Summary One of the profound changes in cellular morphology during mitosis is a massive alteration in the organization of microfilament cytoskeleton. It has been recently discovered that nonmuscle caldesmon, an actin and calmodulin binding microfilament-associatedprotein of relative molecular mass Mr=83 000, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specificphosphorylation. cdc2 kinase, which is a catalytic subunit of MPF (maturation or mitosis promoting factor), is found to be responsible for the mitosis-specific phosphorylation of caldesmon. Because caldesmon is implicated in the regulation of actin myosin interactions and/or microfilament organization, these results suggest that cdc2 kinase directly affects microfilament re-organization during mitosis. Introduction At mitosis in eukaryotic cclls, there are profound changes in cellular morphology. These are accompanied by changes in cellular structures including nuclear envelope breakdown, chromosome condensation, fragmentation of intracellular membrane structures (including the endoplasmic reticulum and Golgi structure), disassembly of cytoplasmic microtubules and formation of mitotic spindles. The molecular mechanisms controlling the changes in cell shape during mitosis are almost entirely obscure. What is known is that there are dramatic changes in the organization of micro filament^('-^). These changes include the disassembly of microfilament bundles during prophase accompanied by the rounding-up of cultured cells, the formation of transient contractile rings during cytokinesis, and, subsequently, the reassembly of microfilament bundles and the respreading of the two daughter cells. It has also been demonstrated by microinjection of antibodies against myosin 11, as well as by disruption of the myosin I1 gene, that myosin I1 is involved in cyt~kinesis(~-~), though its mechanism of action remains unclear. One of the approaches toward understanding the biochemistry of these events, in which the disassembly and reassembly of microfilament appear to play an
important part, is to search for alterations of the molecular constitution of microfilaments during mitosis. Such changes (if any) might be responsible for the alterations of microfilament organization in mitotic cells. We have recently reported the novel finding that non-muscle caldesmon, a protein of relative molecular mass M,=83 000 which binds to actin and calmodulin. is disassociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosph~rylation(~). This process of cell-cycle regulated phosphorylation may contribute to the changes in shape and structure of cells seen during mitosis. Caldesmon is known to inhibit actomyosin ATPase activity, and caldcsmon and tropomyosin together regulatc the actin severing and capping activities of gelsolin. In this essay, we will focus our discussion on possible physiological functions of the mitosis-specific phosphorylation of nonmuscle caldesmon.
Properties of Caldesmon We first briefly summarize the known properties of caldesmon. Caldecmon is a regulatory protein implicated in the control of actomyosin interactions in smooth muccle and nonmuscle cells (see ref. 8 for review). The protein, first isolatcd in smooth muscle, is an actin- and calmodulin-binding protein('). Two major classes of caldesmons have been identified: a set of high molecular wcight form in smooth muscle estimated from SDS gel electrophoresis at M,= 120 000-150 000; and a group of lower molecular weight species found mainly in nonmuscle cells estimated at M,= 70 000-80 OOO(l"). Smooth muscle and nonmuscle caldesmons are localized on thin filaments and stress fibers, respectively, suggesting their involvement in the regulation of cell motility and/or microfilament organization. Both muscle and nonmuscle caldesmons have similar properties including a rod-like molecular shape, hcatstability, calmodulin-regulated actin binding, selfassociation through disulfide bonds, periodic localization along actin filaments, and stimulation of the actin binding properties of trop~myosin('-~~). Furthermore, it has been recently shown that cultured cells do not distinguish between muscle and nonmuscle caldesmons in terms of the incorporation into microfilament structures. when these two types of caldesmon are microinjected into cultured cells("). This similarity is well explained by the recent analysis of cDNA clones showing that the two caldesmons are highly homologous; nonmuscle caldesmon is smaller by virtue of lacking 232 amino acids found in the central region of smooth muscle caldesmon(1',16). The sequence data, together with studies on domain mapping by others (see ref. 17 for review), have placed both the actin- and calmodulin-binding domains on the C-terminal end of the caldesmon molecule and the myosin binding domain on the N-terminus.
Functions of Caldesmon The biological function of caldesmon is uncertain. In reconstituted systems. caldesmon inhibits the tropomyosin-stimulated , actin-activated ATPase of myosin(‘’). This inhibition is attenuated by Ca2+/calmodulin because Ca”/calmodulin reverses actin binding by caldesmon. In addition to binding to actin or calmodulin, caldesmon is also reported to bind to other microfilament-associated froteins including myosin(””’) and tropomyosin( O), although the biological functions of these binding activities remain to be determined. In addition, caldesmon may control microfilament assembly. We, as well as others, found that caldesmon enhances the binding of tropomyosin to actin as much as 10-fold(11.20).This enhancement should lead to the stabilization of actin filament structure because actin filaments become more stable when bound to tropomyosin. Indeed, caldesmon coupled with tropomyosin is found to protect microfilaments against severing by gelsolin, one of the F-actin severing proteins(”). Furthermore, caldesmon together with tropomyosin can anneal gelsolin-severed filaments even in the presence of calcium(22). Actin binding assays have indicated that tropomyosin coupled with caldesmon not only inhibits the binding of gelsolin to actin but also makes gelsolin (probably as a gelsolin/actin complex) dissociate from the barbed ends of actin filaments. These results suggest a potential mechanism by which assembly and disassembly of microfilaments can be controlled in a caldesmon-dependent fashion through the inhibition of both severing and capping activities of gelsolin. While this mechanism requires the dissociation of caldesmon from microfilaments for this mechanism to function in vivo, such a situation appears to exist during mitosis where caldesmon is dissociated from microfilaments as a consequence of mitosis-specific pho~phorylation(~).
