid - EDC 1-Ethyl-3-[3-Dimethylaminopropyl] Carbodiimide - EDTA Ethylenediaminetetraacetic Acid - GTP Guanosine Triphosphate - IBMX 3-isobutyl-1-methylxanthine - kDa Kilodaltons - MgCl₂ Magnesium Chloride - MgAC Magnesium Acetate - NaCl Sodium Chloride - PAGE Polyacrylamide Gel Electrophoresis - PKA cAMP-dependent protein kinase - PKG cGMP-dependent Protein Kinase - PKC Calcium/Phospholipid-dependent Protein Kinase - RIA Radioimmunoassay - SDS Sodium Dodecyl Sulfate - SHR Spontaneously Hypertensive Rat - Tris HCl Tris (Hydroxymethyl) aminomethane Hydrochloride - TPA 12-O-Tetradecanoyl-Phorbol-13-Acetate     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein     

Evidence for selective effects of vanadium on adipose cell metabolism involving actions on cAMP-dependent protein kinase

The insulin-like effects of vanadiumin vivo are likely to be achieved at micromolar concentrations. Demonstrated effects of vanadium on adipose tissue of streptozotocin-diabetic rats include inhibition of basal and stimulated rates of lipolysis and effects on fat cell protein phosphorylation. The studies described below examined the effects of vanadium (to a maximum concentration of 0.5 mM) on adipose cells or tissuein vitro. Vanadium, added as a vanadyl-albumin complex or as sodium orthovanadate, produced a marked (greater than 50%) inhibition of isoproterenol-stimulated lipolysis. Inhibition of lipolysis equivalent to that seen with insulin, was achieved with 100 M vanadium. In contrast, no insulin-like stimulation ofde novo fatty acid biosynthesis was observed with vanadium below 0.5 mM. Surprisingly, the antilipolytic effects of vanadium persisted in the presence of cilostamide, an inhibitor of the insulin-sensitive isoform of cyclic nucleotide phosphodiesterase. Studies with purified preparations of the catalytic subunit of cyclic AMP-dependent protein kinase revealed dose-dependent inhibition with vanadyl-glutathione (to a maximum of 40% inhibition). Equivalent inhibition of cyclic AMP-dependent phosphorylation of Kemptide (50%) was observed upon incubation of freshly-prepared fat-pad supernatant fractions with vanadyl-glutathione. These results suggest that effects of low concentrations of vanadium may be mediated, at least in part, by actions on the catalytic subunit of cyclic AMP-dependent protein kinase.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Purification of a 107 kilodalton (kDa) casein kinase G substrate from thyroid cytosol

Summary A 107 kDa (pp107) casein kinase G (ck-G) substrate has been purified from mouse and beef thyroid cytosol; ck-G was purified from beef thyroid cytosol. Ck-G and pp107 were found to co-elute on DEAE cellulose chromatography at approximately 300 mM NaCl. Ck-G and pp107 were separated by spermine-agarose affinity chromatography; ppl07 is eluted with a stepped gradient at 250 mM NaCl and ck-G is eluted at 500 mM NaCl. Ck-G was subsequently purified by casein-agarose and GTP-agarose affinity chromatography. The 107 kDa protein was purified using heparin-agarose affinity chromatography. Phosphorylation of purified pp107 by ck-G was stimulated by spermine (ED₅₀ = 0.2 mM) and inhibited by low concentrations of heparin (0.1–5 µg/ml). The Km and Vmax for the reaction were 1.46 µM and 32.2 nmoles P transferred/20 min/mg protein, respectively; 1 mole pp107 incorporated 0.81 mole phosphorus. pp107 was found to be an acidic substrate with a pI of 3.87 and was absorbed to wheat-germ agglutinin-agarose. The specificity of pp107 phosphorylation was studied using diacylglycerol-activated calcium/phospholipid-dependent protein kinase C, calcium-activated calmodulin-dependent protein kinase, and the catalytic subunit of cAMP-dependent protein kinase A. Phosphorylation of pp107 by the other protein kinases tested never exceeded 4% of that of ck-G. Our data show that pp107 is an acidic glycoprotein which may serve as a high-affinity and specific substrate for ck-G.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - ck-G Casein Kinase G (casein kinase II) - PK-C Diacylglycerol-Activated Calcium/phospholipid-Dependent Protein Kinase - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - EGTA Ethylene Glycol Bis (B-aminoethylether)-N,N,N,N-tetraacetic Acid - PMSF Phenylmethyl Sulfonyl Floride - TCA Trichloroacetic Acid     

Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes

The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the -subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of ³²P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the -subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn²⁺ (K_a = 1.0 mM) was much better than Mg²⁺ (K_a = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t_1/2 = 3.6 min) proceeded at a much slower rate compared to that of the -subunit of IR (t_1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4 : 1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR -subunit suggest that pp43 was not derived from IR -subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase.     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.     

