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PubMed1998

Modulation of calmodulin function by ubiquitin-calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin.

Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.

Amino Acid Sequence↗
PubMed1982

Effects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates.

Reticulocyte lysates contain two major classes of protein phosphatase activities, designated type 1 and type 2. These designations are based on criteria derived from the analyses of protein phosphatase species in other tissues. The criteria include (i) chromatographic elution profiles on DEAE-cellulose; (ii) specificity of lysate phosphatases toward [(32)P]phosphorylase a and [(32)P]phosphorylase kinase; (iii) sensitivity of lysate phosphatases to Mg(2+) ATP; and (iv) sensitivity to the heat-stable protein phosphatase inhibitor-2. The lysate phosphatase species are similar to those described in rabbit skeletal muscle and rabbit liver. Reticulocyte protein phosphatase type 1, but not type 2, is inhibited by heat-stable protein phosphatase inhibitor-1 and -2 which have been characterized from rabbit skeletal muscle. We have initiated a study on the function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Our findings are as follows. (a) Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. (b) Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). (c) Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the alpha-subunit (38,000 daltons) of eIF-2 (eIF-2alpha) and of two heat-stable polypeptides of 29,000 and 44,000 daltons. (d) The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significance of this modification of inhibitor-2 is not clear. (e) Inhibitor-2 has no effect on the activation in vitro of isolated heme-regulated or double-stranded RNA-dependent eIF-2alpha kinases. We propose that the inhibition of protein synthesis in hemin-supplemented lysates by added inhibitor-2 is due at least in part to the inhibition of a type 1 eIF-2alpha phosphatase activity, which permits a basal eIF-2alpha kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2alpha and an inhibition of protein synthesis.

Adenosine Triphosphate↗
PubMed1993

Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties--e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus E1A proteins, and the alpha subunit of phosphorylase kinase. No phosphotyrosine phosphatase activity was observed. Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity. The name PP-lambda is proposed for this unusual prokaryotic enzyme.

Amino Acid Sequence↗
PubMed1995

A Mg(2+)-dependent, Ca(2+)-inhibitable serine/threonine protein phosphatase from bovine brain.

The Mg(2+)-dependent serine/threonine protein phosphatases, also known as type 2C phosphatases (PP2C), belong to a gene family distinct from the other serine/threonine phosphatases and tyrosine phosphatases. Here we report the purification to apparent homogeneity of a novel Mg(2+)-dependent, Ca(2+)-inhibitable serine/threonine protein phosphatase from bovine brain. It is a type 2C enzyme in view of its Mg2+ requirement, resistance to okadaic acid and calyculin A, inability to use phosphorylase alpha as substrate, and a segment of amino acid sequence typical of all PP2C type phosphatases known to date. However, it differs from the other PP2C enzymes, particularly the mammalian PP2C alpha and -beta isoforms, in that its molecular weight, 76,000, is considerably larger and that it is inhibited by Ca2+, NaF, and polycations, but not by orthovanadate. The Ca2+ inhibition may not be related to its cellular regulation because of Ki values in the 20-90 microM range, but this property permits distinction of this enzyme from the other phosphatases. Although the precise physiological role of this phosphatase is not yet known, its ability to dephosphorylate a wide variety of phosphoproteins and its broad distribution, as shown by a survey of mouse tissues for its activity, suggest that it may serve an important cellular function.

Amino Acid Sequence↗
PubMed1997

Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Animals↗
PubMed1976

Hybrid enzyme of liver phosphorylase and phosphorylase I.

