It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.
Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.Metallothionein (MT) has been studied very extensively since its discovery (1), but the biochemistry of thionein (T), its apoprotein, has received relatively little experimental attention thus far. Efforts to demonstrate its endogenous production have consistently failed, in some measure owing to its lack of any appropriate spectroscopic property that could be a guide to its isolation. A series of manuscripts from this laboratory (2-4) have indicated its transient existence and generation at high local concentrations at the instant of its formation, emphasizing its importance as an endogenous and potent zinc-chelating agent effective at exceedingly low cellular concentrations. We are unaware of any analogous biological substance with corresponding properties. T avidly binds metal ions and is highly susceptible to proteolytic digestion and oxidation. It suppresses the DNA-binding capacity of zincfinger transcription factors in vitro by sequestering zinc and removing it from their structural sites (5-7). We have now established conditions to identify, isolate, and store T so that it completely retains its function including cluster formation through binding by its sulfhydryl groups (4). We have also shown that zinc is transferred from MT to the apoforms of zinc metalloenzymes and that T is indeed transiently formed in situ during this process (3, 4). For the reverse reaction, T itself does not remove significant amounts of zinc from zinc metalloenzymes. Instead, agents such as glutathione or citrate, which can bind zinc themselves but do not remove it from the active site of zinc metalloenzymes, can serve in the transfer process (4).In addition to its catalytic role in more than 300 zinc metalloenzymes and its structural role in an even greater number of nonenzymatic proteins, zinc is also a known inhibitor of enzymes in general, including zinc me...
Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubuleassociated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.Mitogen-activated protein kinase (MAPK), also known as the extracellular signal-regulated kinase (ERK), is involved in the transmission of signals between plasma membrane receptors and the nucleus (1, 2). MAPK has been shown to phosphorylate and regulate cytoskeletal components such as the microtubule-associated proteins in vitro, and it was originally named microtubule-associated protein-2 kinase after its substrate, MAP2 (3). Nonetheless, numerous immunocytochemical analyses have failed to show an association of MAPK with microtubules in proliferating cells (4-9). Thus, despite the often misunderstood nature of the original name, it is generally considered that MAPK is not actually associated with the microtubules in systems where mitogenesis occurs. However, MAPK was shown to associate with microtubules in certain rat brain dendrites in situ (10) and to copolymerize with bovine brain microtubules in vitro (11). MAPK was also shown to associate with the microtubule-organizing centers, but not spindle structures, in mouse oocytes (12). These findings raise the possibility that MAPK physically interacts with and regulates microtubule dynamics under certain unique circumstances such as meiosis and dendritic remodeling in the brain.In proliferating cells, significant evidence suggests that MAPK plays a role in cytoskeletal regulation. In addition to MAPs found only in the brain, such as MAP2 and tau, MAPK also phosphorylates cytoskeletal components present in cycling cells such as MAP4 and caldesmon (13,14). MAPs, which bind to and stabilize microtubules, are phosphorylated in response to cell stimulation by a variety of mitogens. The resulting phosphorylation inhibits their capacity to stabilize the microtubules (15). MAPK, which is activated by these mitogens, has been shown to be causative in MAP inhibition in vitro (13,16). MAPK activation is triggered not only by a large number of mitogens but also upon integrin-extracellular matrix association (17, 18), a first step toward cell spreading. Although these findings collectively imply a possible role for MAPK in the regulation of the cytoskeleton, the abovedescribed immunofluorescence evidence sugge...
Phosphorylase b kinase was purifed from fresh rabbit skeletal muscle by a procedure involving mild acid precipitation (pH 6.1), differential centrifugation, and free electrophoresis. The extent of purzcation achieved was 100-to 150-fold. In the procedure phosphorylase b was separated from the kinase by sedimentation of a glycogen-phosphorylase b complex at 80,000 x g for 1 hour. The kinase itself was sedimented when centrifuged at 100,000 x g for 3 hours, and there was evidence that the activity was associated with a component having a sedimentation coefficient in the range of 22-24 S. The form of phosphorylase b kinase purified in this preparation was referred to as nonactivated phosphorylase b kinase to distinguish it from activated kinase obtained by incubation of the enzyme with ATP or with trypsin. Nonactivated phosphorylase b kinase has a very high K,,, (Michaelis constant) for phosphorylase b which was decreased in the presence of glycogen. The K, for activated phosphorylase b kinase was much lower than that of nonactivated kinase. Activation of phosphorylase b kinase by incubation with ATP occurred more rapidly in the presence of adenosine-3',5'-phosphate but did require this nucleotide. Glycogen and heparin also increased the rate of activation of phosphorylase b kinase by ATP. No gross changes in the sedimentation pattern of purified phosphorylase b kinase were caused by activation.Muscle phosphorylase b kinase catalyzes the conversion of phosphorylase b to phosphorylase a according to the following equation (Krebs et al., 1958):The terminal phosphate of ATP is transferred to a specific seryl residue (Fischer et al., 1959) in the phosphorylase subunit, two of which are present in phosphorylase b and four of which are found in phosphorylase a. The latter has twice the molecular weight of phosphorylase b (Keller, 1955). Phosphorylase a formation can be followed conveniently by measuring phosphorylase activity in absence of AMP or by determining the extent of incorporation of 3*P into the protein Krebs et al., 1958). It is also possible to follow the reaction by coupling it with the pyruvic kinase-lactic dehydrogenase system, so that the ADP produced can be measured spectrophotometrically (Gonzales, 1962). No reversal of the kinase reaction has been demonstrated by any of several means employed .Purification of muscle phosphorylase b kinase was undertaken to facilitate further studies on the mechanism of phosphorylase b to a reaction and as a part of a general program to resolve the numerous components involved in the complex system regulating glycogenolysis. It is known that glycogenolysis is accelerated in muscle under circumstances in which phosphorylase a formation is increased. Thus, when resting muscle containing phosphorylase predominantly in the b form (Krebs and Fischer, 1955) is stimulated electrically, phosphorylase a is formed and glycogenolysis ensues (Cori, 1956). Muscle glycogenolysis produced by epinephrine administration is also associated with phosphorylase a formation (Sutherland, 1951; ...
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