Human Cyclins A and Bl Are Differentially Located in the Cell and Undergo Cell Cycle-Dependent Nuclear Transport

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and Bl during the somatic cell cycle . In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards . Cyclin A may associate with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin BI accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown . In mitotic cells, cyclin BI associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus . CYCLINs are proteins which have been implicated in the control of mitosis in all eukaryotes . They were first identified in rapidly cleaving embryonic cells, and were distinguished by their steady accumulation in interphase, followed by specific and rapid proteolysis at mitosis (Evans et al ., 1983 ; Swenson et al ., 1986 ; Standart et al ., 1987; Westendorf et al ., 1989 ; for review see Hunt, 1989 ; Pines, 1991) . Mitotic cyclin cDNAs have been cloned and sequenced from a variety of organisms, and on the basis of sequence homologies in the most conserved central 200 amino acids (the cyclin box) have been subdivided into two classes, A type and B type . Synthetic cyclin A or B mRNAs will cause meiosis when microinjected into Xenopus oocytes (Swenson et al ., 1986 ; Pines and Hunt, 1987 ; Westendorfet al ., 1989), and periodic cyclin B synthesis and destruction is necessary and sufficient tocause a Xenopus egg cell-free system tooscillate between S and M phases (Minshull et al ., 1989 ; Murray and Kirschner, 1989) . A second class of cyclins have been identified in budding yeast . These are the Gl cyclins which associate with p34cDc" (the functional homologue of p34cdc' in fission yeast) and are required at START in GI phase for the cell to enter S phase (Hadwiger et al ., 1989 ; Richardson et al ., 1989 ; Wittenberg et al ., 1990) . GI cyclins are -25% homologous in the cyclin box to the mitotic cyclins and have been identified in buddingand fission yeast (Hadwiger etal ., 1989 ; Forsburg and Nurse, 1991), and candidate cDNAs have been isolated from mammalian cells (Matsushime et al ., 1991 ; © The Rockefeller University Press, 0021-9525/91/10/1/17 $2 .00 The Journal of Cell Biology, Volume 115, Number l, October 1991 1-17 Cyclin A is degraded during metaphase and cyclin BI is precipitously destroyed at the metaphase-anaphase transition . Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin BI are associated with PSTAIRE-containing proteins . The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein . Cyclin BI is associated with p34cdc' in the cytoplasm . Thus we propose that the different localization of cyclin A and cyclin BI in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIREcontaining protein kinase subunit . Motokura et al ., 1991 ; Xiong et al ., 1991 ; Lew et al ., 1991) . Saccharomyces cerevisiae has recently been shown to have 4 mitotic cyclins (Ghiara et al ., 1991 ; Surana et al ., 1991), thereby demonstrating that Gl cyclins are a separate family from mitotic cyclins . Mitotic cyclins are believed to act through association with the highly conserved protein-serine/threonine kinase p34cdcz, the product of the cell division cycle gene cdc2 in the fission yeast Schizosaccharomyces pombe (reviewed in Norbury and Nurse, 1989 ; Nurse, 1990; Pines and Hunter, 1990b) . There is strong genetic and biochemical evidence for this association for the B-type cyclins . On the basis of sequence comparison the product of the essential Schizosaccharornyces pombe cdc13+ gene was identified as a B-type cyclin (Booher and Beach, 1988; Goebel and Byers, 1988 ; Hagan et al ., 1988 ; Solomon et al ., 1988) . In S. pombe a cdc13 allele suppresses a cold-sensitive cdc2 mutant in its G2 to M function (Booher and Beach, 1987), and conversely overexpression of p34cdc' suppresses a temperature-sensitive cdc13 mutant (Booher and Beach, 1987; Booher and Beach, 1988 ; Hagan et al ., 1988) . There is biochemical evidence that p34cdaz and