Purification of a novel insulin-stimulated protein kinase from rat liver

We previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.     

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms and was investigated. Both SR and PKC were isolated from canine heart. Junctional and free SR vesicles were prepared by calcium-phosphate-loading. The substrate specificities of PKC and PKC were found to be similar in both SR fractions. A high molecular weight junctionally-associated protein was phosphorylated by PKA, PKC and an endogenous Ca²⁺/calmodulin-dependent protein kinase activity: the highest levels of phosphate incorporation being catalysed by the latter kinase. In addition to this high molecular weight junctionally-associated protein, PKC induced phosphorylation of 45, 96 kDa and several proteins of greater than 200 kDa in junctional SR. A protein of 96 kDa was phosphorylated by both isoforms in junctional and free SR. The major substrate for PKA, PKC, PKC and the Ca²⁺/calmodulin-dependent protein kinase, in both junctional and free SR, was phospholamban. Although the phosphorylation of phospholamban by PKC was activated by Ca²⁺, a component of this activity appeared to be independent of Ca²⁺. PKC-mediated phosphorylation of phospholamban was fully activated by 1 M Ca²⁺ whereas the Ca²⁺/calmodulin dependent kinase required concentrations in excess of 5 M Ca²⁺. In the in vitro system employed in these studies, the concentrations of either PKC or the catalytic subunit of PKA required to phosphorylate phospholamban were found to be similar. In addition, in the presence of a 15 kDa sarcolemmal-associated protein, which becomes phosphorylated upon activation of PKC in vivo, phosphorylation of phospholamban by PKC was unaffected. These results demonstrate that, although substrates for both subtypes are found in both junctional and free SR, PKC and PKC do not show differences in selectivity towards these substrates.Abbreviations Ca²⁺ free calcium - CaM kinase Ca²⁺/calmodulin-dependent protein kinase - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(b-aminoethylether)-N,N,N,N-tetraacetic acid - FSR free sarcoplasmic reticulum - JSR junctional sarcoplasmic reticulum - PKC protein kinase C - PS phosphatidylserine - SDS sodium dodecyl sulfate - SAG 1-stearoyl-2-arachidonylglycerol - TPCK L-1-tosylamido-2-phenylethyl chloromethyl ketone - Tris/HCI tris(hydroxymethyl)aminomethane hydrochloride This work was supported by a grant (to S.K.) from the Heart and Stroke Foundation of B.C. and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Recipient of a Studentship form the Heart and Stroke Foundation of Canada.     

Phosphorylation Sites Within α4 Subunits of α4β2 Neuronal Nicotinic Receptors: A Comparison of Substrate Specificities for cAMP-Dependent Protein Kinase (PKA) and Protein Kinase C (PKC)

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human 4 subunit of 42 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 M; Kemptide had a Km of 7.7 M. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 M and 2896 M, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 M; GS 1–8 had a Km of 2.1 M. VRCRSRSI had a comparative affinity for PKC with a Km of 327 M. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 M, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human 4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.     

Effects of phorbol ester on immunoreactive protein kinase C,insulin binding,and glucose uptake in astrocytic glial and neuronal cells from the brain

Phorbol esters, potent stimulators of protein kinase C (PKC), stimulate [³H]2-deoxy-d-glucose (dGlc) uptake and [¹²⁵I] insulin binding in cultured glial cells but not neuronal cells from neonatal rat brains. Using an antibody to the and forms of PKC we have demonstrated that both neuronal and glial cells contain an immunoactive PKC of Mr 80 kD, although the PKC level in neurons is greater than 4-fold that in glia. The majority of immunoactive PKC (63%) is cytosolic in glial cells although the reverse is true in neuronal cells, in which 88% of the PKC is membrane-bound in the basal state. The most potent phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates a redistribution of this enzyme in neuronal and glial cells. The TPA-stimulated translocation of PKC from cytosol to membrane precedes TPA's effecs of [³H]dGlc uptake and insulin binding in glial cells.     