A hybrid enzyme (LI) of liver phosphorylase [EC 2.4.1.1.] (L) and phosphorylase I, which is mainly located in brain, was isolated and its enzymatic and immunological properties were examined. The following results were obtained: (1) AMP stimulated the b forms of the hybrid (LIb), I(Ib), and L(Lb); (2) in the presence of AMP, SO42- stimulated Lb more than LIb and inhibited Ib; (3) in the absence of AMP, SO42- stimulated all three isozymes in the order: Ibeta less than LI less than Lbeta; (4) on conversion to the a forms, the activities of L, LI, and I increased 35.5-fold, 3-fold, and 1.2-fold, respectively; (5) the relative inhibition potencies of anti-Lb antibody with LIa and LIb were 63% and 4%, respectively of that with La, and those of anti-Ib antibody with LIa and LIb were 42% and 88%, respectively of that with Ia. Since the ratios of the specific activities of purified La and Ia and of Lbeta and Ibeta are 70: 82 and 2 : 70, respectively (Schliselfeld, 1973), the present findings suggest a 1 : 1 association of I and L subunits in the hybrid molecule.

Adenosine Monophosphate↗
PubMed1990

Viewing molecules with scanning tunneling microscopy and atomic force microscopy.

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.

Brain↗
PubMed2002

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation.

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Animals↗
PubMed1985

Experimental studies on the adult respiratory distress syndrome: effects of induced DIC; granulocytes and elastase in mini pigs.

The potential role of granulocyte proteinases on experimentally induced ARDS was evaluated. In order to investigate the acute effects on lung function, elastase (330 U kg-1h-1) or thrombin (75-150 U kg-1 h-1) was continuously infused into anaesthetized and mechanically ventilated mini pigs. Both elastase as well as thrombin induced a progressive respiratory failure with prompt increase in pulmonary vascular resistance, and decrease of cardiac output, further a pulmonary leukostasis, and a disturbance of blood coagulation leading to hypocoagulability. High proteolytic activity selectively in the lung indicates a possible role of proteinases released from sequestered polymorphonuclear neutrophils. Similar results following elastase infusion were however obtained in leukopenic animals pretreated with a single dose of dimethylmyleran (5 mg kg-1) which depleted the granulocytes totally. These results offer the possibility that elastase itself may cause respiratory failure and lung tissue damage. On the other hand the digestion pattern of phosphorylase kinase by lung tissue homogenates of thrombin- or elastase-infused mini pigs clearly indicates that elastase is only one of several mediators which may cause experimentally induced ARDS even in the absence of granulocytes.

Animals↗
PubMed1983

The protein phosphatases involved in cellular regulation. 4. Classification of two homogeneous myosin light chain phosphatases from smooth muscle as protein phosphatase-2A1 and 2C, and a homogeneous protein phosphatase from reticulocytes active on protein synthesis initiation factor eIF-2 as protein phosphatase-2A2.

Two homogeneous protein phosphatases, termed 'smooth muscle phosphatase-I' and 'smooth muscle phosphatase-II', isolated from turkey gizzard as enzymes active against the 20-kDa light chain of smooth muscle myosin, and a third homogeneous protein phosphatase from rabbit reticulocytes, purified as an enzyme active against protein synthesis initiation factor eIF-2, were classified using the criteria defined by Ingebritsen and Cohen [Eur. J. Biochem. (1983) 132, 255-261]. All three enzymes were type-2 protein phosphatases based on their specificity for the alpha-subunit of phosphorylase kinase and insensitivity to inhibitor-1 and inhibitor-2. The substrate specificities of smooth muscle phosphatase-I and the eIF-2 phosphatase were similar to the catalytic subunit of protein phosphatase-2A. Smooth muscle phosphatase-I could be designated as protein phosphatase-2A1 and eIF-2 phosphatase as protein phosphatase-2A2 on the basis of their subunit compositions. The substrate specificity, dependence of activity on Mg2+ and subunit composition of smooth muscle phosphatase-II allowed its assignment as protein phosphatase-2C.

Animals↗
PubMed1985

Insulin-induced increases in the activity of the spontaneously active and ATP.Mg-dependent forms of phosphatase-1 in alloxan-diabetic rat liver.