Phosphorylation of Caldesmon Before describing mitosis-specific phosphorylation of caldesmon, we would like to briefly review what is known about the phosphorylation of caldesmon by various kinases. Caldesmon is phosphorylated in vitro by a variety of kinases including C-kinase, casein kinase. Ca2+ calmodulin-dependent kinase 11, and A - k i n a ~ e ( ),~ ~However, -~ the biological relevance of such phosphorylation has been obscure. Phosphorylation by these kinases does not appear to change the actin binding properties of caldesmon, unlike the mitosis-specific phosph~rylation(~). The only reports on the effects of such phosphorylation are those by Walsh’s group, who showed that the phosphorylation of smooth muscle caldesmon by endogenous Ca’+/calmodulindependent kinase reduces the inhibition of ATPase activity by caldesmon. This phosphorylation does not reduce the actin binding properties of smooth muscle
1
caldesmon but rather inhibits its myosin-binding activity(’4). Dissociation of 83 kDa Nonmuscle Caldesmon from Microfilaments by Mitosis-specific Phosphorylation Analyses of intact microfilaments isolated from both mitotic and non-mitotic cells have revealed that 83 kDa nonmuscle caldesmon is almost entirely missing from microfilaments purified from mitotic cells, while caldesmon is tightly associated with microfilaments from non-mitotic cells. The levels of total caldesmon are, however, indistinguishable in mitotic and nonmitotic cells, and caldesmon purified from mitotic cells shows much lower affinity for actin than does caldesmon from non-mitotic cells. These results indicate that some modification of caldefmon during mitosis causes the reduction of actin affinity of caldesmon. It was concluded that mitosis-specific phosphorylation causes the reduction in the actin binding affinity of caldesmon seen during mitosis based on the following results(’): First, inimunoprecipi tation experiments have shown that thc extent of in vivo phosphorylation of caldesmon is higher in mitotic cells by one order of magnitude than in non-mitotic cells. Second, this in vivo mitosis-specific phosphorylation of caldesmon can be reconstituted in v i m with mitotic cell extracts from HeLa cells. Finally. this mitosis-specific in vitro phosphorylation can reduce the actin binding affinity of non-mitotic caldesmon by two orders of magnitude. Cdc2 Kinase is Responsible for the Mitosisspecific Phosphorylation of Caldesmon It is quite important to identify kinase(s) responsible for the mitosis-specific phosphorylation of caldesmon. Recent studies havc shown that a variety of mitotic events are all induced by a single molecular complex called MPF, maturation or mitosis promoting facMPF consists of at least two subunits: 45000-62000 M , polypeptide known as cyclin, and 34 000 M , catalytic subunit, a serine/threonine kinase known as cdc2 kina~e(*~-”). Because cdc2 kinase is a major kinase for cell cycle control in mitotic cells, and because the phosphorylation of caldesmon by known kinases does not reduce its actin binding affinity, it was decided to test whether cdc2 kinase phosphorylates caldesmon in vitro. In general, three criteria should be met to identify caldesmon as an in iiivu substrate of cdc2 kinase during mitosis. First. does purified cdc2 kinase phosphorylate caldesmon? Second, are the in vitro phosphorylation sites identical to those observed in vivo during mitosis? Third. does phosphorylation elicit changes in the biochemical properties of caldesmon during mitosis? Caldesmon appears to satisfy all these criteria as described below(32).
(1) In vitro phosphorylation: Both human and Xenopus cdc2 kinase preparations phosphorylate caldesmon in vitro. Human cdc2 kinase was prepared by immunoprecipitation from HeLa mitotic extracts using anti-cdc2 antibody (raised against C-terminus of human cdc2(j3)).The specificity of the antibody was confirmed by the observation that the phosphorylation of caldesmon as well as histoiie H1 is effectively blocked when human cdc2 kinase was prepared in the presence of competing antigenic peptide. Because the human and Xenopus cdc2 kinase preparations were totally independent. it is unlikely that caldesinon was phosphorylated by impurities contaminating these cdc2 preparations. It is worthy of note that the antibody against C-terminus, unlike the antibody against a peptide with a conservative amino acid sequence of PSTAIR (one letter amino acid code), does not precipitate cdc2-like kinases. Another piece of supporting evidence for the in vitro phosphorylation of caldesmon by cdc2 is that the histone H1 kinase activity of cdc2 is always co-eluted with the caldesmon kinase activity following each step of sequential column chromatographies (SephacrylS-300, poly-L-lysine Agarose, phenyl-Sepharose, and reactive yellow Y-86 column in this order). (2) Sites of phosphorylation: The two-dimensional phosphopeptide map has revealed that six out of seven phosphopeptide spots produced by phosphorylation with cdc2 kinase match the spots generated from in vivo phosphorylated caldesmon during mitosis, strongly suggesting that phosphorylation sites of cdc2 are the same as those phosphorylated in vivo. This notion is reinforced by the obqervation5 that (a) rat nonmuscle and chick smooth muscle caldesmons have seven and six consensus sequences (TP or SP) for cdc2 kinase, respectively; (b) among these, five are found in exactly the same positions of the sequenccs when the two caldesmon sequences are aligned for maximum homology; (c) these five sites are all located within the 20-25 kDa C-terminal actin- and calmodulin-binding domain, which was found to be the major phosphorylation site both in vitro by cdc2 kinase and in vwo. (3) Biochemical effects by phosphorylation with cdc2 kinase: The phosphorylation of caldesmon by cdc2 reduces both actin and calmodulin binding affinities of caldesmon, which are the most important biochemical properties of caldesmon. The reduction in the actin affinity seems to explain the dissociation of caldesmon From microfilaments during mitosis which we observed previously(7). The findings of caldesmon phosphorylation by cdc2 kinase strongly suggest that cdc2 kinase directly controls the changes in microfilament assembly seen during mitosis. Other examples of direct action of cdc2 kinase on cellular structures have been recently reported in the cases of lamin and vimentin; thc phosphorylation of nuclear lamin and vimentin by cdc2 kinase induces thc disassembly of nuclear lamina and intermediate filaments, r e s p e ~ t i v e l y ( ~ ~Because . ~ ~ ) . microfilament re-
