Calcium-dependent regulator protein: Localization in mitotic apparatus of eukaryotic cells

Welsh, Michael J.; Dedman, John R.; Brinkley, B. R.; Means, Anthony R.

doi:10.1073/pnas.75.4.1867

Cited by 235 publications

(82 citation statements)

References 27 publications

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“…Furthermore, microinjection of calcium chelators inhibits mitosis (57-59), again implicating calcium in this process. Finally, indirect immunofluorescence has localized calmodulin to the kinetochore microtubules of the mitotic apparatus (60,61), and, in a related approach, fluorescently labeled calmodulin microinjected into cells binds to the kinetochore microtubules (62,63). Taken together, these studies strongly implicate calcium and calmodulin in the controlled depolymerization of microtubules that is necessary for anaphase A.…”

Section: Fig 7 Western Blot Analysis Of P62 Phosphorylated In Vivomentioning

confidence: 62%

Molecular Characterization of p62, a Mitotic Apparatus Protein Required for Mitotic Progression

Ye¹,

Sloboda

1997

Journal of Biological Chemistry

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Controlled microtubule disassembly occurs during anaphase A, the stage of mitosis characterized by the movement of the chromosomes toward the poles, an event thought to be powered by molecular motors that reside at the kinetochores. The kinetochore microtubules selectively disassemble during anaphase A, and if the microtubules are prevented from disassembling, e.g. by treatment with the microtubule stabilizing, anti-tumor drug Taxol, then mitosis ceases. Microtubule depolymerization can be induced by calcium, and calcium has been shown to result in the shortening of the distance separating the poles and movement of the chromosomes toward the poles (1). Furthermore, microtubule disassembly is necessary for the reactivation of chromosome movement in lysed cells (2), and isolated mitotic chromosomes can be translocated in association with a single microtubule in vitro as the result of microtubule depolymerization (3, 4). Thus, a critical step in anaphase is the controlled disassembly of the kinetochore microtubules.We have previously demonstrated that a protein of 62 kDa (p62), a substrate of a calcium/calmodulin-dependent protein kinase, is associated with the mitotic apparatus (5, 6). Both p62 and the kinase responsible for its phosphorylation copurify with mitotic apparatuses isolated from first cell cycle sea urchin embryos (5, 7). Importantly, the phosphorylation of p62 has been shown to correlate directly with the disassembly of microtubules in isolated mitotic apparatuses (5). Furthermore, microinjection of sea urchin embryos with affinity-purified antibodies to p62 inhibits progression through the cell cycle, specifically at the metaphase to anaphase transition (6). Because p62 is present at constant levels through at least two cell cycles (8), p62 dephosphorylation must be due to the action of a specific phosphatase and not to the degradation of the protein, as occurs, for example, with the cyclins. In fact, the existence of protein phosphatase 1 in the mitotic apparatus isolated from sea urchin embryos that dephosphorylates p62 has been demonstrated by pharmacological studies with phosphatase inhibitors (9). Finally, the subcellular localization of p62 varies with the cell cycle. During mitosis, p62 is associated with the mitotic apparatus, particularly with the microtubules (6,8,10), while during interphase, p62 resides in the nucleus (8, 10).Based on this background, our working hypothesis is that phosphorylation and dephosphorylation of p62 play key roles in controlling cell division, particularly during anaphase A, by permitting kinetochore microtubule disassembly at the appropriate time during mitosis. As the logical next step in elucidating the mode of action of p62 in mitosis, we present here a characterization of the polypeptide based on its deduced amino acid sequence obtained from cloned cDNA. The p62 polypeptide is highly acidic, yet contains distinct subdomains of alternating clusters of basic and acidic residues. Furthermore, confocal microscopy reveals that during interphase the protein bind...

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Section: Fig 7 Western Blot Analysis Of P62 Phosphorylated In Vivomentioning

confidence: 62%

Molecular Characterization of p62, a Mitotic Apparatus Protein Required for Mitotic Progression

Ye¹,

Sloboda

1997

Journal of Biological Chemistry

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“…There is also a coincident localization of cold-stable microtubules and CaM to kinetochore-to-pole fibers of the mitotic apparatus (31). Preliminary evidence (unpublished observations) indicates the STOP145 antibody binds selectively to the cold-stable kinetochore-to-pole microtubules of cultured cells.…”

Section: Discussionmentioning

confidence: 91%

Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

Margolis¹,

Rauch²,

Job³

1986

Proc. Natl. Acad. Sci. U.S.A.