Liver supernatant from normal and alloxan-diabetic rats was fractionated by DEAE-cellulose chromatography and the separated phosphoprotein phosphatase fractions were assayed with [32P]histone f2b, [32P]phosphorylase a and [32P]phosphorylase kinase as substrates. In diabetic rat liver, one of the phosphatase fractions found in the normal liver was significantly reduced. This fraction was identified as a mixture of the spontaneously active form and the ATP . Mg-dependent form of phosphoprotein phosphatase-1 (Fc) based on sensitivity to inhibitor-2, substrate specificity, and the fact that it could be activated 42-70% by glycogen synthase kinase-3 in the presence of ATP . Mg. Further analysis of this fraction showed that liver cytosol from diabetic rats contained 62-79% lower spontaneously active phosphatase-1 activity and 40-51% lower combined spontaneously active and ATP . Mg-dependent protein phosphatase-1 (Fc) activity. Insulin administration increased the spontaneously active and the ATP . Mg-dependent protein phosphatase-1 activities approximately 45% and 36%, respectively, in alloxan-diabetic rats. These data imply that the lower levels of spontaneously active phosphatase-1 activity in diabetic rat liver cannot be explained by presuming phosphatase-1 to have been present as Fc, the inactive form. Moreover, insulin restored the total activity of the spontaneously active and activatable forms of phosphatase-1 to those present in normal liver implying that both forms of phosphatase-1 activity are under hormonal control.

Adenosine Triphosphate↗
PubMed1992

The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.

Amino Acid Sequence↗
PubMed1993

Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme.

Three isoforms of mammalian protein phosphatase-1 (PP1 alpha, PP1 beta and PP1 gamma) were expressed in Escherichia coli and purified to near homogeneity. The activities of all isoforms towards phosphorylase, phosphorylase kinase and myosin and their sensitivities to inhibitor-2 were similar to the native PP1 catalytic subunit (PP1C) isolated from vertebrate tissues. Like PP1C, they each formed a complex with the glycogen-targetting(G) subunit which directs PP1C to glycogen particles in skeletal muscle. However, other properties differed strikingly from native PP1C. The expressed isoforms were 100-600-fold less sensitive to inhibitor-1, 3-5-fold less sensitive to okadaic acid, 5-100-fold less sensitive to microcystin-LR and approximately 20-fold more active in dephosphorylating histone H1 than native PP1C. Although PP1 gamma (like PP1C) was active in the absence of Mn2+, expressed PP1 alpha and PP1 beta were completely dependent on Mn2+ for activity. PP1 beta, like PP1C, interacted with the myofibrillar-targetting(M) complexes from skeletal-muscle and smooth-muscle producing species with enhanced myosin-phosphatase activity, whereas expressed PP1 alpha and PP1 gamma did not. The expressed isoforms of PP1 combined with inhibitor-2 to form an inactive complex (PP1I) that could be reactivated by the glycogen-synthase-kinase-3(GSK3)-catalysed phosphorylation of inhibitor-2. This procedure transformed the properties of all three expressed isoforms to those of native PP1C. Their sensitivities to inhibitor-1, okadaic acid and microcystin-LR were increased greatly, their histone-phosphatase activities decreased and the activities of PP1 alpha and PP1 beta became independent of Mn2+. Furthermore PP1 alpha and PP1 gamma now interacted with the M complexes in a similar manner to PP1 beta and PP1C. Conversely, incubation of native PP1C with 50 mM NaF caused conversion to a Mn(2+)-dependent form with properties similar to those of the expressed isozymes. The G subunit from skeletal muscle or the M complex from smooth muscle could displace PP1C from activated PP1I, but not inactive PP1I, to form G-subunit/PP1C and M-complex/PP1C heterodimeric complexes. Inhibitor-2 was also found to be essential for the reactivation of PP1C from 6 M guanidinium chloride in the absence of Mn2+. Taken together, the results suggest that inhibitor-2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PP1C molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.

Animals↗