arrangement, as well as the disassembly of intermediate filaments and nuclear lamin are all observed in early stages of mitosis, the direct involvement of cdc2 kinase in these events appears reasonable. These studies have revealed a strong correlation between caldesmon phosphorylation and cell rounding during mitosis although we can not state definitively at present that caldesmon phosphorylation is the cause of cell rounding rather than the consequence of it. However, two reports are relevant to this notion. Lamb and co-workers have shown that injection of ~ 3 4 " ~ " kinase into fibroblasts results in cell rounding and detachment from the substratum without completion of many of the other changes associated with entry into mitosis(36). It is possiblc that phosphorylation of caldesmon by the injected cdc2 kinase causes cell rounding. It would thus be interesting to see, either by immunofluorescence or by immunoelectron microscopy, whether the caldesmon in these injected cells is dissociated from microfilaments. The other relevant report is that okadaic acid causes a rounded morphology similar to that seen during mitosis(37).Because okadaic acid is a potent inhibitor of protein phosphatase, it is possible that caldesmon could not be dephosphorylated by the treatment with okadaic acid. leading to the observed cell rounding. Unlike other cultured cells, PtK cells stay spread during mitosis. Docs this mcan that caldesmon in PtK cells may stay dephosphorylated during mitosis? If so. is the caldesmon in PtK cells a mutant form lacking the phosphorylation sitcs? Answering these questions will also provide us with information as to whether caldesmon phosphorylation has causal effects on cell rounding during mitosis. Physiological Functions of Caldesmon Phosphorylation by cdc2 Kinase during Mitosis We have two working hypothcses. First, the dissociation may affect the contractility of the actomyosin system during mitosis (Fig. 1A). Previous work has shown that caldesrnon inhibits actin-activatcd myosin ATPase activity. Ca2+/calmodulin releases caldesmon's inhibition of myosin ATPase as it reduces the actin binding affinity of caldesmon. It is thus likely that the dissociation of caldesmon from microfilaments, resulting from its mitosis-specific phosphorylation, also releases the inhibition of actomyosin ATPase by caldesmon. This release may lead to contraction of the actin-myosin system in mitotic cells in the following two ways: First, such contraction may cause rounding-up of cell shape when cells enter prophase. Second. the dissociation of nonmuscle caldesmon may be needed at a later stage to activate contractile rings during cytokinesis (our recent results have shown that caldesmon is still phosphorylated during cytokinesis and is not associated with contractile rings). Our second hypothesis is that caldesmon may function in the reorganization of microfilaments by
lnterphase
Mitosis
F - -P
+-8 w - -
p v “
B
latch ”
lnterphase
n
“
contraction ”
Mitosis
0
changes in tropomyosin expression lower expression of caldesmon
Transformed cells
phosphoryfatedrn
Myosin Fig. 1. Hypotheses for physiological functions of the mitosis-specific phosphorylation of caldesmon (CDM), and a hypothesis for changes in microfilament organization upon cell transformation. A . The dissociation of caldesmon from microfilaments induced by phosphorylation releases caldesmon’s inhibition of actomyosin ATPase. ’rliis allows actom yosin to contract, thereby leading to rounding-up of cell shape during prophase. B. Thc dissociation of caldesmon releases caldesmon’s inhibition of actin severing activity of gelsolin. This will lead to severing of inicrofilaincnts into short ones, leading to disassembly of stress fibers and rounding-up of cell shape. Upon cell transformation. caldesmon expression is decreased and tropomyosin (TM) isoforms are switched from normal cell troponiyosins to transformed cell tropomynsins. Both of these changes make microfilaments unprotected against severing action of gelsolin. See the text for detail.
regulating the severing activity of gelsolin-like proteins (Fig. 1B). As described previously, caldesmon couplcd with tropomyosin inhibits both severing and capping activities of gelsolin(” ,22). The dissociation of caldesmon from microfilaments by the mitosis-specific kinase activity may thus release the inhibition of gelsolin activity, resulting in the severing of microfilamcnts into short filaments. This severing may lead to the disassembly of stress fibers and the concomitant morphological alterations during prophase. Moreover, such actin severing activity may also be needed for cytokinesis, because contractile rings should be simul-
taneously disassembled as they contract for cytoplasmic division. In addition to the binding to actin and calmodulin. caldesmon is also known to bind to myosin. The binding of caldesmon to both actin and myosin enables caldesmon to cross-link actin and myosin in the presence of Mg-ATP. This cross-linking, together with caldesmon’s inhibition of actomyosin ATPase suggests that caldesmon may play functions in the so called ‘latch’ state contraction of smooth muscle, where smooth muscle maintains tension without consuming much ATP. It is possible that stress fibers of cultured cells are in a state analogous to this Llatch‘state, which may function to sustain a spread-out morphology. The dissociation of caldesmon resulting from its mitosisspecific phosphorylation may release this *latch’state, leading to the contraction of actomyosin and rounding of cell shape seen during prophase (Fig. 1A). Is Cell Rounding during Mitosis Biochemically Related to the Morphological Changes Caused by Cell Transformation? Cell transformation and cell cycle control are intimately related phenomena(“.’Y). One of the definitive characteristics of cancer cells is the loss of regulation of cell division. This is evident from the recent findings that many oncogenes play functions in perturbing normal cell cycle controls: a sis oncogene encodes a growth factor (PDGF) and stimulates cell proliferation. Others (such as erhB. fms) alter intracellular signal transduction pathways in ways that may mimic the actions of activated growth factor receptors. The similarity in cell shape and structure between mitotic and transformed cells reinforces this association between normal cell cycle control and oncogenic transformation. Mitotic cells show rounded morphology as do most transformed cells. Microfilament bundles arc disassembled when cells enter into prophase just as many transformed cells with rounded morphology exhibit dispersed microfilament patterns. Microfilament bundles that are anchored to focal contacts arc also disrupted, causing reduced adhesion to substrate by both mitotic and transformed cells. These changes in actin cables, as well as in adhesiveness are closely coupled to cell division and proliferation in both normal and transformed cells. It is thus tempting to see how these two morphological alterations are related to each other at a molecular level. In cell transformation, we previously found changes in tropomyosin expression(4o).Our results have demonstrated that: (1)normal cultured rat cells have as many as five variants of tropomyosin; (2) of these, the tropomyosin variants with higher affinity for actin are replaced with tropomyosin variants with lower affinity for actin upon transformation; and (3) the changes in tropomyosin expression are regulated at the level of mRNA. Similar rcsults have also been reported by others (reviewed in ref. 40). It was suggested that the