p63cd°" can be co-immunoprecipitated (Booher et al ., 1989 ; Moreno et al ., 1989), and B-type cyclins can also be co-immunoprecipitated with p34cd° 2 homologues from clams, Xenopus, and human cells (Draetta et al ., 1989 ; Pines and Hunter, 1989 ; Gautier et al ., 1990) . Evidence for the physiological significance ofthe association between cyclin B and p34cdc' comes from the identification on D ecem er 2, 2017 jcb.rress.org D ow nladed fom of both these proteins as components of purified M phase promoting factor (MPF)', and of the cell cycle-dependent histone Hl kinase (Draetta et al ., 1989 ; Labbts et al ., 1989 ; Meijer et al ., 1989 ; Chambers and Langan, 1990 : Gautier et al ., 1990) . In Xenopus two types of cyclin B have been cloned (Minshull et al ., 1990), designated cyclins BI and B2 . By sequence homology, the human cyclin B clone we isolated is a cyclin Bl, and a human cyclin B2 has since been cloned by S . Reed and colleagues (S . Reed, personal communication) . We have recently shown that human cyclin A associates with p34cdc2 and with a related protein of rv 33 kD that we have termed p33 (Pines and Hunter, 1990a) . p33 is related in structure to p34edc2 ; it contains the PSTAIRE epitope found in the cdc2 family of protein kinases, and N-chlorosuccinimide treatment of p33 generates the same size fragments as p34°dc 2 . These data suggest that p33 is likely to be a protein kinase. Both Aand B-type cyclin immunocomplexes display in vitro protein kinase activity and have very similar in vitro substrate specificities (Minshull et al ., 1990) . Cyclin A levels rise and fall in advance of cyclin B; cyclin A levels increase throughout S and G2 phases and decline as cells begin metaphase, whereas cyclin B accumulates in G2 phase and persists to mid-mitosis (Minshull et al ., 1990 ; Pines and Hunter, 1990a) . The associated in vitro protein kinase activities of both cyclin A and cyclin B peak during late G2/M phase, which might suggest that they serve similar, if not interchangeable functions . Some support for this idea is the observation that either cyclin A or cyclin B alone can cause Xenopus oocytes to mature and can elicit repeated rounds of mitosis in a Xenopus cell-free system . However, there is genetic evidence from Drosophila that cyclin A and B cannot substitute for each other in the early embryonic cell cycle (Lehner and O'Farrell, 1990) . As yet no A-type cyclin homologs have been identified in yeast to confirm this observation . It seemed possible that Aand B-type cyclins might perform different, essential roles by localizing their respective associated protein kinases to different parts of the cell . Therefore we undertook a study by immmunofluorescence staining of the intracellular distribution of human cyclins A and Bl throughout the cell cycle . We show here that in human cells cyclin Bl is predominantly cytoplasmic untiljust before mitosis, whereas cyclin A accumulates in the nucleus . In metaphase, cyclin Bl binds to the spindle and to condensed chromosomes. Materials andMethods Cell Culture and Synchronization HeLa S3 TKcells were cultured at 37°C on plates in DME supplemented with 10% calfserum (complete medium) . Human foreskin fibroblasts (gift of Dr. R . Allen, Salk Institute) were cultured at 37°C, 7.5% C02 in DME/ F12 medium . Cells were synchronized at the Gl/S boundary by sequential thymidine (Sigma Chemical Co ., St . Louis, MO) and aphidicolin (Sigma Chemical Co.) treatment according to Heintz et al . (1983), and as previously described (Pines and Hunter, 1989) . Cells were synchronized in G2 phase by the addition of0.15 jAg/ml Hoechst 33342 dye (Calbiochem-Behring Corp ., La Jolla, CA) according to Tobey et al . (1990), or in pseudo-metaphase by 1 . Abbreviations used in this paper: MPF, M phase promoting factor ; SRP, signal recognition particle . The Journal of Cell Biology, Volume 115, 1991 2 the addition of 0.4 pg/ml nocodazole (Sigma Chemical Co.), for 12 h after release from a thymidine block.