Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID: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. 838 92

Smooth muscle myosin bound phosphatase (MBP) purified from chicken gizzard, which is a holoenzyme of type 1 delta protein phosphatase and dephosphorylated intact myosin, catalyzed the dephosphorylation of calponin phosphorylated by protein kinase C (PK-C). The Km of MBP for calponin was 0.6 microM and the Vmax was 350 nmol/min/mg. All of the multiple sites of phosphorylation by PK-C of calponin were completely dephosphorylated by MBP. Functionally, calponin dephosphorylated by MBP recovered its inhibitory effect on the actin-activated Mg(2+)-ATPase activity of myosin. Therefore, these results suggest that a type 1 delta protein phosphatase causes relaxation of smooth muscle by the dephosphorylation not only of myosin but also of calponin.
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PMID:Calponin phosphatase from smooth muscle: a possible role of type 1 protein phosphatase in smooth muscle relaxation. 839 7

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
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PMID:Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase. 839 39

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 microM TM or 0.1 microM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.
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PMID:Effects of the protein phosphatase inhibitors, tautomycin and calyculin-A, on protein phosphorylation and cytoskeleton of human platelets. 858 89

The specificity of the catalytic subunit of protein phosphatase-1 (PP1c) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1c-binding domains on GL and GM, the subunits that target PP1c to hepatic and muscle glycogen, respectively, and on M110, the subunit that targets PP1c to smooth muscle myosin. GM-(G63-T93) interacted with PP1c and prevented GL from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate GL from PP1c or affect other characteristic properties of the PP1GL complex. These results indicate that GL contains two PP1c-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, GM-(G63-N75) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates GM from PP1c because phosphate is inserted into the PP1c-binding domain of GM. M110-(M1-E309) and M110-(M1-F38), but not M110-(D39-E309), mimicked the M110 subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M110 subunit and M110-(M1-E309), neither M110-(M1-F38) nor M110-(D39-E309) suppressed the PP1c-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M110 subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M110-(M1-F38) displaced GL from PP1c, while GM-(G63-T93) displaced M110 from PP1c in vitro. These observations indicate that the region(s) of PP1c that interact with GM/GL and M110 overlap, explaining why different forms of PP1c contain just a single targetting subunit.
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PMID:Identification of protein-phosphatase-1-binding domains on the glycogen and myofibrillar targetting subunits. 870 35

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.
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PMID:Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments. 884 29

A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase- and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca2+-independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10-20-fold after preincubation with 5-50 microM of CoCl2; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (Ki = 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 microM) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.
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PMID:Purification and characterization of a kinase-associated, myofibrillar smooth muscle myosin light chain phosphatase possessing a calmodulin-targeting subunit. 905 93

We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SP-Sepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase- and myosin-associated protein phosphatase (KAMPPase). The KAMPPase multienzyme complex was composed of a 37-kDa catalytic (PC) subunit, a 67-kDa targeting (PT) subunit, and MLCKase with or without CaM. The approximate molar ratio of the PC and PT subunits was 1:2 with a variable and usually higher molar content of MLCKase. The targeting role of the PT subunit was directly demonstrated in binding experiments in which the PT subunit bound to both the kinase and to CaM. Its binding to CaM was, however, Ca2+-independent. MLCKase and the PT subunit potentiated activity of the PC subunit when intact myosin was used as the substrate. These data indicated that there is a Ca2+-independent interaction among the MLCPase, MLCKase, and CaM that are involved in the regulation of phosphatase activity.
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PMID:Purification and characterization of a smooth muscle myosin light chain kinase-phosphatase complex. 905 94

Upon platelet activation by a high shear stress (108 dyne/cm2), actin and actin-binding protein increased rapidly into the Triton-insoluble cytoskeleton, whereas the association of myosin increased gradually. The amounts of cytoskeleton-associated myosin depended on the extent of aggregation. Preceding the maximal aggregation and ATP secretion, the 20 kDa light chain of myosin (MLC) is rapidly phosphorylated to approx. 45% of 20 kDa MLC and is then dephosphorylated. Cytoskeletal association of myosin and phosphorylation of 20 kDa MLC was inhibited by OP-41483, a prostaglandin I2 analog, which inhibited the full aggregation response to shear stress. Exposure to high shear stress resulted in an increased association of myosin light chain kinase and protein phosphatase types 1 and 2A with the cytoskeleton, while the cytoskeletal association of protein kinase C was not evident. These results indicate that 20 kDa MLC phosphorylation is involved in shear stress-induced platelet activation, and that cytoskeletal association of protein phosphatases may regulate the phosphorylation level of cytoskeletal elements such as myosin together with myosin light chain kinase.
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PMID:Shear stress-induced myosin association with cytoskeleton and phosphorylation in human platelets. 907 28

We have previously isolated a form of protein phosphatase-1 (PP1M) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PP1C) bound to an M-complex consisting of 110-kDa (M110) and 21-kDa (M21) subunits. The interaction of PP1C with an N-terminal region of the M110 subunit enhances the dephosphorylation of myosin and suppresses the dephosphorylation of other substrates [Alessi, D. R., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035; Chen, Y. H., Chen, M. X., Alessi, D. R., Campbell, D. G., Shanahan, C., Cohen, P. & Cohen, P. T. W. (1994) FEBS Lett. 356, 51-56; Johnson, D. F., Moorhead, G., Caudwell, F. B., Cohen, P., Chen, Y. H., Chen, M. X. & Cohen, P. T. W. (1996) Eur. J. Biochem. 239, 317-325]. In this paper, we establish that PP1M accounts for nearly all the myosin phosphatase activity in myofibrils, that the M110 and M21 subunits are present at similar concentrations in the myofibrillar fraction, and that these subunits are entirely bound to PP1. We demonstrate that the M21 subunit does not interact with PP1C, but with the C-terminal 72 residues of the M110 subunit, a region which is 43% identical to residues 87-161 of the M21 subunit. A fragment of the M21 subunit, M21-(M1-L146), which lacks the C-terminal leucine zipper, also bound to the M110 subunit, but two other fragments M21-(M1-E110) and M21-(E110-K186) did not. The M110 and M21 subunits were both found to be myosin-binding proteins. The C-terminal 291 residues of the M110 subunit, but not the C-terminal 72 residues, bound to myosin, but the N-terminal fragments M110-(M1-E309) and M110-(M1-S477) did not. Thus, the region of the M110 subunit that stimulates the dephosphorylation of myosin by PP1C is distinct from the region that targets PP1M to myosin. Remarkably, each myosin dimer was capable of binding about 20 mol M21 subunit and many of the M21-binding sites were located in the myosin rod domain. The potential significance of this observation is discussed.
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PMID:Identification of the regions on the M110 subunit of protein phosphatase 1M that interact with the M21 subunit and with myosin. 910 68


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