lower affinity in actin binding of tropomymin in transformed cells may result in a more unstable structure of microfilaments, leading to the disassembly of stress fibers in many transformed cells. The expression of caldesmon is reported to be reduced in transformed cells relative to normal cells(4'). which would probably further weaken the binding of tropomyosin to actin in transformed cells. During mitosis, on the other hand. caldesmon is found to be dissociated as a consequence of mitosisspecific phosphorylation (there appears no obvious change in tropomyosin expression during mitosis). Although these two events (changes in tropomyosin expression and phosphorylation of caldesmon) appear different, it is worthy of notc that both events lcad to the same biochemical change, i.e. the reduction of the actin-binding affinity of tropomyosin. In transformation. tropomyosin isoforms with lower affinity for actin become predominant. In mitosis, phosphorylation causes caldesinon to dissociate from microfilaments, Because caldesmon enhances actin binding of tropomyosin, the dissociation of caldcsmon may also result in a decrease in the actin binding affinity of tropomyosin. This suggests that the reduction of the actin binding affinity of tropomyosin is a key biochemical event that controls thc alterations in actin assembly observed in both cases. It is also worthy of note that changes in gene expression of tropomyosin are probably involved in permanent morphological alterations such as those in cell transformation, while post-translational modification such as phosphorylation may control transient morphological alterations such as those during mitosis. Perspective The best way to probe the physiological functions of caldesmon phosphorylation during mitosis is perhaps to do transfection or microinjection experiments with mutant caldesmons lacking phosphorylation sites. If such mutant caldesmons stay bound to microfilaments during mitosis, and inhibit actomyosin ATPase and/or gelsolin activities, perturbation on microfilament organization would be predicted during mitosis. For example, the cells having mutant caldcsmons might not bc rounded during prophase. Other possible effects in these manipulated cells might be blockage or retardation of cytokinesis. It should be emphasized that the phosphorylation of caldesmon must be one of many biochemical events that control re-organization of microfilaments during mitosis. A variety of microfilament- and microtubuleassociated proteins are most likely to be modified, at various stages of mitosis, for the precise control of cytoskeletal organization in both spatial and temporal ways. It is therefore important to elucidate what components are modified at which stages of mitosis and how such modifications lead to massive alterations in thc cytoskeletal organization such as cytokinesis. Such studies will help us to understand one of the most
fascinating of biological problems, how cells integrate temporal and spatial information for cytokinesis. Acknowledgments We would like to thank Dr. Natsumi Hosoya for her excellent artwork of Fig. 1. This work was supported in part by Grant R37 CA42742 from the National Cancer Institute, Grant CD-442 from the American Cancer Society. Biomedical Research Support Grant PHS RR 07058-24, and Johnson and Johnson Discovery Fund. FM is a recipient of Research Career Development Award KO4 CA01304 from thc National Cancer Institute. References 1 SCHROEDER, T. E. (1976). Actin in dividing cells: evidence for its role in
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Shigeko Yamashiro and Fumio Matsumura arc at the Department of Molecular Biology and Biochemistry, Rutgers University Piscataway, New Jersey 088541059, USA.
hnnoi mc-ement
THE NEK:'\I'ORK ACADl?hfI7 OF SCIENCES
Conference on The Melanotropic Peptides Septerriber 6 to 9. 1092 Palais des Congres, Place cie la Catlieclralc, Rourn, Francc The conference will evaluate thc recent findings on biochcmistry, physiology and pharmacology on melanocyte-stimulating hormones (MSH) and melanin-concentrating hormone (MCH). The current knowledge of hormonal and neuromodulator/neurotransmittcr functions of mclanotropins will be presented. The conference will covcr all rcccnt developrncnts concerning thc melanotropic peptides, from basic research to clinical perspectives. There will be contributed poster scssions in conjunction with this conference. The deadline for submission of poster abstracts is April 15, 1992.
Conference Chairmen : Alex N. Eberlk, Ph.D., D.Sc. Hubert Vaudry, Ph.D., D.Sc. Department of Research (ZLF) Laboratory of Molecular Eiidocrinology University Hospital University of Rouen, B.P. 118 76134 Mont-Saint-Aignan Hebelstrasse 20 France CH-4031 Basel, Switzerland For abstract spccification,r and for further inlormation please contact: Conference Department, New York Academy of Sciences, 2 East 63rd Street, New York, NY 1002 , USA TEL: 212-838-0230, FAX: 212-888-2894
important part, is to search for alterations of the molecular constitution of microfilaments during mitosis. Such changes (if any) might be responsible for the alterations of microfilament organization in mitotic cells. We have recently reported the novel finding that non-muscle caldesmon, a protein of relative molecular mass M,=83 000 which binds to actin and calmodulin. is disassociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosph~rylation(~). This process of cell-cycle regulated phosphorylation may contribute to the changes in shape and structure of cells seen during mitosis. Caldesmon is known to inhibit actomyosin ATPase activity, and caldcsmon and tropomyosin together regulatc the actin severing and capping activities of gelsolin. In this essay, we will focus our discussion on possible physiological functions of the mitosis-specific phosphorylation of nonmuscle caldesmon.
Properties of Caldesmon We first briefly summarize the known properties of caldesmon. Caldecmon is a regulatory protein implicated in the control of actomyosin interactions in smooth muccle and nonmuscle cells (see ref. 8 for review). The protein, first isolatcd in smooth muscle, is an actin- and calmodulin-binding protein('). Two major classes of caldesmons have been identified: a set of high molecular wcight form in smooth muscle estimated from SDS gel electrophoresis at M,= 120 000-150 000; and a group of lower molecular weight species found mainly in nonmuscle cells estimated at M,= 70 000-80 OOO(l"). Smooth muscle and nonmuscle caldesmons are localized on thin filaments and stress fibers, respectively, suggesting their involvement in the regulation of cell motility and/or microfilament organization. Both muscle and nonmuscle caldesmons have similar properties including a rod-like molecular shape, hcatstability, calmodulin-regulated actin binding, selfassociation through disulfide bonds, periodic localization along actin filaments, and stimulation of the actin binding properties of trop~myosin('-~~). Furthermore, it has been recently shown that cultured cells do not distinguish between muscle and nonmuscle caldesmons in terms of the incorporation into microfilament structures. when these two types of caldesmon are microinjected into cultured cells("). This similarity is well explained by the recent analysis of cDNA clones showing that the two caldesmons are highly homologous; nonmuscle caldesmon is smaller by virtue of lacking 232 amino acids found in the central region of smooth muscle caldesmon(1',16). The sequence data, together with studies on domain mapping by others (see ref. 17 for review), have placed both the actin- and calmodulin-binding domains on the C-terminal end of the caldesmon molecule and the myosin binding domain on the N-terminus.