Interaction of creatine kinase and hexokinase with the mitochondrial membranes,and self-association of creatine kinase: crosslinking studies

Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu⁺⁺-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK Adenylate Kinase (EC 2.7.4.3) - CKm Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2) - DMS Dimethyl Suberimidate - DTT Dithiothreitol - EGS Ethylene Glycol bis (succinimidyl succinate) - EGTA Ethylene Glycol bis (aminoethyl ether) - N,N,N,N Tetraacetic acid - G6P Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid - HK Hexokinase (EC 2.7.1.1) - MABI methyl 4-Azido Benzoimidate - NaPi Sodium Phosphate - SANPAH N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate - SDS Sodium Dodecyl Sulfate (sodium lauryl sulfate) - Tris Tris (hydroxymethyl) Aminomethane     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Effects of glucagon and insulin on the cyclic AMP binding capacity of hepatocyte cyclic AMP-dependent protein kinase

Extracts obtained from rat hepatocytes incubated with saline, glucagon or insulin were electrophoresed on polyacrylamide gels and then assayed for cyclic (³H)AMP binding capacity. Analysis of the binding patterns demonstrated that glucagon dissociated a holoenzyme of cyclic AMP-dependent protein kinase in a dose-dependent manner. The increase in free regulatory subunits and, hence, in free catalytic subunits explains the activation of this enzyme by glucagon in the liver. Insulin decreased both the amount of cyclic (³H)AMP bound to the holoenzyme and the capacity of the enzyme to be dissociated when the extracts were incubated with increasing concentrations of this cyclic nucleotide. We propose that these insulin-induced effects are determined by an inhibition of the cyclic AMP binding capacity of this protein kinase. This mechanism could account for the inactivation of cyclic AMP-dependent protein kinase that insulin causes in the liver.Abbreviations cAMP (cyclic AMP), Adenosine 3,5 monophosphate - (³H)cAMP cyclic (³H)AMP - MIX 1-methyl-3-isobutylxanthine     

Insulin signal transduction through protein kinase cascades

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.     

Characterization of three novel members of the Arabidopsis SHAGGY-related protein kinase (ASK) multigene family

In this paper we report the characterization of three novel members of the Arabidopsis shaggy-related protein kinase (ASK) multigene family, named ASKdzeta (ASK), ASKetha (ASK) and ASKiota (ASK). The proteins encoded by the ASK genes share a highly conserved catalytic protein kinase domain and show about 70% identity to SHAGGY (SGG) and glycogen synthase kinase-3 (GSK-3) from Drosophila and rat respectively. SGG is an ubiquitous intracellular component of the wingless signalling pathway that establishes cell fate and/or pattern formation in Drosophila. At least ten different ASK genes are expected to be present per haploid genome of A. thaliana. Different amino- and carboxy-terminal extensions distinguish different ASK family members. Five ASK gene sequences were analysed and shown to be present as single-copy genes in the Arabidopsis genome. A comparison based on the highly conserved catalytic domain sequences of all known sequences of the GSK-3 subfamily of protein kinases demonstrated a clear distinction between the plant and the animal kinases. Furthermore, we established the presence of at least three distinct groups of plant homologues of SGG/GSK-3. These different groups probably reflect biochemical and/or biological properties of these kinases. The differential expression patterns of five ASK genes were accessed by northern and in situ hybridization experiments using gene-specific probes. While ASK is expressed in the whole embryo during its development, ASK expression is limited to the suspensor cells. No signal was detected for ASK, ASK and ASK in developing embryos.     

Induction of apoptosis by a calpain stimulator, ONO-3403

The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 g/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 g/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified -calpain at a concentration higher than 100 g/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of -calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Identification of proteins that associate with protein kinase CK2

In order to aid in an understanding of the cellular functions of protein kinase CK2, a search for interacting proteins was carried out using a 32P-labeled CK2 overlay method. Several proteins were found to associate with CK2 by this assay; among them, one protein of 110 kDa appeared to be the most prominent one. The possible association of CK2 with p110 was suggested by experiments involving the co-immunoprecipitation using anti-CK2 antibodies. Further analysis using GST-CK2 fusion proteins demonstrated that the CK2-p110 interaction occurred through the CK2/ subunits. To identify p110, it was purified using a GST-CK2 affinity column, and internal amino acid sequencing was then performed. p110 was found to be nucleolin, a nucleolar protein that may be important for rRNA synthesis; a possible role of CK2 in the control of this process is suggested. Using the same CK2 overlay technique, another interacting protein, insulin receptor substrate 1 (IRS-1), was also identified. By applying a modified overlay method using individual 35S-labeled CK2 subunits, obtained by in vitro translation in rabbit reticulate lysates, it was determined that CK2 associates with IRS-1 through its / subunits; i.e. in keeping with the fact that IRS-1 is a known substrate for CK2. However, further work is needed to examine the association of CK2 with IRS-1 in vivo in order to fully understand the significance of the interaction.