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The capacity of microtubules to disassemble in vitro is profoundly affected by a protein factor designated STOP (stable tubule only polypeptide). Here we report the isolation of STOP protein and confirm that its activity is, as predicted, highly substoichiometric to the tubulin in microtubules. The isolation of the 145-kDa STOP (STOP145) protein has been effected from isolated cold-stable microtubules by two column steps: DEAE ion-exchange and a calmodulin affinity column. To confirm the protein's activity we have produced an antibody against STOP145 and have used the antibody to specifically remove the protein and the activity using an antibody-linked affinity column. We conclude that the STOP14S protein accounts for the observed in vitro stabilization of microtubules.The diverse functions that microtubules perform in cells often require the precise control of assembly equilibrium and of polymer stability (1). These properties are invested in the polymer, we believe, by specialized proteins that selectively associate with microtubules for specific purposes. Among these proteins, STOP (stable tubule only polypeptide) is one of particular interest, since it displays stabilizing and motility behavior on the polymer.STOP induces stability in the polymer by preventing the endwise dissociation of subunits. Microtubules in the presence of STOP are indefinitely stable to low temperature, to millimolar calcium, or to assembly-inhibiting drugs. STOP protein is remarkably potent in effect; we have inferred previously that it induced polymer stability at concentrations highly substoichiometric to tubulin (2). Also embodied in the STOP protein is the capacity to slide on microtubules (3). It is not known at this time which activity, stabilization or sliding, predominates in the cell. It is possible that both aspects of its behavior coordinately act to promote intracellular motility. Recent evidence from our laboratory indicates that STOP protein is present among cold-stable microtubules in the spindle of dividing cells (unpublished observations). We have postulated, in this case, that sliding and stabilization may act coordinately to create the poleward movement of chromosomes in anaphase (4).In the initial work on microtubule cold stability, it was reported that the activity was not intrinsic to tubulin but was due to a protein factor associated substoichiometrically with microtubules (5). We tentatively identified a protein of 145 kDa and a few smaller polypeptides as candidates for STOP protein based on their unique association with cold-stable microtubules (2).Here we report on the purification to near homogeneity of a STOP protein of 145 kDa and confirm its substoichiometric activity. Stable microtubules readily respond to simple metabolic signals. They become labile when exposed to ATP (6)(7)(8) Margolis and Wilson (10) and Asnes and Wilson (11), except that all procedures were carried out in MME buffer. After the third cycle of polymerization, the preparation was cleaned by centrifugation of microtubules ...

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“…Those rats that received ACTH for a total of 16 (24,25) and Andersen et al (26) found calmodulin staining associated with a stress cable (actinlike) pattern in quiescent cells, but no nuclear staining was observed. Dividing cultured cells, however, had certain areas of the mitotic apparatus that were heavily stained for calmodulin.…”

Section: Resultsmentioning

confidence: 99%

Localization of calmodulin in rat tissues.

Harper¹,

Cheung²,

Wallace³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

102

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The localization of calmodulin, a calciumdependent modulator of many enzymes, was studied in rat liver, skeletal muscle, and adrenal slices. Calmodulin is found in liver cytoplasm, nucleus, and plasma membrane. Much of the cytoplasmic calmodulin is associated with glycogen particles presumably bound to enzymes involved in g ycogen metabolism. Skeletal muscle calmodulin is found on the I-band, also associated with glycogen particles. Intermyofibrillar staining that is not glycogen associated is also observed. Calmodulin is localized in the cytoplasm and nucleus of adrenal cortex cells. Injection of corticotropin leads to a greatly increased localization of calmodulin in nuclei of the adrenal cortex. These observations suggest that one role of calmodulin may be the regulation of hormone effects on nuclear processes.Calcium ions regulate many cellular processes that are usually mediated through their interaction with high-affinity calcium-binding proteins. One such protein, calmodulin, mediates the effect of calcium on a wide variety of cellular enzymes. Calmodulin was originally described as an activator of cyclic nucleotide phosphodiesterase (1, 2). Subsequently, calmodulin was found to regulate many other enzymes, including the Ca2+,Mg2+-ATPase (or calcium transport) in several tissues (3-7), one class of brain adenylate cyclase (8-10), and the protein kinases that phosphorylate myosin light chain (11, 12), glycogen synthase (13,14), and phosphorylase (15 Tissue was usually obtained from male rats (150-300 g, Charles River CD) that had been killed by cervical dislocation. Rats providing skeletal muscle samples were injected with 50 mg of pentobarbital instead, and those providing adrenal tissue were injected with dexamethasone (0.5 mg intraperitoneally) 21, 16, and 11 hr'prior to sacrifice. Some rats were also given 20 units of corticotropin (ACTH) respository gel subcutaneously 16, 11, and 1 hr before sacrifice. Liver or adrenal pieces were placed in optimal cutting temperature compound (OCT compound) prior to freezing in a dry ice/acetone bath; skeletal muscle was prepared differently (see below).Immunofluorescence studies were performed with 4-,um-thick tissue slices by using an indirect "sandwich" technique (18). Briefly, tissue slices were washed twice for 5 min with phosphate-buffered saline and then incubated for 30 min with the Ig fraction of anticalmodulin antiserum. The antiserum was diluted 1:12 from the original serum volume with phosphate-buffered saline. This incubation was followed by a phosphate-buffered saline wash (three times at 3 min each), a 30-min incubation with fluorescein-conjugated goat anti-rabbit IgG (1:15), and a final wash in phosphate-buffered saline (three times at 3 min each), before the tissue was overlaid with 50% glycerol/phosphate-buffered saline and a coverslip was applied. The buffer contained neither added calcium nor chelator. In some experiments tissue slices were incubated with Bacillus subtilis a-amylase for 30 min prior to immunofluorescent staining in order ...

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Calcium-dependent regulator protein: Localization in mitotic apparatus of eukaryotic cells

Cited by 235 publications

References 27 publications

Molecular Characterization of p62, a Mitotic Apparatus Protein Required for Mitotic Progression

Molecular Characterization of p62, a Mitotic Apparatus Protein Required for Mitotic Progression

Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

Localization of calmodulin in rat tissues.

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