Functions of Caldesmon The biological function of caldesmon is uncertain. In reconstituted systems. caldesmon inhibits the tropomyosin-stimulated , actin-activated ATPase of myosin(‘’). This inhibition is attenuated by Ca2+/calmodulin because Ca”/calmodulin reverses actin binding by caldesmon. In addition to binding to actin or calmodulin, caldesmon is also reported to bind to other microfilament-associated froteins including myosin(””’) and tropomyosin( O), although the biological functions of these binding activities remain to be determined. In addition, caldesmon may control microfilament assembly. We, as well as others, found that caldesmon enhances the binding of tropomyosin to actin as much as 10-fold(11.20).This enhancement should lead to the stabilization of actin filament structure because actin filaments become more stable when bound to tropomyosin. Indeed, caldesmon coupled with tropomyosin is found to protect microfilaments against severing by gelsolin, one of the F-actin severing proteins(”). Furthermore, caldesmon together with tropomyosin can anneal gelsolin-severed filaments even in the presence of calcium(22). Actin binding assays have indicated that tropomyosin coupled with caldesmon not only inhibits the binding of gelsolin to actin but also makes gelsolin (probably as a gelsolin/actin complex) dissociate from the barbed ends of actin filaments. These results suggest a potential mechanism by which assembly and disassembly of microfilaments can be controlled in a caldesmon-dependent fashion through the inhibition of both severing and capping activities of gelsolin. While this mechanism requires the dissociation of caldesmon from microfilaments for this mechanism to function in vivo, such a situation appears to exist during mitosis where caldesmon is dissociated from microfilaments as a consequence of mitosis-specific pho~phorylation(~).
Phosphorylation of Caldesmon Before describing mitosis-specific phosphorylation of caldesmon, we would like to briefly review what is known about the phosphorylation of caldesmon by various kinases. Caldesmon is phosphorylated in vitro by a variety of kinases including C-kinase, casein kinase. Ca2+ calmodulin-dependent kinase 11, and A - k i n a ~ e ( ),~ ~However, -~ the biological relevance of such phosphorylation has been obscure. Phosphorylation by these kinases does not appear to change the actin binding properties of caldesmon, unlike the mitosis-specific phosph~rylation(~). The only reports on the effects of such phosphorylation are those by Walsh’s group, who showed that the phosphorylation of smooth muscle caldesmon by endogenous Ca’+/calmodulindependent kinase reduces the inhibition of ATPase activity by caldesmon. This phosphorylation does not reduce the actin binding properties of smooth muscle
1
caldesmon but rather inhibits its myosin-binding activity(’4). Dissociation of 83 kDa Nonmuscle Caldesmon from Microfilaments by Mitosis-specific Phosphorylation Analyses of intact microfilaments isolated from both mitotic and non-mitotic cells have revealed that 83 kDa nonmuscle caldesmon is almost entirely missing from microfilaments purified from mitotic cells, while caldesmon is tightly associated with microfilaments from non-mitotic cells. The levels of total caldesmon are, however, indistinguishable in mitotic and nonmitotic cells, and caldesmon purified from mitotic cells shows much lower affinity for actin than does caldesmon from non-mitotic cells. These results indicate that some modification of caldefmon during mitosis causes the reduction of actin affinity of caldesmon. It was concluded that mitosis-specific phosphorylation causes the reduction in the actin binding affinity of caldesmon seen during mitosis based on the following results(’): First, inimunoprecipi tation experiments have shown that thc extent of in vivo phosphorylation of caldesmon is higher in mitotic cells by one order of magnitude than in non-mitotic cells. Second, this in vivo mitosis-specific phosphorylation of caldesmon can be reconstituted in v i m with mitotic cell extracts from HeLa cells. Finally. this mitosis-specific in vitro phosphorylation can reduce the actin binding affinity of non-mitotic caldesmon by two orders of magnitude. Cdc2 Kinase is Responsible for the Mitosisspecific Phosphorylation of Caldesmon It is quite important to identify kinase(s) responsible for the mitosis-specific phosphorylation of caldesmon. Recent studies havc shown that a variety of mitotic events are all induced by a single molecular complex called MPF, maturation or mitosis promoting facMPF consists of at least two subunits: 45000-62000 M , polypeptide known as cyclin, and 34 000 M , catalytic subunit, a serine/threonine kinase known as cdc2 kina~e(*~-”). Because cdc2 kinase is a major kinase for cell cycle control in mitotic cells, and because the phosphorylation of caldesmon by known kinases does not reduce its actin binding affinity, it was decided to test whether cdc2 kinase phosphorylates caldesmon in vitro. In general, three criteria should be met to identify caldesmon as an in iiivu substrate of cdc2 kinase during mitosis. First. does purified cdc2 kinase phosphorylate caldesmon? Second, are the in vitro phosphorylation sites identical to those observed in vivo during mitosis? Third. does phosphorylation elicit changes in the biochemical properties of caldesmon during mitosis? Caldesmon appears to satisfy all these criteria as described below(32).
(1) In vitro phosphorylation: Both human and Xenopus cdc2 kinase preparations phosphorylate caldesmon in vitro. Human cdc2 kinase was prepared by immunoprecipitation from HeLa mitotic extracts using anti-cdc2 antibody (raised against C-terminus of human cdc2(j3)).The specificity of the antibody was confirmed by the observation that the phosphorylation of caldesmon as well as histoiie H1 is effectively blocked when human cdc2 kinase was prepared in the presence of competing antigenic peptide. Because the human and Xenopus cdc2 kinase preparations were totally independent. it is unlikely that caldesinon was phosphorylated by impurities contaminating these cdc2 preparations. It is worthy of note that the antibody against C-terminus, unlike the antibody against a peptide with a conservative amino acid sequence of PSTAIR (one letter amino acid code), does not precipitate cdc2-like kinases. Another piece of supporting evidence for the in vitro phosphorylation of caldesmon by cdc2 is that the histone H1 kinase activity of cdc2 is always co-eluted with the caldesmon kinase activity following each step of sequential column chromatographies (SephacrylS-300, poly-L-lysine Agarose, phenyl-Sepharose, and reactive yellow Y-86 column in this order). (2) Sites of phosphorylation: The two-dimensional phosphopeptide map has revealed that six out of seven phosphopeptide spots produced by phosphorylation with cdc2 kinase match the spots generated from in vivo phosphorylated caldesmon during mitosis, strongly suggesting that phosphorylation sites of cdc2 are the same as those phosphorylated in vivo. This notion is reinforced by the obqervation5 that (a) rat nonmuscle and chick smooth muscle caldesmons have seven and six consensus sequences (TP or SP) for cdc2 kinase, respectively; (b) among these, five are found in exactly the same positions of the sequenccs when the two caldesmon sequences are aligned for maximum homology; (c) these five sites are all located within the 20-25 kDa C-terminal actin- and calmodulin-binding domain, which was found to be the major phosphorylation site both in vitro by cdc2 kinase and in vwo. (3) Biochemical effects by phosphorylation with cdc2 kinase: The phosphorylation of caldesmon by cdc2 reduces both actin and calmodulin binding affinities of caldesmon, which are the most important biochemical properties of caldesmon. The reduction in the actin affinity seems to explain the dissociation of caldesmon From microfilaments during mitosis which we observed previously(7). The findings of caldesmon phosphorylation by cdc2 kinase strongly suggest that cdc2 kinase directly controls the changes in microfilament assembly seen during mitosis. Other examples of direct action of cdc2 kinase on cellular structures have been recently reported in the cases of lamin and vimentin; thc phosphorylation of nuclear lamin and vimentin by cdc2 kinase induces thc disassembly of nuclear lamina and intermediate filaments, r e s p e ~ t i v e l y ( ~ ~Because . ~ ~ ) . microfilament re-
arrangement, as well as the disassembly of intermediate filaments and nuclear lamin are all observed in early stages of mitosis, the direct involvement of cdc2 kinase in these events appears reasonable. These studies have revealed a strong correlation between caldesmon phosphorylation and cell rounding during mitosis although we can not state definitively at present that caldesmon phosphorylation is the cause of cell rounding rather than the consequence of it. However, two reports are relevant to this notion. Lamb and co-workers have shown that injection of ~ 3 4 " ~ " kinase into fibroblasts results in cell rounding and detachment from the substratum without completion of many of the other changes associated with entry into mitosis(36). It is possiblc that phosphorylation of caldesmon by the injected cdc2 kinase causes cell rounding. It would thus be interesting to see, either by immunofluorescence or by immunoelectron microscopy, whether the caldesmon in these injected cells is dissociated from microfilaments. The other relevant report is that okadaic acid causes a rounded morphology similar to that seen during mitosis(37).Because okadaic acid is a potent inhibitor of protein phosphatase, it is possible that caldesmon could not be dephosphorylated by the treatment with okadaic acid. leading to the observed cell rounding. Unlike other cultured cells, PtK cells stay spread during mitosis. Docs this mcan that caldesmon in PtK cells may stay dephosphorylated during mitosis? If so. is the caldesmon in PtK cells a mutant form lacking the phosphorylation sitcs? Answering these questions will also provide us with information as to whether caldesmon phosphorylation has causal effects on cell rounding during mitosis. Physiological Functions of Caldesmon Phosphorylation by cdc2 Kinase during Mitosis We have two working hypothcses. First, the dissociation may affect the contractility of the actomyosin system during mitosis (Fig. 1A). Previous work has shown that caldesrnon inhibits actin-activatcd myosin ATPase activity. Ca2+/calmodulin releases caldesmon's inhibition of myosin ATPase as it reduces the actin binding affinity of caldesmon. It is thus likely that the dissociation of caldesmon from microfilaments, resulting from its mitosis-specific phosphorylation, also releases the inhibition of actomyosin ATPase by caldesmon. This release may lead to contraction of the actin-myosin system in mitotic cells in the following two ways: First, such contraction may cause rounding-up of cell shape when cells enter prophase. Second. the dissociation of nonmuscle caldesmon may be needed at a later stage to activate contractile rings during cytokinesis (our recent results have shown that caldesmon is still phosphorylated during cytokinesis and is not associated with contractile rings). Our second hypothesis is that caldesmon may function in the reorganization of microfilaments by
lnterphase
Mitosis
F - -P
+-8 w - -
p v “
B
latch ”
lnterphase
n
“
contraction ”
Mitosis
0
changes in tropomyosin expression lower expression of caldesmon
Transformed cells
phosphoryfatedrn
Myosin Fig. 1. Hypotheses for physiological functions of the mitosis-specific phosphorylation of caldesmon (CDM), and a hypothesis for changes in microfilament organization upon cell transformation. A . The dissociation of caldesmon from microfilaments induced by phosphorylation releases caldesmon’s inhibition of actomyosin ATPase. ’rliis allows actom yosin to contract, thereby leading to rounding-up of cell shape during prophase. B. Thc dissociation of caldesmon releases caldesmon’s inhibition of actin severing activity of gelsolin. This will lead to severing of inicrofilaincnts into short ones, leading to disassembly of stress fibers and rounding-up of cell shape. Upon cell transformation. caldesmon expression is decreased and tropomyosin (TM) isoforms are switched from normal cell troponiyosins to transformed cell tropomynsins. Both of these changes make microfilaments unprotected against severing action of gelsolin. See the text for detail.
regulating the severing activity of gelsolin-like proteins (Fig. 1B). As described previously, caldesmon couplcd with tropomyosin inhibits both severing and capping activities of gelsolin(” ,22). The dissociation of caldesmon from microfilaments by the mitosis-specific kinase activity may thus release the inhibition of gelsolin activity, resulting in the severing of microfilamcnts into short filaments. This severing may lead to the disassembly of stress fibers and the concomitant morphological alterations during prophase. Moreover, such actin severing activity may also be needed for cytokinesis, because contractile rings should be simul-
taneously disassembled as they contract for cytoplasmic division. In addition to the binding to actin and calmodulin. caldesmon is also known to bind to myosin. The binding of caldesmon to both actin and myosin enables caldesmon to cross-link actin and myosin in the presence of Mg-ATP. This cross-linking, together with caldesmon’s inhibition of actomyosin ATPase suggests that caldesmon may play functions in the so called ‘latch’ state contraction of smooth muscle, where smooth muscle maintains tension without consuming much ATP. It is possible that stress fibers of cultured cells are in a state analogous to this Llatch‘state, which may function to sustain a spread-out morphology. The dissociation of caldesmon resulting from its mitosisspecific phosphorylation may release this *latch’state, leading to the contraction of actomyosin and rounding of cell shape seen during prophase (Fig. 1A). Is Cell Rounding during Mitosis Biochemically Related to the Morphological Changes Caused by Cell Transformation? Cell transformation and cell cycle control are intimately related phenomena(“.’Y). One of the definitive characteristics of cancer cells is the loss of regulation of cell division. This is evident from the recent findings that many oncogenes play functions in perturbing normal cell cycle controls: a sis oncogene encodes a growth factor (PDGF) and stimulates cell proliferation. Others (such as erhB. fms) alter intracellular signal transduction pathways in ways that may mimic the actions of activated growth factor receptors. The similarity in cell shape and structure between mitotic and transformed cells reinforces this association between normal cell cycle control and oncogenic transformation. Mitotic cells show rounded morphology as do most transformed cells. Microfilament bundles arc disassembled when cells enter into prophase just as many transformed cells with rounded morphology exhibit dispersed microfilament patterns. Microfilament bundles that are anchored to focal contacts arc also disrupted, causing reduced adhesion to substrate by both mitotic and transformed cells. These changes in actin cables, as well as in adhesiveness are closely coupled to cell division and proliferation in both normal and transformed cells. It is thus tempting to see how these two morphological alterations are related to each other at a molecular level. In cell transformation, we previously found changes in tropomyosin expression(4o).Our results have demonstrated that: (1)normal cultured rat cells have as many as five variants of tropomyosin; (2) of these, the tropomyosin variants with higher affinity for actin are replaced with tropomyosin variants with lower affinity for actin upon transformation; and (3) the changes in tropomyosin expression are regulated at the level of mRNA. Similar rcsults have also been reported by others (reviewed in ref. 40). It was suggested that the
lower affinity in actin binding of tropomymin in transformed cells may result in a more unstable structure of microfilaments, leading to the disassembly of stress fibers in many transformed cells. The expression of caldesmon is reported to be reduced in transformed cells relative to normal cells(4'). which would probably further weaken the binding of tropomyosin to actin in transformed cells. During mitosis, on the other hand. caldesmon is found to be dissociated as a consequence of mitosisspecific phosphorylation (there appears no obvious change in tropomyosin expression during mitosis). Although these two events (changes in tropomyosin expression and phosphorylation of caldesmon) appear different, it is worthy of notc that both events lcad to the same biochemical change, i.e. the reduction of the actin-binding affinity of tropomyosin. In transformation. tropomyosin isoforms with lower affinity for actin become predominant. In mitosis, phosphorylation causes caldesinon to dissociate from microfilaments, Because caldesmon enhances actin binding of tropomyosin, the dissociation of caldcsmon may also result in a decrease in the actin binding affinity of tropomyosin. This suggests that the reduction of the actin binding affinity of tropomyosin is a key biochemical event that controls thc alterations in actin assembly observed in both cases. It is also worthy of note that changes in gene expression of tropomyosin are probably involved in permanent morphological alterations such as those in cell transformation, while post-translational modification such as phosphorylation may control transient morphological alterations such as those during mitosis. Perspective The best way to probe the physiological functions of caldesmon phosphorylation during mitosis is perhaps to do transfection or microinjection experiments with mutant caldesmons lacking phosphorylation sites. If such mutant caldesmons stay bound to microfilaments during mitosis, and inhibit actomyosin ATPase and/or gelsolin activities, perturbation on microfilament organization would be predicted during mitosis. For example, the cells having mutant caldcsmons might not bc rounded during prophase. Other possible effects in these manipulated cells might be blockage or retardation of cytokinesis. It should be emphasized that the phosphorylation of caldesmon must be one of many biochemical events that control re-organization of microfilaments during mitosis. A variety of microfilament- and microtubuleassociated proteins are most likely to be modified, at various stages of mitosis, for the precise control of cytoskeletal organization in both spatial and temporal ways. It is therefore important to elucidate what components are modified at which stages of mitosis and how such modifications lead to massive alterations in thc cytoskeletal organization such as cytokinesis. Such studies will help us to understand one of the most
fascinating of biological problems, how cells integrate temporal and spatial information for cytokinesis. Acknowledgments We would like to thank Dr. Natsumi Hosoya for her excellent artwork of Fig. 1. This work was supported in part by Grant R37 CA42742 from the National Cancer Institute, Grant CD-442 from the American Cancer Society. Biomedical Research Support Grant PHS RR 07058-24, and Johnson and Johnson Discovery Fund. FM is a recipient of Research Career Development Award KO4 CA01304 from thc National Cancer Institute. References 1 SCHROEDER, T. E. (1976). Actin in dividing cells: evidence for its role in
cleavage but not mitosis, pp, 265-278. In: Cell Mordily (edited by R. Goldman, Pollard and J. Rosenbaum). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2 SANGER.J. W. AND SANGER.J. M. (1976). Actin localization during cell division, pp. 1295-1336. In: Cell Moiii~iy(edited b y R . Goldman, T. Pollard and .1. Rosenbaum). Cold Spring Harbor L.alioratory, Cold Spring Harbor, NY. 3 MARUCHI? J. (1986). Biochemical aspects of cytokinesis. fnt. Rev. Cpol. 101, 175-213. 4 MABUCHI. 1. AND OWNO,M. (1977). The effect of myosin antibody on thc division of starfish blastomeres. J. Celi Biol. 74, 251-263. 5 KNKHI, D. A. A N D LUOMIS, W. F. (1987). Antiscnsc RNA inactivation of myosin heavy chain gene expression in Dict,vosfeizuni discoideurn. Science 236. 1081- 1086. 6 DELULANNE. A. AND SPUDICH,J. A . (1987). Disruption of the Dictyosteliuni myosin heavy chain gene by homologous rcconibination. Science 236, 1086-1091. 7 YAMASHIRO. S., YAMAKITA. Y., TSHIKAWA. R . AUT) MATSUMURA. F. (1990). Mitosis-specific phosphorylation causes 83K non-muscle caldesmon to dissociate irorn microfilaments. Nature 344, 675-678. 8 BRETSCHER. A. (1986). Thin fiiament regulatory proteins of smooth- and nonmuscle cells. Nature 321, 726-727. 9 SOBUB,K.; MURAMOTO. Y., FUJITA, M. AND KAKIUCHI, S . (1981). Purification of a calmodulin-binding protcin from chicken eizzard that interacts with Factin. Proc. Nut1 Acad. Sci. USA 78, 5652-565. 10 BRFTSCHER. A. ANT) I,Y.NCH. U'.(1985). Identification and localization of initnunoreactive forms of caldesmon in smooth and non-muscle cells: a comparison with the distributions of tropomyosin and a-actinin. .I. Cell Biol. 100. 1656-1663. 11 Y.4hlASHIRO-MATSCMURA, s. AND MATSLMURA, F. (1988). Characterization of 83-kilodalton nonmusclc caldcsmon from cultured rat cell: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin. J . C M l Biol. 106, 1973-1983. 12 DINGUS, J.. Hwo, S. AND BRY~XN, J. (1Y86). Identification by nionoclonal antibodm and characlerifittion of human platelet caldesmon. J. CeN Bml. 102, 1748--1757. 13 SOSUE, K . , 1'4N.hK.4. T.. KANDA.K., ASHINO,N. 4 N D KAKIIJCHI, S. (1985). Purification and characterization of caldesrnoni7: a calniodul i n-binding protein that interacts with actin filamcnts from bovinc adrenal medula. Proc. Nor/ Read. Sci. U S A 82, 5025-5029. 14 YAMAKITA, Y., YAMASHIRO, S. AUD MATSIJMIJRA, F. (1990). Microinjection of nonmuscle and smooth muscle caldesmon into fibroblads and muscle cells. J. Cell Biol. 111, 2487-2498. 15 BRYAA-.J., IMAI,M.. LF.R R., MOORE,E., COOK.K. G. A N D LIN. W.-G. (1989). Cloning and erpresqion u1 a smooth muscle caldesmon. J. Bid. C h m . 264, 13 873-13 879. 16 HAYASHI, K., FIJJIO,Y . , KAI.U.I. AND SOSUE,K. (1991). Structural and functional relationships between h- and 1-caldesmons. .I. B i d . Chern. 266: 355-361. 17 BRYAN. J. (1990). Caldcsmon: fragments, sequence. and domain mapping. Ann. N. Y Acad. Sci. 599, 100-110. 28. 18 CHALOYICH, J. M., HEMRIC, M. E . AND VOLAZ,L. (1990). Regulation of ATP hydrolysis b y caldesmon. A novel change in thc intcraction of myosin with aclin. A n n . A'. Y . Acad. Sci. 599. 85-99. 19 I K L BM. ~ ,AND R l ; i ~S.~(1988). ~ ~ ~Binding , of caldesmon lo smooth muscle myosin. J . Bid. Cltem. 263, 3055-3058. 20 ~ ~ K A C E T I A ,P. (1987). Evidence for interaction between smooth muscle tropomposiii and caldesmon. FEBS Lett. 218. 139-142. '[.
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33 DRAETT.~, G.. BPACH. D., D R A L r m . G. A X D BEACH,D. (1988). Activation of cdr2 protein kiriase dunng mitosis in human cells: cell cycle-dependcnt phosphorylation and subunit rearrangement. Cell 54, 17-26. 34 PTTER.M..NAKAGAWA, J . . UORLL,M.. L>\BEE.J . C. .AND NIGG, E. A. (1990). in vilro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinasc. Cell 61, 591-602. 35 CHOL,Y . - H . , Brscrrorr. J. R., BPACH,D. AND GOLDMh', R. D. (1990). Intermediate filament reorganization during mitosis is mcdiatecl hy ~34"": Phosphorylation of vimentin. Celi 62, 1063-1071. 36 LAMB.N . J . C.. FLKNAXDEZ, A,. WATRIN, A,. LABBE, J-C. ANT) CAVADORE, J-C. (199U). Microinjection of p34cdc2 kinasc induccs marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts. Cell 60. 151-165. 37 KIPREOS. E. AND \VANG. .T. Y. J. (1990). Differential phosphorylation of cAbl in cell cycle determined by cdc2 kinase and phorphatase acrivily. ,E.imt.e 248, 217-220. 38 B ~ A C HD., . BASILIC~, C. AND NEWPORT, J.. cds. (1988). Cell Cyde Control in Eukaryores. Cold Spring Harbor Laboratory, Cold Spring Harhor. NY. 39 KAAN.P. AND C+RAF. T.. eds. (1986). Oncogenes and Growth Control. Berlin, Springer. 40 MATSLMLJKA, k. .4hD YAM.\SHlRo, s. (1986). Tropomyosiii ill CCll transtormation. Cancer Rev. 6 , 21-39. 41 K0rl-O\h'AI1.4, M., HAKIJKA, h.,I l L l A , K.. YAHAKA, I., SOEUE, K. A N D KAKIUCHI, S. (1984). Occurrence ot caldesmon (a calmodulin-binding protcin). in cultured cells: comparisoii of normal and transfnrmcd cclls. Proc. Nafl Acnd. Sci. USA 81. 3133-3137.
Shigeko Yamashiro and Fumio Matsumura arc at the Department of Molecular Biology and Biochemistry, Rutgers University Piscataway, New Jersey 088541059, USA.
hnnoi mc-ement
THE NEK:'\I'ORK ACADl?hfI7 OF SCIENCES
Conference on The Melanotropic Peptides Septerriber 6 to 9. 1092 Palais des Congres, Place cie la Catlieclralc, Rourn, Francc The conference will evaluate thc recent findings on biochcmistry, physiology and pharmacology on melanocyte-stimulating hormones (MSH) and melanin-concentrating hormone (MCH). The current knowledge of hormonal and neuromodulator/neurotransmittcr functions of mclanotropins will be presented. The conference will covcr all rcccnt developrncnts concerning thc melanotropic peptides, from basic research to clinical perspectives. There will be contributed poster scssions in conjunction with this conference. The deadline for submission of poster abstracts is April 15, 1992.
Conference Chairmen : Alex N. Eberlk, Ph.D., D.Sc. Hubert Vaudry, Ph.D., D.Sc. Department of Research (ZLF) Laboratory of Molecular Eiidocrinology University Hospital University of Rouen, B.P. 118 76134 Mont-Saint-Aignan Hebelstrasse 20 France CH-4031 Basel, Switzerland For abstract spccification,r and for further inlormation please contact: Conference Department, New York Academy of Sciences, 2 East 63rd Street, New York, NY 1002 , USA TEL: 212-838-0230, FAX: 212-888-2894
